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Colon_Cancer

craigpattersontelecare“toj rajani r perry m the reality of medical work the case for a new perspectiveon telemedicine virtual real “ bf01421807 hoek pd schers hj bronkhorst em vissers kcp hasselaar jgj the eï¬ectof weekly specialist palliative care teleconsultations in patients with advancedcancer a randomized clinical trial bmc med s1291601708669 ministero della salute telemedicina linee diavailabledocumentazionep6_2_2_1jsplinguaitalianoid2129 onlineatindirizzo nazionalihttpwwwsalutegovitportaleaccessed april aiom indicazioni aiom cipomo su covid19 per l™oncologia wwwaiomitwpcontentuploads202003accessedavailable20200313_covid19_indicazioni_aiomcipomocomupdfapril onlineat cox a lucas g marcu a piano m grosvenor w mold f cancer survivors™ experience with telehealth a systematic review andthematic synthesis j med internet res 19e11 102196jmir rogante m giacomozzi c grigioni m kairy d telemedicine in palliativecare a review of systematic reviews ann ist super sanita “ 104415ann_16_03_16 kruse cs krowski n rodriguez b tran l vela j brooks mtelehealth and patient satisfaction a systematic review and narrativeanalysis bmj open 7e016242 101136bmjopen2017 holzner b giesinger jm pinggera j zugal s sch¶pf f oberguggenbergeras the computerbased health evaluation software ches aelectronic patientreported outcome monitoring bmcsoftwaremedinform decis makfor gutteling jj busschbach jj de man ra darlington as logistic feasibilityin clinical practiceof health related quality ofresults of a prospective study in a large population of chronic liverpatients health qual life outcomes life measurement taenzer p bultz bd carlson le speca m degagne t olson k impact of computerized quality of life screening on physician behaviourand patient satisfaction in lung cancer outpatients psychooncology “ wright ep selby pj crawford m gillibrand a johnston c perren tj feasibility and compliance of automated measurement of quality of life inoncology practice j clin oncol “ 101200jco200311 jazieh ar alenazi th alhejazi a alofoncoloncologypatients“safif al olayaninfected with coronavirus101200go20a outcomejcoglobtelemed flodgren ginteractivehealthsandrevrachas afarmer ajtelemedicinecareoutcomes2015cd002098eï¬ectsoninzitari mprofessionalshepperdpracticesyst10100214651858cd0databasecochrane ye z hong y wu x hong d zhang y dong x[management of a colon cancer patientdisease ] zhejiang da xue xue bao yi xue banalinfected with corona viruset o™gorman ld hogenbirkin northern ontario astelemedicineunitstohealthcare telemed j e health “ 101089tmj20a measure of potentialjc driving distanceaccessto perri fc ottaiano acancertranslionna f longo f della vittoria scarpati g de angelisagainst head and necktherapy101016jtranon2019immunebiological mechanismsoncolresponseand“implicationonaletfrontiers in oncology wwwfrontiersinaugust volume 0ccrispo covid19 emergency and postemergency hisada y mackman n cancerassociated pathways and biomarkers of venousthrombosis blood “ 101182blood20170374 xu x lai y hua zc apoptosis and apoptotic body disease message andtherapeutic target potentials biosci rep 39bsr20180992 bsr20180992 zhao z bai h duan j wang j recommendations ofandlungtreatmentcancermedicalforepidemic thorac cancerpatientsindividualizedevents managementof covid19 “ commonduringadversetheoutbreakconflict of interest the authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestcopyright crispo montagnese perri grimaldi bimonte augustin amorecelentano di napoli cascella and pignata this is an openaccess distributedunder the terms of the creative commons attribution license cc by the usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this is cited in accordance with accepted academic practice no usedistribution or reproduction is permitted which does not comply with these termsfrontiers in oncology wwwfrontiersinaugust volume 0c'

cancer7500

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: craigpattersontelecare“toj rajani r perry m the reality of medical work the case for a new perspectiveon telemedicine virtual real “ bf01421807 hoek pd schers hj bronkhorst em vissers kcp hasselaar jgj the eï¬ectof weekly specialist palliative care teleconsultations in patients with advancedcancer a randomized clinical trial bmc med s1291601708669 ministero della salute telemedicina linee diavailabledocumentazionep6_2_2_1jsplinguaitalianoid2129 onlineatindirizzo nazionalihttpwwwsalutegovitportaleaccessed april aiom indicazioni aiom cipomo su covid19 per l™oncologia wwwaiomitwpcontentuploads202003accessedavailable20200313_covid19_indicazioni_aiomcipomocomupdfapril onlineat cox a lucas g marcu a piano m grosvenor w mold f cancer survivors™ experience with telehealth a systematic review andthematic synthesis j med internet res 19e11 102196jmir rogante m giacomozzi c grigioni m kairy d telemedicine in palliativecare a review of systematic reviews ann ist super sanita “ 104415ann_16_03_16 kruse cs krowski n rodriguez b tran l vela j brooks mtelehealth and patient satisfaction a systematic review and narrativeanalysis bmj open 7e016242 101136bmjopen2017 holzner b giesinger jm pinggera j zugal s sch¶pf f oberguggenbergeras the computerbased health evaluation software ches aelectronic patientreported outcome monitoring bmcsoftwaremedinform decis makfor gutteling jj busschbach jj de man ra darlington as logistic feasibilityin clinical practiceof health related quality ofresults of a prospective study in a large population of chronic liverpatients health qual life outcomes life measurement taenzer p bultz bd carlson le speca m degagne t olson k impact of computerized quality of life screening on physician behaviourand patient satisfaction in lung cancer outpatients psychooncology “ wright ep selby pj crawford m gillibrand a johnston c perren tj feasibility and compliance of automated measurement of quality of life inoncology practice j clin oncol “ 101200jco200311 jazieh ar alenazi th alhejazi a alofoncoloncologypatients“safif al olayaninfected with coronavirus101200go20a outcomejcoglobtelemed flodgren ginteractivehealthsandrevrachas afarmer ajtelemedicinecareoutcomes2015cd002098eï¬ectsoninzitari mprofessionalshepperdpracticesyst10100214651858cd0databasecochrane ye z hong y wu x hong d zhang y dong x[management of a colon cancer patientdisease ] zhejiang da xue xue bao yi xue banalinfected with corona viruset o™gorman ld hogenbirkin northern ontario astelemedicineunitstohealthcare telemed j e health “ 101089tmj20a measure of potentialjc driving distanceaccessto perri fc ottaiano acancertranslionna f longo f della vittoria scarpati g de angelisagainst head and necktherapy101016jtranon2019immunebiological mechanismsoncolresponseand“implicationonaletfrontiers in oncology wwwfrontiersinaugust volume 0ccrispo covid19 emergency and postemergency hisada y mackman n cancerassociated pathways and biomarkers of venousthrombosis blood “ 101182blood20170374 xu x lai y hua zc apoptosis and apoptotic body disease message andtherapeutic target potentials biosci rep 39bsr20180992 bsr20180992 zhao z bai h duan j wang j recommendations ofandlungtreatmentcancermedicalforepidemic thorac cancerpatientsindividualizedevents managementof covid19 “ commonduringadversetheoutbreakconflict of interest the authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestcopyright crispo montagnese perri grimaldi bimonte augustin amorecelentano di napoli cascella and pignata this is an openaccess distributedunder the terms of the creative commons attribution license cc by the usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this is cited in accordance with accepted academic practice no usedistribution or reproduction is permitted which does not comply with these termsfrontiers in oncology wwwfrontiersinaugust volume 0c' Answer:

Colon_Cancer

acute myeloid leukemia aml is the most common type ofacute leukemia in adults caused by the clonal expansion ofundiï¬erentiated myeloid progenitor cells although mostpatients with aml can achieve complete remission by inductionchemotherapy the recurrence rate remains high and thus is themain factor aï¬ecting the outcomes of aml patients relapseoften develops from minimal residual disease in the protectivebone marrow bm microenvironment a comprehensiveunderstanding of the bm microenvironment is conducive to thediagnosis and personalized treatment of aml leukemic cellscytogenetics and molecular aberrations are the main factorsfor risk stratification in patients with aml in additionthe bm microenvironment also plays a very important role thein the homing and survival ofbm microenvironment contains various componentssuchas immune cells stromal cells endothelial progenitor cellsextracellular matrix growth factors and cytokines theinteraction between leukemic cells and bm microenvironmentaï¬ects resistance to chemotherapy in aml the modulationof bm microenvironment in aml is currently undergoingpreclinical research and early clinical trials molecular inhibitorssuch as cxcr4 inhibitors vla4 inhibitors and eselectininhibitors are currently undergoing clinical trials immunecells and stromal cells are important components of the bmmicroenvironment and are also the main ‚uencing factorsof leukemia development the estimate program isa common method to explore the microenvironment of manytumors recently it has also been used to explore theprognostic genes in the microenvironment of aml patients“ most studies have focused on diï¬erentially expressedgenes degs however the interaction and relationship betweengenes are open to investigate moreover the coding genes wereextensively explored but regions that encoded lncrnas andmirnas were less wellstudiedweighted gene coexpression network analysis wgcnais an algorithm commonly used in systems biology toexplore the correlation between gene sets and the clinic functionalization is achieved by constructing a freescalecoexpression gene network wgcna can identify highlyrelated genes and aggregate them into the same genetic modulecurrently wgcna is used in multiple fields such as cancer andnervous system or to identify potential biomarker candidates ornew therapeutic targets from genetic data “the competition endogenous rna cerna hypothesis was anew regulatory mechanism between noncoding rna ncrnaand messenger rna mrna proposed by salmena in in this theory crosstalk between cernas is achieved byabbreviations aml acute myeloid leukemia auc area under curve bmbone marrow cam cell adhesion molecules cerna competing endogenousrnas deg diï¬erentially expressed gene david the database for annotationvisualization and integrated discovery go gene ontology icam1 intercellularadhesion molecule il10ra interleukin receptor subunit alpha keggkyoto encyclopedia of genes and genomes ppi proteinprotein interactiontcga the cancer genome atlas tlr6 toll like receptor tlr8 toll likereceptor tom topological overlap matrix wgcna weighted correlationnetwork analysisimmunerelated cerna network of amlcompetitively combining shared mirnas in recent yearsthe cernas hypothesis has attracted widespread attention in thestudy of molecular and biological mechanisms of tumorigenesisand development for example previous studies have foundthat lncrnarelated cernas were involved in the biologicalprocesses of glioblastoma and breast cancer theresearch on cernas of leukemia was generally based on thediï¬erential genes screened by leukemia and normal controls but no known module based on cernas network related tomicroenvironment in leukemia has been set upinformation ofthe aml cohortin this study mrnas mirnas and lncrnas data andclinicalfrom the cancergenome atlas tcga database were used to calculate theimmune and stromal scores of these aml cases using theestimate algorithm diï¬erentially expressed mrnas andlncrnas were applied to wgcna to identify the modulesmost relevant to the aml immune microenvironment thenthe immunerelated lncrnamirnamrna cerna networkwas established to screen genes with clinical significance thesefindings will help to better understand the role oftumormicroenvironment in aml and shed light on the developmentand progression of amlmaterials and methodsdata acquisitionall data sets of aml patients were downloaded from tcgadatabase1 the data used in this study met the following criteria excluding samples combined with other malignancies samples with lncrnas and mirnas and mrnas detection datafinally all lncrnas mrnas and mirnas expression profilesof aml specimens and the corresponding clinical followupdata were downloadedidentification of differentially expressedgenesthe estimate algorithm2 was used to calculate the immunescores and stromal scores of aml samples diï¬erentiallyexpressed genes degs such as lncrnas mirnas and mrnaswere identified between high and low score groups stratified bythe median value of immune scores and stromal scores usinglimma package all q values use fdr to correct the statisticalsignificance of the multiple test lncrnas and mrnas withlog fc and fdr were regarded as diï¬erentiallyexpressed while mirnas with log fc and fdr were regarded as diï¬erentially expressed then all the degs wereentered into r version auckland nz united states forcluster analysis based on the expression value of each sample inits respective data set the results were expressed in a clustergrameach column represents a sample and each row represents theexpression level of a given gene1portalgdccancergov2sourcefenetprojectsestimateprojectfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amldavid3 wasgo and pathway enrichment analysesthe database for annotation visualization and integrateddiscoveryenrichedbiological themes of degs functions particularly go geneontology terms and kegg kyoto encyclopedia of genes andgenomes pathway enrichment p was set as thecutoï¬ criterionapplied to analyzeweighted gene coexpression networkanalysiswgcna is an algorithm for identification of gene coexpressionnetworksthrough highthroughput expression profiles ofmrnas or lncrnas with diï¬erent characteristics pairwisepearson correlation analysis was used to evaluate the weightedcoexpression relationship between all datasettopics in theadjacency matrix in this study wgcna was used to analyzemrnas and lncrnas to obtain the mrnas or lncrnas mostrelevant to aml immune microenvironmentcerna network construction andanalysisaccording to the results of wgcna we selected all mrnasand lncrnas in the most relevant module turquoise anddiï¬erentially expressed mirnas to construct a cerna networkbriefly the associated cerna network in aml was constructedfollowing three stages prediction of lncrnamirna inorder to make lncrnas and mirnas map into the interactionssuccessfully we used the miranda4 and pita5 to get targetedlncrnas that mirnas may regulate prediction of mirnamrna three online databases miranda4 targetscan6 andmirwalk7 were used simultaneously for target mrna prediction construction of lncrnamirnamrna cerna networkthe cerna network was constructed based on the negativelyregulating target relationships of mirna“mrna and mirna“lncrna correlation pairsppi network constructionthe retrieval of interacting genes string database8 wasutilized to construct a protein“protein interaction ppi networkof the degs identified in the cerna network the interactingpairs with a confidence score greater than were considered assignificant and were retained the degree represents the numberof interaction partnerssurvival analysiskaplan“meier plots weretherelationship between the overall survival of aml patientsand the expression level of mrnas lncrnas and mirnasthe statistical significance of the correlation was tested by theconstructed to illuminatelogrank test and p was considered significant theanalysis was conducted using the r package of survivalstatistical analysisgraphpad prismtm san diego ca united states or rversion auckland nz united states software was usedfor all data analyses diï¬erences across groups were comparedusing kruskal“wallis test for continuous variables diï¬erenceswere considered significant when p resultsimmune and stromal scores areassociated with aml clinicalparameterswe obtained gene expression profiles and clinical informationof aml patients from tcga database supplementarymaterial among them were male and were female with a median age range “ years oldaccording to the fab classification there were cases of m0 m1 cases m2 cases m3 cases m4 cases m5 cases m6 cases and m7 case the estimate algorithm was used tocalculate the immune scores and stromal scores of aml patientsthe median immune score was range from to and the median stromal score wasˆ’ range fromˆ’ to we analyzed the relationship between immune scores andstromal scores and clinical parameters of aml patients caseswith m4 subtype aml had the highest immune scores while caseswith m3 subtype had the lowest immune scores p figure 1a similarly m4 cases had the highest stromal scoreswhereas m0 subtypes had the lowest p figure 1baccording to cytogenetics aml patients were divided intothree groups favorable intermediatenormal and poor therewas an obvious correlation between the cytogenetic risk andthe immune scores p figure 1cfavorable vsintermediate p favorable vs poor p intermediate vs poor p but no significant correlationbetween the cytogenetic risk and the stromal scores was observedp figure 1dscoreand high immunestromalusing the median immune or stromal score as a thresholdaml patients were divided into two groups with lowimmunestromalscoresurvival analysis showed that the survival rate of aml patientswith low immune scores was significantly higher than that ofpatients with high immune scores p figure 1ehowever there was no significant diï¬erence in survival betweenpatients with low stromal scores and those with high stromalscores p figure 1f3httpdavidncifcrfgov4httpwwwmicrorna5genieweizmannacilpubsmir07mir07_exehtml6httpwwwtargetscan7http12920671508tringdbidentification of differentially expressedgenes based on immune scoresand stromal scoressetting the cutoï¬ criteria as log fc and fdr we identified mrnas figure 2a and lncrnasfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure immune and stromal scores are associated with aml clinical parameters ab distribution of immune scores a and stromal scores b for aml fabsubtypes cd the correlation between immune scores c and stromal scores d and aml cytogenetic risk ef kaplan“meier survival curve based on immunese and stromal scores f aml acute myeloid leukemiafigure heatmap of differentially expressed genes in the high and low immunestromal score groups a mrnas b lncrnas and e mirnas based onimmune scores c mrnas d lncrnas and f mirnas based on stromal scoresfigure 2b based on immune scores and mrnasfigure 2c and lncrnasfigure 2d based onstromal scores setting the cutoï¬ criteria as log fc and fdr we identified and mirnas based onimmune scores figure 2e and stromal scores figure 2frespectively the degs oflow vs high immunethefrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top go terms in each of biological process were performed for functional enrichment clustering analysis a top25 significant go terms based onupregulated genes in immune scores b top25 significant go terms based on upregulated genes in stromal scores c top25 significant go terms based ondownregulated genes in immune scores d top25 significant go terms based on downregulated genes in stromal scores go gene ontologyscore or stromal score groups were illustrated in the heatmap figure aml and thus require further research to determine theirbiological contributionfunctional enrichment analysis of degsbased on the david the databasefor annotationvisualization and integrated discovery gene annotation tool weperformed go analyses of both upregulated and downregulateddegs the top go biological process indicated that theupregulated degs based on immune or stromal scores wereprimarily enriched in neutrophil degranulation regulationof immune response signal transduction and ‚ammatoryresponse figures 3ab while the downregulated degs basedon immunestromal scores were primarily enriched in rrnaprocessing regulation of translation regulation of transcriptionand cell diï¬erentiation figures 3cd subsequently weperformed kegg kyoto encyclopedia of genes and genomespathway enrichment and interrelation analysis kegg analysisrevealed that the upregulated degs were mainly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingpathway hematopoietic cell lineage and cell adhesion moleculescams pathways figures 4ab while the downregulateddegs were mainly enriched in ribosome metabolism pi3kaktsignaling pathway transcriptional dysregulation in cancer andmirnas in cancer figures 4cd above analyses revealedthatthese degs play a vital role in the development ofconstruction of weighted correlationnetwork analysis and identification ofkey modulesbased on the results of survival analysis degs based on immunescores were selected for subsequent analysis the best β valuein the lncrnamrna coexpression network was which wascalculated using the diï¬erential lncrnas diï¬erentialmrnas and their expression data in leukemia samples nextthe method of dynamic tree cutting was used to producecoexpression modules finally modules of lncrnamrnacoexpression networks were generated and the heat map plot oftopological overlap matrix tom was shown figure 5a eachmodule was calculated and plotted with its corresponding clinicalcharacteristics correlation analysis showed that turquoisemodule displayed the highest relationship with aml immunescores r which included mrnas and lncrnasfigure 5b these mrnas were further used to performthe gene enrichment analysis the genes were most relatedto neutrophil degranulation immune response ‚ammatoryresponse signal transduction and tolllike receptor signalingpathway figure 5c in addition genes were highly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top kegg pathway analysis were performed for functional enrichment clustering analysis a top25 significant kegg pathways based onupregulated genes in immune scores b top25 significant kegg pathways based on upregulated genes in stromal scores c top25 significant kegg pathwaysbased on downregulated genes in immune scores d top25 significant kegg pathways based on downregulated genes in stromal scores kegg kyotoencyclopedia of genes and genomespathway metabolic pathways and hematopoietic cell lineage bykegg analysis figure 5dcerna network constructionsince the turquoise module showed the highest relationshipwith aml immune scores we selected lncrnas and mrnas inthe turquoise module and diï¬erentially expressed mirnasbased on immune scores to construct a cerna network firstlybased on the pita and mircode online database that matchespotential mirnas with lncrnas a total of lncrnamirnapairs contained lncrnas and mirnas then we searchedfor the mrnas targeted by the diï¬erentially expressed mirnasusing three target gene prediction websites miranda mirwalkand targetscan using these websites we detected and target mrnas respectively based on the venn intersectionanalysis target mrnas were selected subsequently wematched the predicted target genes with the mrnas in theturquoise module then we constructed the cerna networkby integrating the mirnalncrnamrna interactions at lasta final lncrnamirnamrna cerna network was constructedwith lncrnas mirnas and mrnas figure 6aprotein“protein interaction ppinetwork analysisto further explore the interplay among the mrnas in cernawe constructed a ppi network based on the string theretrieval of interacting genes online database figure 6b inthe network tlr8 toll like receptor icam1 intercellularadhesion molecule tlr6 toll like receptor and il10rainterleukin receptor subunit alpha had higher degrees and respectively supplementary table the genes encoding these proteins have been confirmed tobe associated with immune microenvironment and leukemiaprogression “association between mrnas mirnasand lncrnas in cerna and overall amlsurvivalwe further analyzed the prognostic values of mrnas mirnasand lncrnas in the cerna network subjects were dividedinto highexpression and lowexpression cohorts accordingto the median value ofthese genes for overall survivalfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure wgcna was used to analyze genetic modules a cluster diagram of coexpression network modules based on topological overlap in mrna andlncrna b study the relationship between each module with their corresponding clinical characteristics c go analysis showed the gene symbols and geneinteractions in the turquoise module d kegg analysis was used to study the pathway enrichment in the turquoise module wgcna weighted correlation networkanalysis go gene ontology kegg kyoto encyclopedia of genes and genomesthrough the package survivalthe highexpression and lowexpression cohorts were splitfor the logrank testin rsoftware out of lncrnas ac0099485 cmahp cta331p31 fcgr2c grk6p1 linc00539 linc01272 mipepp3psmb8as1 rp11266l95 rp11320g101 rp11421f163rp11439e1910 rp11792a83 and stag3l2 out of mirnas hsamir125b5p and hsamir3383p and out of mrnas agpat3 ankrd27 cbx2 ccnd2cd300lb cyth1 erg gdf11 igf1r kiaa0513 kiaa0930larp1 lfng lpcat1 nrep nudt16 pou2f2 ppm1hptafr qsox2 rab3d ralgps2 siglec7 slc43a2 srsf6tnfaip2 tns3 trib1 zbtb5 znf70 and znrf1 wereassociated with overall survival according to the logrank testp representative genes are shown in figure age ‰¥ years vs years and risk group favorablevsintermediatenormal vs poor were also associated withoverall survival according to the logrank test p and respectively the above mentioned lncrnas mrnas and mirnas were brought into further multivariatecox proportional hazard regression analysis with age and riskgroup finally mrnas ccnd2 erg lpcat1 nudt16ralgps2 tnfaip2 and znf70 lncrnas cmahp fcgr2cpsmb8as1 rp11266l95 rp11320g101 rp11792a83 andstag3l2 and mirnas hsamir125b5p and hsamir3383p were independently associated with overall survival table discussionin recent years studies about the roles of gene mutationsand chromosomalin the occurrence anddevelopment of aml and their prognostic values have madesignificant progress however the bm microenvironmentwhich also plays an important role in the pathophysiologytranslocationsfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure cerna network construction and protein“protein interaction network a a lncrnamirnamrna cerna network was constructed by lncrnas mirnas and mrnas b a ppi network was constructed based on the string database the rectangle represents micrornas the circle represents mrnasand the triangle represents lncrnas the red represents upregulation in ishigh and the green represents downregulation in ishigh the size of the dot representsthe regulatory capacity of the mrna and larger points indicate stronger regulatory capability cerna competing endogenous rnas ppi protein“protein interactionprocess in aml are poorly understood therefore mosttreatments previously targeted only tumor cells butfewtargeted the tumor microenvironmentindepth researchon the microenvironment of leukemia will help to furtherunderstand the mechanism of leukemia development and mayfind new targets for microenvironment treatment this studyscreened microenvironmentrelated genes based on the tcgadatabase and further established microenvironmentrelatedlncrnamirnamrna cerna networks through wgcnafirst we calculated the immune scores and stromal scores ofaml patients based on the estimate algorithm and found thatthese scores were related to the fab typing of aml in additionimmune scores were significantly correlated with cytogenetic riskand overall survival estimate is a new algorithm to inferthe level of stromal and immune cells in tumor tissues andtumor purity using gene expression data high immunescores in the bm samples from patients with poor prognosisindicated that more immune cells were recruited in their bmfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure correlation between mrnas mirnas and lncrnas in cerna and overall aml survival in tcga kaplan“meier survival curves with the logrank testwere performed for the representative mrnas mirnas and lncrnas cerna competing endogenous rnas aml acute myeloid leukemia tcga the cancergenome atlasmicroenvironment this may be due to that aml cells activelyshape the bm environment and immune cells to promote diseaseprogression through cellular structural and functional changes however there was no significant correlation betweenstromal scores and cytogenetic risk or survival of aml patientssuggesting that the proportion of stromal cells were comparablein diï¬erent group possible explanation may be that stromal cellsplay an important role in solid tumors while its role inleukemia is not as strong as in solid tumors “then we identified diï¬erentially expressed mrnas mirnasstromaland lncrnas based on the immune scores orscores functional enrichment analysis indicated thatthesedegs were mainly involved in immune and ‚ammatoryresponses consistent with these results previous studies haveshown that the biology of the immune system is essentialfor the formation of a complex bm microenvironment in recent yearsthe immunologicalcharacteristics of aml has increased and the developmentof eï¬ective aml immunotherapy strategies has attractedwidespread attention the understanding ofin recent years important advances in cerna coexpressionnetwork research have developed rapidly the disruption offrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amltable multivariate cox proportional hazard regression analysis of lncrnas mrnas and mirnasgenesccnd2erglpcat1nudt16ralgps2tnfaip2znf70cmahpfcgr2cpsmb8as1rp11266l95rp11320g101rp11792a83stag3l2hsamir125b5phsamir3383phr 95ci “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “pthe cerna network balance is a major cause for tumorigenesis therefore understanding the complex interactions betweendiï¬erent cerna networks will lead to an indepth understandingof gene regulatory networks and has implications for cancertreatment in addition the lncrnamirnamrna cernanetwork can predict the prognosis of the disease for examplea lncrnamirnamrna cerna network was established basedon rnaseq data of breast cancer from tcga which consistsof mirnas lncrnas and mrnas multiplexcox regression analyses showed that four of these lncrnasadamts9as1 linc00536 al3914211 and linc00491 havesignificant prognostic value wang identified lncrnas mirnas and mrnas from a database toconstruct a lncrnamirnamrna cerna network inthe network a univariate and multivariate cox proportionalhazard regression analysis was used to establish a survivalmodel with target mrnas hoxa9 insr krit1 mybspry2 ube2v1 wee1 and znf711 where auc area undercurve is indicating the sensitivity and specificity ofprognostic prediction however the screening of diï¬erentialgenes in this study was based on leukemia patients andnormal people and did not focus on tumor microenvironmentso far there is no cerna research based on the leukemiamicroenvironment in our research the screening of diï¬erentialgenes was based on the immune score then wgcna wasused to identify the modules most relevantto the amlimmune microenvironment then using wgcna and mirnaprediction websites a lncrnamirnamrna cerna networkconsisting of lncrnas mirnas and mrnaswas constructedsubsequently we built a ppi network predicting theinteraction among the proteins encoded by the degs inthe cerna network tlr8 icam1 tlr6 and il10ra hadhigher degrees tlr8 and tlr6 are members of the tolllike receptor family which is upstream to the transcriptionittransportationthe innate immune system andfactor nfκb and part ofplays an importantin progression of aml roleicam1 is one of the cams a large class of transmembraneproteinsinvolved in the binding of cells to another cellor extracellular matrix and involved in cell proliferationdiï¬erentiation movementandtissue structure the protein encoded by il10ra is areceptor for interleukin which has been shown to mediatethe immunosuppressive signal ofinterleukin and thusinhibits the synthesis of pro‚ammatory cytokines and isreported to promote survival of progenitor myeloid cellsthrough the insulin receptor substrate2pi3kakt pathway these results indicated that this novel cerna networkwere closely associated with immune microenvironment andprogression of amlapoptosisfurthermore lncrnas mirnas and mrnas withprognostic significance were screened out which could be usedas biomarkers for prognosis among the genes with prognosticsignificance in our module of immunerelated cerna networkthere were aml related reports about cbx2 ccnd2 ergigf1r larp1 lfng nudt16 pou2f2 ptafr rab3dsiglec7 srsf6 tnfaip2 trib1 zbtb5 and znrf1 themost reported of which were erg ccnd2 and ifg1r ergtranslocation was involved in the occurrence and developmentof aml and high expression of erg was a poor prognosticfactor for patients with normal karyotype aml ccnd2mutations were more common in cbfaml and it was also afrequent mutation event in t aml ccnd2 leadedto increased phosphorylation of retinoblastoma proteins leadingto significant cell cycle changes and increased proliferation ofaml cell lines nicolas chapuis found that igf1spontaneous lesions played a key role in pi3kakt activation ofaml cells providing strong evidence for targeting igf1r as apotential new therapy for aml the functions of agpat3ankrd27 cd300lb cyth1 gdf11 kiaa0513 kiaa0930lpcat1 nrep ppm1h qsox2 ralgps2 slc43a2 tns3and znf70 in aml have not been reported we identified lncrnas with clinical significance among them only cmahpwas reported to be related to mllpositive aml andother lncrnas have not been reported in leukemia twomirnas including hsamir125b5p and hsamir3383p havebeen reported to be associated with a variety of cancers “ but no studies have been reported related to aml all theseunreported mrnas mirnas and lncrnas may be potentialnovel biomarkers or therapeutic targets for amlis importantto note that our current research haslimitations we selected the target data from the tcga publicdatabase only through the biological algorithm method weshould further verify the results of this in clinical patientsin further studyconclusionin summary a comprehensive bioinformatics analysis wasperformed on the aml dataset in tcga with an emphasison the immune microenvironment using wgcna andfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlmirna prediction programs an immunerelated lncrnamirnamrna cerna network was established and degs withprognostic value were further identified further studies of thesegenes are needed in the clinic and may provide new insights intothe pathogenesis of aml this study increases our understandingof the complex interactions between aml tumor cells and the bmmicroenvironment and may provide novel prognostic factors andtherapeutic targetsdata availability statementall data sets of aml patients were downloaded from the cancergenome atlas tcga database portalgdccancergovauthor contributionsyfl cw and zj conceptualization and design sw dataacquisition and writing “ original draft sw ly yx and dzmethodology sw and cw data analysis and interpretationyjl and yfl writing “ review and editing yfl cw and zjproject administration all authors contributed to the andapproved the submitted versionfundingthis work was supported by the national natural sciencefoundation of china grant numbers and u1804191and the henan medical science and technology research projectgrant number acknowledgmentswe thank the tcga database for the availability of the datasupplementary materialthe supplementary materialonline202001579fullsupplementarymaterialfor this can be foundwwwfrontiersins103389foncatreferences ferrara f schiï¬er ca acute myeloid leukaemia in adults lancet “ doi 101016s0140673612617279 zeng z shi yx samudio ij wang ry ling x frolova o targetingthe leukemia microenvironment by cxcr4 inhibition overcomes resistanceto kinase inhibitors and chemotherapy in aml blood “doi 101182blood200805158311 shafat ms gnaneswaran b bowles km rushworth sa the bone marrowmicroenvironment“home of the leukemic blasts blood rev “ doi 101016jblre201703004 bullinger l döhner k döhner h genomics of acute myeloid leukemiadiagnosis and pathways j clin oncol “ doi 101200jco ayala f dewar r kieran m kalluri r contribution of bonemicroenvironment to leukemogenesis and leukemia progression leukemia “ doi 101038leu2009175 uy gl rettig mp motabi ih mcfarland k trinkaus km hladnik lm a phase study of chemosensitization with the cxcr4 antagonist plerixaforin relapsed or refractory acute myeloid leukemia blood “doi 101182blood201110383406 rashidi a uy gl targeting the microenvironmentin acute myeloidleukemia curr hematol malig rep “ doi 101007s11899 austin r smyth mj lane sw harnessing the immune system in acutemyeloid leukaemia crit rev oncol hematol “ doi 101016jcritrevonc201604020 yehudairesheï¬ s attiasturgeman s sabbah r gabay t musallam rfridmandror a abnormal morphological and functional nature ofbone marrow stromal cells provides preferential support for survival of acutemyeloid leukemia cells int j cancer “ doi 101002ijc yoshihara k shahmoradgoli m martínez e vegesna r kim h torresgarciaw inferring tumour purity and stromal and immune cell admixture fromexpression data nat commun doi 101038ncomms3612 yan h qu j cao w liu y zheng g zhang e identification of prognosticgenes in the acute myeloid leukemia immune microenvironment based ontcga data analysis cancer immunol immunother “ doi101007s00262019024087 huang s zhang b fan w zhao q yang l xin w identification ofprognostic genes in the acute myeloid leukemia microenvironment aging “ doi 1018632aging102477 ni j wu y qi f li x yu s liu s screening the cancer genome atlasdatabase for genes of prognostic value in acute myeloid leukemia front oncol doi 103389fonc201901509 langfelder p horvath s wgcna an r package for weighted correlationnetwork analysis bmc bioinformatics doi yao y zhang t qi l zhou c wei j feng f integrated analysis of coexpression and cerna network identifies five lncrnas as prognostic markersfor breast cancer j cell mol med “ doi 101111jcmm14721 spiers h hannon e schalkwyk lc smith r wong ccy o™donovan mc methylomic trajectories across human fetal brain development genomeres “ doi 101101gr180273114 liu q jiang c xu j zhao mt van bortle k cheng x genomewide temporal profiling of transcriptome and open chromatin of earlycardiomyocyte diï¬erentiation derived from hipscs and hescs circulat res “ doi 101161circresaha116310456 salmena l poliseno l tay y kats l pandolfi pp a cerna hypothesisthe rosetta stone of a hidden rna language cell “ doi101016jcell201107014 karreth fa pandolfi pp cerna crosstalk in cancer when cebling rivalriesgo awry cancer discov “ doi 10115821598290cd13 zhang k li q kang x wang y wang s identification and functionalcharacterization of lncrnas acting as cerna involved in the malignantprogression of glioblastoma multiforme oncol rep “ doi103892or20165070 wang jd zhou hs tu xx he y liu qf liu q prediction ofcompeting endogenous rna coexpression network as prognostic markers inaml aging “ doi 1018632aging101985 ritchie me phipson b wu d hu y law cw shi w limma powersdiï¬erential expression analyses for rnasequencing and microarray studiesnucleic acids res 43e47 doi 101093nargkv007 kanehisa m goto s kegg kyoto encyclopedia of genes and genomes nucleicacids res “ brenner ak bruserud ø functional tolllike receptors tlrs are expressed

cancer7501

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: acute myeloid leukemia aml is the most common type ofacute leukemia in adults caused by the clonal expansion ofundiï¬erentiated myeloid progenitor cells although mostpatients with aml can achieve complete remission by inductionchemotherapy the recurrence rate remains high and thus is themain factor aï¬ecting the outcomes of aml patients relapseoften develops from minimal residual disease in the protectivebone marrow bm microenvironment a comprehensiveunderstanding of the bm microenvironment is conducive to thediagnosis and personalized treatment of aml leukemic cellscytogenetics and molecular aberrations are the main factorsfor risk stratification in patients with aml in additionthe bm microenvironment also plays a very important role thein the homing and survival ofbm microenvironment contains various componentssuchas immune cells stromal cells endothelial progenitor cellsextracellular matrix growth factors and cytokines theinteraction between leukemic cells and bm microenvironmentaï¬ects resistance to chemotherapy in aml the modulationof bm microenvironment in aml is currently undergoingpreclinical research and early clinical trials molecular inhibitorssuch as cxcr4 inhibitors vla4 inhibitors and eselectininhibitors are currently undergoing clinical trials immunecells and stromal cells are important components of the bmmicroenvironment and are also the main ‚uencing factorsof leukemia development the estimate program isa common method to explore the microenvironment of manytumors recently it has also been used to explore theprognostic genes in the microenvironment of aml patients“ most studies have focused on diï¬erentially expressedgenes degs however the interaction and relationship betweengenes are open to investigate moreover the coding genes wereextensively explored but regions that encoded lncrnas andmirnas were less wellstudiedweighted gene coexpression network analysis wgcnais an algorithm commonly used in systems biology toexplore the correlation between gene sets and the clinic functionalization is achieved by constructing a freescalecoexpression gene network wgcna can identify highlyrelated genes and aggregate them into the same genetic modulecurrently wgcna is used in multiple fields such as cancer andnervous system or to identify potential biomarker candidates ornew therapeutic targets from genetic data “the competition endogenous rna cerna hypothesis was anew regulatory mechanism between noncoding rna ncrnaand messenger rna mrna proposed by salmena in in this theory crosstalk between cernas is achieved byabbreviations aml acute myeloid leukemia auc area under curve bmbone marrow cam cell adhesion molecules cerna competing endogenousrnas deg diï¬erentially expressed gene david the database for annotationvisualization and integrated discovery go gene ontology icam1 intercellularadhesion molecule il10ra interleukin receptor subunit alpha keggkyoto encyclopedia of genes and genomes ppi proteinprotein interactiontcga the cancer genome atlas tlr6 toll like receptor tlr8 toll likereceptor tom topological overlap matrix wgcna weighted correlationnetwork analysisimmunerelated cerna network of amlcompetitively combining shared mirnas in recent yearsthe cernas hypothesis has attracted widespread attention in thestudy of molecular and biological mechanisms of tumorigenesisand development for example previous studies have foundthat lncrnarelated cernas were involved in the biologicalprocesses of glioblastoma and breast cancer theresearch on cernas of leukemia was generally based on thediï¬erential genes screened by leukemia and normal controls but no known module based on cernas network related tomicroenvironment in leukemia has been set upinformation ofthe aml cohortin this study mrnas mirnas and lncrnas data andclinicalfrom the cancergenome atlas tcga database were used to calculate theimmune and stromal scores of these aml cases using theestimate algorithm diï¬erentially expressed mrnas andlncrnas were applied to wgcna to identify the modulesmost relevant to the aml immune microenvironment thenthe immunerelated lncrnamirnamrna cerna networkwas established to screen genes with clinical significance thesefindings will help to better understand the role oftumormicroenvironment in aml and shed light on the developmentand progression of amlmaterials and methodsdata acquisitionall data sets of aml patients were downloaded from tcgadatabase1 the data used in this study met the following criteria excluding samples combined with other malignancies samples with lncrnas and mirnas and mrnas detection datafinally all lncrnas mrnas and mirnas expression profilesof aml specimens and the corresponding clinical followupdata were downloadedidentification of differentially expressedgenesthe estimate algorithm2 was used to calculate the immunescores and stromal scores of aml samples diï¬erentiallyexpressed genes degs such as lncrnas mirnas and mrnaswere identified between high and low score groups stratified bythe median value of immune scores and stromal scores usinglimma package all q values use fdr to correct the statisticalsignificance of the multiple test lncrnas and mrnas withlog fc and fdr were regarded as diï¬erentiallyexpressed while mirnas with log fc and fdr were regarded as diï¬erentially expressed then all the degs wereentered into r version auckland nz united states forcluster analysis based on the expression value of each sample inits respective data set the results were expressed in a clustergrameach column represents a sample and each row represents theexpression level of a given gene1portalgdccancergov2sourcefenetprojectsestimateprojectfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amldavid3 wasgo and pathway enrichment analysesthe database for annotation visualization and integrateddiscoveryenrichedbiological themes of degs functions particularly go geneontology terms and kegg kyoto encyclopedia of genes andgenomes pathway enrichment p was set as thecutoï¬ criterionapplied to analyzeweighted gene coexpression networkanalysiswgcna is an algorithm for identification of gene coexpressionnetworksthrough highthroughput expression profiles ofmrnas or lncrnas with diï¬erent characteristics pairwisepearson correlation analysis was used to evaluate the weightedcoexpression relationship between all datasettopics in theadjacency matrix in this study wgcna was used to analyzemrnas and lncrnas to obtain the mrnas or lncrnas mostrelevant to aml immune microenvironmentcerna network construction andanalysisaccording to the results of wgcna we selected all mrnasand lncrnas in the most relevant module turquoise anddiï¬erentially expressed mirnas to construct a cerna networkbriefly the associated cerna network in aml was constructedfollowing three stages prediction of lncrnamirna inorder to make lncrnas and mirnas map into the interactionssuccessfully we used the miranda4 and pita5 to get targetedlncrnas that mirnas may regulate prediction of mirnamrna three online databases miranda4 targetscan6 andmirwalk7 were used simultaneously for target mrna prediction construction of lncrnamirnamrna cerna networkthe cerna network was constructed based on the negativelyregulating target relationships of mirna“mrna and mirna“lncrna correlation pairsppi network constructionthe retrieval of interacting genes string database8 wasutilized to construct a protein“protein interaction ppi networkof the degs identified in the cerna network the interactingpairs with a confidence score greater than were considered assignificant and were retained the degree represents the numberof interaction partnerssurvival analysiskaplan“meier plots weretherelationship between the overall survival of aml patientsand the expression level of mrnas lncrnas and mirnasthe statistical significance of the correlation was tested by theconstructed to illuminatelogrank test and p was considered significant theanalysis was conducted using the r package of survivalstatistical analysisgraphpad prismtm san diego ca united states or rversion auckland nz united states software was usedfor all data analyses diï¬erences across groups were comparedusing kruskal“wallis test for continuous variables diï¬erenceswere considered significant when p resultsimmune and stromal scores areassociated with aml clinicalparameterswe obtained gene expression profiles and clinical informationof aml patients from tcga database supplementarymaterial among them were male and were female with a median age range “ years oldaccording to the fab classification there were cases of m0 m1 cases m2 cases m3 cases m4 cases m5 cases m6 cases and m7 case the estimate algorithm was used tocalculate the immune scores and stromal scores of aml patientsthe median immune score was range from to and the median stromal score wasˆ’ range fromˆ’ to we analyzed the relationship between immune scores andstromal scores and clinical parameters of aml patients caseswith m4 subtype aml had the highest immune scores while caseswith m3 subtype had the lowest immune scores p figure 1a similarly m4 cases had the highest stromal scoreswhereas m0 subtypes had the lowest p figure 1baccording to cytogenetics aml patients were divided intothree groups favorable intermediatenormal and poor therewas an obvious correlation between the cytogenetic risk andthe immune scores p figure 1cfavorable vsintermediate p favorable vs poor p intermediate vs poor p but no significant correlationbetween the cytogenetic risk and the stromal scores was observedp figure 1dscoreand high immunestromalusing the median immune or stromal score as a thresholdaml patients were divided into two groups with lowimmunestromalscoresurvival analysis showed that the survival rate of aml patientswith low immune scores was significantly higher than that ofpatients with high immune scores p figure 1ehowever there was no significant diï¬erence in survival betweenpatients with low stromal scores and those with high stromalscores p figure 1f3httpdavidncifcrfgov4httpwwwmicrorna5genieweizmannacilpubsmir07mir07_exehtml6httpwwwtargetscan7http12920671508tringdbidentification of differentially expressedgenes based on immune scoresand stromal scoressetting the cutoï¬ criteria as log fc and fdr we identified mrnas figure 2a and lncrnasfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure immune and stromal scores are associated with aml clinical parameters ab distribution of immune scores a and stromal scores b for aml fabsubtypes cd the correlation between immune scores c and stromal scores d and aml cytogenetic risk ef kaplan“meier survival curve based on immunese and stromal scores f aml acute myeloid leukemiafigure heatmap of differentially expressed genes in the high and low immunestromal score groups a mrnas b lncrnas and e mirnas based onimmune scores c mrnas d lncrnas and f mirnas based on stromal scoresfigure 2b based on immune scores and mrnasfigure 2c and lncrnasfigure 2d based onstromal scores setting the cutoï¬ criteria as log fc and fdr we identified and mirnas based onimmune scores figure 2e and stromal scores figure 2frespectively the degs oflow vs high immunethefrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top go terms in each of biological process were performed for functional enrichment clustering analysis a top25 significant go terms based onupregulated genes in immune scores b top25 significant go terms based on upregulated genes in stromal scores c top25 significant go terms based ondownregulated genes in immune scores d top25 significant go terms based on downregulated genes in stromal scores go gene ontologyscore or stromal score groups were illustrated in the heatmap figure aml and thus require further research to determine theirbiological contributionfunctional enrichment analysis of degsbased on the david the databasefor annotationvisualization and integrated discovery gene annotation tool weperformed go analyses of both upregulated and downregulateddegs the top go biological process indicated that theupregulated degs based on immune or stromal scores wereprimarily enriched in neutrophil degranulation regulationof immune response signal transduction and ‚ammatoryresponse figures 3ab while the downregulated degs basedon immunestromal scores were primarily enriched in rrnaprocessing regulation of translation regulation of transcriptionand cell diï¬erentiation figures 3cd subsequently weperformed kegg kyoto encyclopedia of genes and genomespathway enrichment and interrelation analysis kegg analysisrevealed that the upregulated degs were mainly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingpathway hematopoietic cell lineage and cell adhesion moleculescams pathways figures 4ab while the downregulateddegs were mainly enriched in ribosome metabolism pi3kaktsignaling pathway transcriptional dysregulation in cancer andmirnas in cancer figures 4cd above analyses revealedthatthese degs play a vital role in the development ofconstruction of weighted correlationnetwork analysis and identification ofkey modulesbased on the results of survival analysis degs based on immunescores were selected for subsequent analysis the best β valuein the lncrnamrna coexpression network was which wascalculated using the diï¬erential lncrnas diï¬erentialmrnas and their expression data in leukemia samples nextthe method of dynamic tree cutting was used to producecoexpression modules finally modules of lncrnamrnacoexpression networks were generated and the heat map plot oftopological overlap matrix tom was shown figure 5a eachmodule was calculated and plotted with its corresponding clinicalcharacteristics correlation analysis showed that turquoisemodule displayed the highest relationship with aml immunescores r which included mrnas and lncrnasfigure 5b these mrnas were further used to performthe gene enrichment analysis the genes were most relatedto neutrophil degranulation immune response ‚ammatoryresponse signal transduction and tolllike receptor signalingpathway figure 5c in addition genes were highly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top kegg pathway analysis were performed for functional enrichment clustering analysis a top25 significant kegg pathways based onupregulated genes in immune scores b top25 significant kegg pathways based on upregulated genes in stromal scores c top25 significant kegg pathwaysbased on downregulated genes in immune scores d top25 significant kegg pathways based on downregulated genes in stromal scores kegg kyotoencyclopedia of genes and genomespathway metabolic pathways and hematopoietic cell lineage bykegg analysis figure 5dcerna network constructionsince the turquoise module showed the highest relationshipwith aml immune scores we selected lncrnas and mrnas inthe turquoise module and diï¬erentially expressed mirnasbased on immune scores to construct a cerna network firstlybased on the pita and mircode online database that matchespotential mirnas with lncrnas a total of lncrnamirnapairs contained lncrnas and mirnas then we searchedfor the mrnas targeted by the diï¬erentially expressed mirnasusing three target gene prediction websites miranda mirwalkand targetscan using these websites we detected and target mrnas respectively based on the venn intersectionanalysis target mrnas were selected subsequently wematched the predicted target genes with the mrnas in theturquoise module then we constructed the cerna networkby integrating the mirnalncrnamrna interactions at lasta final lncrnamirnamrna cerna network was constructedwith lncrnas mirnas and mrnas figure 6aprotein“protein interaction ppinetwork analysisto further explore the interplay among the mrnas in cernawe constructed a ppi network based on the string theretrieval of interacting genes online database figure 6b inthe network tlr8 toll like receptor icam1 intercellularadhesion molecule tlr6 toll like receptor and il10rainterleukin receptor subunit alpha had higher degrees and respectively supplementary table the genes encoding these proteins have been confirmed tobe associated with immune microenvironment and leukemiaprogression “association between mrnas mirnasand lncrnas in cerna and overall amlsurvivalwe further analyzed the prognostic values of mrnas mirnasand lncrnas in the cerna network subjects were dividedinto highexpression and lowexpression cohorts accordingto the median value ofthese genes for overall survivalfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure wgcna was used to analyze genetic modules a cluster diagram of coexpression network modules based on topological overlap in mrna andlncrna b study the relationship between each module with their corresponding clinical characteristics c go analysis showed the gene symbols and geneinteractions in the turquoise module d kegg analysis was used to study the pathway enrichment in the turquoise module wgcna weighted correlation networkanalysis go gene ontology kegg kyoto encyclopedia of genes and genomesthrough the package survivalthe highexpression and lowexpression cohorts were splitfor the logrank testin rsoftware out of lncrnas ac0099485 cmahp cta331p31 fcgr2c grk6p1 linc00539 linc01272 mipepp3psmb8as1 rp11266l95 rp11320g101 rp11421f163rp11439e1910 rp11792a83 and stag3l2 out of mirnas hsamir125b5p and hsamir3383p and out of mrnas agpat3 ankrd27 cbx2 ccnd2cd300lb cyth1 erg gdf11 igf1r kiaa0513 kiaa0930larp1 lfng lpcat1 nrep nudt16 pou2f2 ppm1hptafr qsox2 rab3d ralgps2 siglec7 slc43a2 srsf6tnfaip2 tns3 trib1 zbtb5 znf70 and znrf1 wereassociated with overall survival according to the logrank testp representative genes are shown in figure age ‰¥ years vs years and risk group favorablevsintermediatenormal vs poor were also associated withoverall survival according to the logrank test p and respectively the above mentioned lncrnas mrnas and mirnas were brought into further multivariatecox proportional hazard regression analysis with age and riskgroup finally mrnas ccnd2 erg lpcat1 nudt16ralgps2 tnfaip2 and znf70 lncrnas cmahp fcgr2cpsmb8as1 rp11266l95 rp11320g101 rp11792a83 andstag3l2 and mirnas hsamir125b5p and hsamir3383p were independently associated with overall survival table discussionin recent years studies about the roles of gene mutationsand chromosomalin the occurrence anddevelopment of aml and their prognostic values have madesignificant progress however the bm microenvironmentwhich also plays an important role in the pathophysiologytranslocationsfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure cerna network construction and protein“protein interaction network a a lncrnamirnamrna cerna network was constructed by lncrnas mirnas and mrnas b a ppi network was constructed based on the string database the rectangle represents micrornas the circle represents mrnasand the triangle represents lncrnas the red represents upregulation in ishigh and the green represents downregulation in ishigh the size of the dot representsthe regulatory capacity of the mrna and larger points indicate stronger regulatory capability cerna competing endogenous rnas ppi protein“protein interactionprocess in aml are poorly understood therefore mosttreatments previously targeted only tumor cells butfewtargeted the tumor microenvironmentindepth researchon the microenvironment of leukemia will help to furtherunderstand the mechanism of leukemia development and mayfind new targets for microenvironment treatment this studyscreened microenvironmentrelated genes based on the tcgadatabase and further established microenvironmentrelatedlncrnamirnamrna cerna networks through wgcnafirst we calculated the immune scores and stromal scores ofaml patients based on the estimate algorithm and found thatthese scores were related to the fab typing of aml in additionimmune scores were significantly correlated with cytogenetic riskand overall survival estimate is a new algorithm to inferthe level of stromal and immune cells in tumor tissues andtumor purity using gene expression data high immunescores in the bm samples from patients with poor prognosisindicated that more immune cells were recruited in their bmfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure correlation between mrnas mirnas and lncrnas in cerna and overall aml survival in tcga kaplan“meier survival curves with the logrank testwere performed for the representative mrnas mirnas and lncrnas cerna competing endogenous rnas aml acute myeloid leukemia tcga the cancergenome atlasmicroenvironment this may be due to that aml cells activelyshape the bm environment and immune cells to promote diseaseprogression through cellular structural and functional changes however there was no significant correlation betweenstromal scores and cytogenetic risk or survival of aml patientssuggesting that the proportion of stromal cells were comparablein diï¬erent group possible explanation may be that stromal cellsplay an important role in solid tumors while its role inleukemia is not as strong as in solid tumors “then we identified diï¬erentially expressed mrnas mirnasstromaland lncrnas based on the immune scores orscores functional enrichment analysis indicated thatthesedegs were mainly involved in immune and ‚ammatoryresponses consistent with these results previous studies haveshown that the biology of the immune system is essentialfor the formation of a complex bm microenvironment in recent yearsthe immunologicalcharacteristics of aml has increased and the developmentof eï¬ective aml immunotherapy strategies has attractedwidespread attention the understanding ofin recent years important advances in cerna coexpressionnetwork research have developed rapidly the disruption offrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amltable multivariate cox proportional hazard regression analysis of lncrnas mrnas and mirnasgenesccnd2erglpcat1nudt16ralgps2tnfaip2znf70cmahpfcgr2cpsmb8as1rp11266l95rp11320g101rp11792a83stag3l2hsamir125b5phsamir3383phr 95ci “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “pthe cerna network balance is a major cause for tumorigenesis therefore understanding the complex interactions betweendiï¬erent cerna networks will lead to an indepth understandingof gene regulatory networks and has implications for cancertreatment in addition the lncrnamirnamrna cernanetwork can predict the prognosis of the disease for examplea lncrnamirnamrna cerna network was established basedon rnaseq data of breast cancer from tcga which consistsof mirnas lncrnas and mrnas multiplexcox regression analyses showed that four of these lncrnasadamts9as1 linc00536 al3914211 and linc00491 havesignificant prognostic value wang identified lncrnas mirnas and mrnas from a database toconstruct a lncrnamirnamrna cerna network inthe network a univariate and multivariate cox proportionalhazard regression analysis was used to establish a survivalmodel with target mrnas hoxa9 insr krit1 mybspry2 ube2v1 wee1 and znf711 where auc area undercurve is indicating the sensitivity and specificity ofprognostic prediction however the screening of diï¬erentialgenes in this study was based on leukemia patients andnormal people and did not focus on tumor microenvironmentso far there is no cerna research based on the leukemiamicroenvironment in our research the screening of diï¬erentialgenes was based on the immune score then wgcna wasused to identify the modules most relevantto the amlimmune microenvironment then using wgcna and mirnaprediction websites a lncrnamirnamrna cerna networkconsisting of lncrnas mirnas and mrnaswas constructedsubsequently we built a ppi network predicting theinteraction among the proteins encoded by the degs inthe cerna network tlr8 icam1 tlr6 and il10ra hadhigher degrees tlr8 and tlr6 are members of the tolllike receptor family which is upstream to the transcriptionittransportationthe innate immune system andfactor nfκb and part ofplays an importantin progression of aml roleicam1 is one of the cams a large class of transmembraneproteinsinvolved in the binding of cells to another cellor extracellular matrix and involved in cell proliferationdiï¬erentiation movementandtissue structure the protein encoded by il10ra is areceptor for interleukin which has been shown to mediatethe immunosuppressive signal ofinterleukin and thusinhibits the synthesis of pro‚ammatory cytokines and isreported to promote survival of progenitor myeloid cellsthrough the insulin receptor substrate2pi3kakt pathway these results indicated that this novel cerna networkwere closely associated with immune microenvironment andprogression of amlapoptosisfurthermore lncrnas mirnas and mrnas withprognostic significance were screened out which could be usedas biomarkers for prognosis among the genes with prognosticsignificance in our module of immunerelated cerna networkthere were aml related reports about cbx2 ccnd2 ergigf1r larp1 lfng nudt16 pou2f2 ptafr rab3dsiglec7 srsf6 tnfaip2 trib1 zbtb5 and znrf1 themost reported of which were erg ccnd2 and ifg1r ergtranslocation was involved in the occurrence and developmentof aml and high expression of erg was a poor prognosticfactor for patients with normal karyotype aml ccnd2mutations were more common in cbfaml and it was also afrequent mutation event in t aml ccnd2 leadedto increased phosphorylation of retinoblastoma proteins leadingto significant cell cycle changes and increased proliferation ofaml cell lines nicolas chapuis found that igf1spontaneous lesions played a key role in pi3kakt activation ofaml cells providing strong evidence for targeting igf1r as apotential new therapy for aml the functions of agpat3ankrd27 cd300lb cyth1 gdf11 kiaa0513 kiaa0930lpcat1 nrep ppm1h qsox2 ralgps2 slc43a2 tns3and znf70 in aml have not been reported we identified lncrnas with clinical significance among them only cmahpwas reported to be related to mllpositive aml andother lncrnas have not been reported in leukemia twomirnas including hsamir125b5p and hsamir3383p havebeen reported to be associated with a variety of cancers “ but no studies have been reported related to aml all theseunreported mrnas mirnas and lncrnas may be potentialnovel biomarkers or therapeutic targets for amlis importantto note that our current research haslimitations we selected the target data from the tcga publicdatabase only through the biological algorithm method weshould further verify the results of this in clinical patientsin further studyconclusionin summary a comprehensive bioinformatics analysis wasperformed on the aml dataset in tcga with an emphasison the immune microenvironment using wgcna andfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlmirna prediction programs an immunerelated lncrnamirnamrna cerna network was established and degs withprognostic value were further identified further studies of thesegenes are needed in the clinic and may provide new insights intothe pathogenesis of aml this study increases our understandingof the complex interactions between aml tumor cells and the bmmicroenvironment and may provide novel prognostic factors andtherapeutic targetsdata availability statementall data sets of aml patients were downloaded from the cancergenome atlas tcga database portalgdccancergovauthor contributionsyfl cw and zj conceptualization and design sw dataacquisition and writing “ original draft sw ly yx and dzmethodology sw and cw data analysis and interpretationyjl and yfl writing “ review and editing yfl cw and zjproject administration all authors contributed to the andapproved the submitted versionfundingthis work was supported by the national natural sciencefoundation of china grant numbers and u1804191and the henan medical science and technology research projectgrant number acknowledgmentswe thank the tcga database for the availability of the datasupplementary materialthe supplementary materialonline202001579fullsupplementarymaterialfor this can be foundwwwfrontiersins103389foncatreferences ferrara f schiï¬er ca acute myeloid leukaemia in adults lancet “ doi 101016s0140673612617279 zeng z shi yx samudio ij wang ry ling x frolova o targetingthe leukemia microenvironment by cxcr4 inhibition overcomes resistanceto kinase inhibitors and chemotherapy in aml blood “doi 101182blood200805158311 shafat ms gnaneswaran b bowles km rushworth sa the bone marrowmicroenvironment“home of the leukemic blasts blood rev “ doi 101016jblre201703004 bullinger l döhner k döhner h genomics of acute myeloid leukemiadiagnosis and pathways j clin oncol “ doi 101200jco ayala f dewar r kieran m kalluri r contribution of bonemicroenvironment to leukemogenesis and leukemia progression leukemia “ doi 101038leu2009175 uy gl rettig mp motabi ih mcfarland k trinkaus km hladnik lm a phase study of chemosensitization with the cxcr4 antagonist plerixaforin relapsed or refractory acute myeloid leukemia blood “doi 101182blood201110383406 rashidi a uy gl targeting the microenvironmentin acute myeloidleukemia curr hematol malig rep “ doi 101007s11899 austin r smyth mj lane sw harnessing the immune system in acutemyeloid leukaemia crit rev oncol hematol “ doi 101016jcritrevonc201604020 yehudairesheï¬ s attiasturgeman s sabbah r gabay t musallam rfridmandror a abnormal morphological and functional nature ofbone marrow stromal cells provides preferential support for survival of acutemyeloid leukemia cells int j cancer “ doi 101002ijc yoshihara k shahmoradgoli m martínez e vegesna r kim h torresgarciaw inferring tumour purity and stromal and immune cell admixture fromexpression data nat commun doi 101038ncomms3612 yan h qu j cao w liu y zheng g zhang e identification of prognosticgenes in the acute myeloid leukemia immune microenvironment based ontcga data analysis cancer immunol immunother “ doi101007s00262019024087 huang s zhang b fan w zhao q yang l xin w identification ofprognostic genes in the acute myeloid leukemia microenvironment aging “ doi 1018632aging102477 ni j wu y qi f li x yu s liu s screening the cancer genome atlasdatabase for genes of prognostic value in acute myeloid leukemia front oncol doi 103389fonc201901509 langfelder p horvath s wgcna an r package for weighted correlationnetwork analysis bmc bioinformatics doi yao y zhang t qi l zhou c wei j feng f integrated analysis of coexpression and cerna network identifies five lncrnas as prognostic markersfor breast cancer j cell mol med “ doi 101111jcmm14721 spiers h hannon e schalkwyk lc smith r wong ccy o™donovan mc methylomic trajectories across human fetal brain development genomeres “ doi 101101gr180273114 liu q jiang c xu j zhao mt van bortle k cheng x genomewide temporal profiling of transcriptome and open chromatin of earlycardiomyocyte diï¬erentiation derived from hipscs and hescs circulat res “ doi 101161circresaha116310456 salmena l poliseno l tay y kats l pandolfi pp a cerna hypothesisthe rosetta stone of a hidden rna language cell “ doi101016jcell201107014 karreth fa pandolfi pp cerna crosstalk in cancer when cebling rivalriesgo awry cancer discov “ doi 10115821598290cd13 zhang k li q kang x wang y wang s identification and functionalcharacterization of lncrnas acting as cerna involved in the malignantprogression of glioblastoma multiforme oncol rep “ doi103892or20165070 wang jd zhou hs tu xx he y liu qf liu q prediction ofcompeting endogenous rna coexpression network as prognostic markers inaml aging “ doi 1018632aging101985 ritchie me phipson b wu d hu y law cw shi w limma powersdiï¬erential expression analyses for rnasequencing and microarray studiesnucleic acids res 43e47 doi 101093nargkv007 kanehisa m goto s kegg kyoto encyclopedia of genes and genomes nucleicacids res “ brenner ak bruserud ø functional tolllike receptors tlrs are expressed Answer:

Colon_Cancer

sarcomatoid carcinomas scs are extremely rare aggressivemalignant tumors characterized by distinct cellular morphology the features of this tumor were first described in by snover scs can occur in a wide variety of sitesincluding the respiratory tract digestive tract genitourinary tractbreast and thyroid glands however these tumors are rarein the digestive tract especially in the stomach as of april there are only six cases of gastric sarcomatoid carcinomagsc reported in the english medical literature these previousreports focused on the pathological and clinical manifestationsthem have not systematically described the radiologic appearanceof the tumor due to the more invasive nature and poorerprognosis of gsc than pure gastric adenocarcinoma gacand gastric lymphoma gl it is clinically beneficial to narrowdown the diï¬erential diagnoses by understanding the computedtomography ct characteristics of gsc the present studyanalyzed our experience in diagnosing five patients with gscin terms of the imaging findings and clinical features to thebest of our knowledge our study represents the largest seriesof gscs to datein addition due to the rarity of gsc the diï¬erential diagnosisbetween gsc and other types of malignant gastric tumors hasnot received much attention so we also initially explored thediï¬erential diagnosis of gsc from gac and glmaterials and methodsthe protocol was approved by the medical ethics committeeof zhengzhou universityinformed consent was obtainedfrom all patientspatient selectionfrom august to january we searched the pathologyrecords and the picture archiving and communication systemspacs of our hospital the search terms included stomachand sarcomatoid carcinomas a total of five patients wereidentified as having sc and were enrolled in the present study weretrospectively reviewed all clinical data demographic featureslaboratory findings clinical interventions and the histologicfindings of the five biopsy or operation specimensct evaluationfive gsc patients underwent ct examinations the ctscans were acquired with a 64row multidetector devicediscoveryct750hd ge healthcare waukesha wiunited states conventional axial scanning was performedbefore and after an intravenous iv injection of nonioniciohexol iopromide mgml ge medical systems mlkgand mls through a dualhead pump injector medradwarrendale pa united states the imaging parameters wereas follows tube voltage kv tube current ma fieldof view fov mm matrix — mm and sectionthickness mm finally a 20ml saline flush was performedat a rate of mlsgastric sarcomatoid carcinomacontrastenhanced ct scans were acquired with scanningdelays of s arterial phase ap and s portal venous phasepp after the iv injection of the contrast agent started the ctdose index volume for the three phases was msvimage analysistwo experienced radiologists and years of abdominal ctexperience performed a retrospective analysis of the ct imagesall analyses were performed with an aw47 workstation gehealthcare and the radiologists were blinded to the clinicalinformation of the patients the evaluated parameters includedthe tumor location gastric cardia gastric fundus gastric bodygastric angle and gastric antrum longaxis diameter shapegrowth pattern serosa condition attenuation and enhancementcharacteristics such as the enhancement pattern and degreeof enhancement the enhancement pattern of the tumor wasclassified as homogeneous or heterogeneous based on the apimage the degree of enhancement of the tumor was based ondynamic ct imaging using hu attenuation where œobviousenhancement was defined as hu œmoderate enhancementas hu and œmildly enhancement as huthe gscs were staged with the union for internationalcancer control uicc tnm staging standard all imagingfindings were compared with the postoperative pathologicalfindings the accuracy rate the number of cts coincidentwith the pathological diagnosisthe number of actual pathologicaldiagnoses — pathological evaluationthree patients underwent gastrectomy and two underwentendoscopic biopsy the three gastrectomy specimens measured cm — cm — cm cm — cm — cmcm — cm — cm respectively in two of theseand tumors the mucosal surface of the excised specimen showedulcerative masses of approximately cm — cm — cmand cm — cm the remaining specimen was a soft massmeasuring cm — cm — cm for biopsy multiple sampleswere acquired and the diameter of each sample was cmaccording to the relevant literature the diagnostic criteria fsc were generally as follows the tumor originated fromthe stomach and the tumor consisted of both carcinomatousand sarcomatoid components and the sarcomatoid componentaccounted for more than of the tissue in addition if biopsywas performed the sarcomatoid component can be seen in everysample furthermore sarcomatoid regions express epithelialmarkers such as ck or emathe specimens were fully stretched fixed and soaked in formaldehyde solution for h all biopsy specimenswere analyzed the specimens underwent routine dehydrationparaffin embedding sectioning into µm thick sectionsand hematoxylin eosin he staining immunohistochemicalstaining was performed using a roche benchmark xt automaticimmunohistochemical detector the antibodies used in thisstudy included ae1ae3 ckl ck818 epithelial membraneantigen ema vimentin p40 p63 and antigen ki67 ki67all antibodies listed above were purchased from dako dakoglostrup denmarkfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable comparison between gsc and gac glage median age rangemain symptomsepigastric discomfortpainintermittent vomitingacute hematemesisbloody stooldysphagialocationcardia and fundusbodyantrumthe longaxis diameter median size rangeshapefocal thickeningdiffuse thickeningmassserosal surfacebare areaclearunclearulcersyesnodensity characteristicsheterogeneoushomogeneousenhancement patterheterogeneoushomogeneouslymph node involvementyesnoliver involvementyesnotherapyresectionchemotherapyresection and chemotherapyneoadjuvant chemotherapyradiation therapygscgacgl “ years “ years “ years “ cm “ cm “ cm comprehensive comparative analysiseach patient with gsc was matched by age ± years year ofdiagnosis and sex to four patients with gac gl patients witheach disease were retrieved from pacs patients with gsc werecompared with those with gac gl in terms of demographicclinical and ct characteristics table resultspatient characteristicsthe patients included four men and one woman ranging inage from to years with a median age of years theclinical and ct features of these patients are summarized intables all patients had nonspecific symptoms includingabdominal discomfort epigastric discomfort nausea or vomitingthe other presenting symptoms included hematemesis or weightloss three patients underwent radical resectionin whichonly one patient was treated with adjuvant chemotherapyafter surgery and two patients chose to deny treatment inaddition we also reviewed the upper gastrointestinal radiographyresults figure the laboratory findings revealed that patient was positivefor tumor abnormal protein tap and patient was positivefor carbohydrate antigen ca125 before treatmenthemoglobin and erythrocyte count decreased in three patientsfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and pathological factors of the five gsc patientscasesexage yearscomplaintlocationmaximumdiametertumor markercmanemiatherapymetastasismmfmmsuddenhematemesisepigastricdiscomfortintermittentvomitingepigastric painepigastric painepigastric painlesser curvatureremnant stomachcardia andfunduscardia and fundusfunduscardia and fundusnormaltap ca125 normalna““rrnnonenonerc“““œ yespresentpositive œ“ noabsentnegative f female m male age in years r radical gastrectomy rn remnant gastrectomy c chemotherapyna not availabletable computed tomography features of the five gsc patientscasegross features of the tumorulcersgrowth modedensity characteristicsenhancement patterfocal thickeningfocal thickeningmassfocal thickeningfocal thickening“intracavityintracavityintracavityintracavityintracavityheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousmarginunclearunclearunclearclearunclearœ yespresentpositive œ“ noabsentnegativefigure characteristics of xray examinations of a 65yearold male patient with gsc ab reveals that there is a huge niche with irregular shapes at the smallcurvature of the stomach the niche is located inside the outline of the stomach the niche is surrounded by transparent bands with different widths that is ringembankments with irregular outlines the surrounding mucosa is thickened interrupted and the local gastric cavity is narrowedpatients and and platelet count was elevated in fourpatients patients and pathological featuresmicropathologically the gastric tumor cells showed infiltrativegrowth the cytological characteristics ofthe tumor cellsshowed obvious malignant characteristics microscopicallythe spindle cell structure and the nucleus were obviouslyatypical pleomorphic and enlarged mitotic figures were visiblefigures 2ab on immunohistochemical examination thetumor cells showed positive staining for ae1ae3 cklck818 ema p40 vimentin the ki67 index was higher than figures 2c“i all five tumors were diagnosed as gscin addition the sarcomatoid component showed spindle cellsarcomatoid morphologyct findingsof the cases of gsc were in the gastric fundus and cardiafigure was in the gastric body and was in the gastricfundus of these tumors one was a recurrence in the remnantstomach the ct manifestations of this tumor included localthickening n mass formation n the longaxisfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure histological and immunohistochemical features of gsc ab hematoxylineosin he staining showing tumor cells demonstrated spindleshapedstructures significant atypical nuclei pleomorphic nuclei and giant nuclei mitotic figures visible tumor cells showed infiltrative growth cells were stained withhematoxylin and eosin stain magnification a — b — by immunohistochemistry the tumor cells were positive for ae1ae3 c ckl d ck818 e emaf p40 g and vimentin h moreover of them were positive for ki67 i the final diagnosis was sc [magnification c“i —]diameter of the lesions ranged from to cm mean size cm in addition ulcers with an irregular base and slightlyraised borders were observed in of cases among the threepatients who underwent surgery two lesions invaded the gastricserosa and the remaining lesion invaded the gastric bare areaamong the two patients with biopsyproven gsc one patientexhibited tumor invasion of the gastric bare area the majorchanges in the ct imaging characteristics were an irregularouter layer of the gastric wall and obscuration of the perigastricfat initially the ct findings were interpreted as gac in fourcases and gl in the tumor showed predominantly inhomogeneous densityand the radiodensity values were “ hu in the noncontrastphase heterogeneous enhancement was seen in four casesdue to necrotic or cystic areas and the other tumor revealedhomogeneous enhancement the radiodensity values on the apimages ranged from to hu and to hu in thevenous phase after contrast medium injection two tumorsshowed obvious enhancement and moderate enhancementwas seen in the other three tumors the peak tumor valuewas observed in the portal phase one of the three patientswho underwentlymphsurgery demonstrated evidence ofin one patientthe boundary betweennode involvementthe lesion and the left lobe of the liver was unclear andthe area with low attenuation was confirmed by pathologythe liver withas a metastatic lesion in the rightcircular enhancement the remaining patientshowed noevidence of metastasis among the two patients with biopsyspecimens one patient was identified as having lymph nodemetastasis on ctlobe ofct staging versus pathological stagingof gscnone of the gscs were staged as t1t2 by ct or pathologythe accuracy of ct staging t3 and t4 gsc was and respectively the overall diagnostic accuracy of ctfor determining the t stage of gsc was none of the gscs were staged as n2n3 by ct or pathologythe accuracy of ct in staging n3 and n4 gsc was and respectively the overall diagnostic accuracy of ct fordetermining the n stage of gsc was the comparison of tn staging based on ct and pathology isshown in table frontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure sarcomatoid carcinoma of the stomach in 62yearold women a unenhanced ct image of stomach reveals an intraluminal mass of homogeneousattenuation with an irregular surface at the gastric fundus and cardiac region b“d contrastenhanced ct image shows obvious homogeneous enhancement ofmass with the peak value of the tumor on the portal phase in perigastric lymph nodes an enlarged and significantly enhancement lymph node can be seenb arterial phase of contrast enhancement image c portal phase of contrast enhancement image d portal phase of contrast enhancement coronal imagediscussiontable ct and pathological tn staging for comparisonsarcomatoid carcinoma is an extremely rare and complicatedmalignant tumor composed of malignant epithelial componentsand atypical spindle cells however the spindle cells of scsappear to show evidence of epithelial diï¬erentiationforexample showing epithelial markers or epithelial ultrastructuralinstead of a specific line of mesenchymalcharacteristicsdiï¬erentiation moreoverliteratureemphasizes that the sarcomatous components occupy ofthe elements involved in the present study our patients™tumor cells displayed atypical spindle shapes that expressed theepithelial phenotypethe currentsome ofsarcomatoid carcinomas can occur in almost any an wherecarcinoma can occur in the digestive system the incidencesof scs in the esophagus and liver are relatively high but scsare exceedingly rare in the stomach we could find only sixprevious reports in the english literature table between and patients with sc confirmed by pathologywere retrospectively analyzed with only five tumors occurringin the stomach the average age of the reported patientswas years range “ and that in our series was years range “ a previous study reported that the sexcaseno no no no no ctt4an0t3n0t3n1t3n0t4an1na not available t tumor n nodepathological staget4an1t3n0nanat4an0distribution of male to female gsc patients was and thecorresponding proportion in our patients was “ ithas been noticed that scs are more common in male patientsand sex is a probable risk factor gsc patients may present withepigastric pain or discomfort dysphagia nausea and vomitinghematemesis and emaciation due to thickening of the gastricwall pain or discomfort in the upper abdomen is common thesymptoms can last from a few days to several years withoutobvious specificityin the present study of the cases of gsc were recognizedin the proximal stomach and the remaining tumor was founddistal to the stomach four cases of gsc in the present study hadfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and imaging features of six previously reported cases of gcscasegenderageyearslocationsize cmshapeulcersenhanceappearancerecurrencemetastasistherapyprognosis mmffmmremnant stomachgreater curvaturelesser curvaturegastroesophageal junctionremnant stomachdistal stomachpolypoidpolypoidpolypoidulceratedpolypoidmass“““nenenenenehyperne“““““nonesurgerysurgerysurgeryendoscopysurgeryna mo d mo d mo d mo d mo dthe patient succumbed to heart failure before the surgical treatment an autopsy was performed œ yespresentpositive œ“ noabsentnegative hyper hyperdensene no evaluate mo month d dieareregarded asa longaxis diameter less than cm and the remaining tumorhad the largest longaxis diameter among our patients cmthe location distribution and longaxis diameters of the gscs inour patients were similar to those in previous reports “in the actual diagnosis processthe diagnosis of sc has always been difficult for cliniciansand pathologists especially the diï¬erential diagnosis fromcarcinosarcoma carcinosarcomastrulybiphasic neoplasms composed of distinct malignant epithelialcarcinomatous and mesenchymal sarcomatous componentsthe sarcoma components show typical specialized diï¬erentiation howeverthe termsœsarcomatoid carcinoma and œcarcinosarcoma have been usedinterchangeably in some cases therefore the understanding ofthese tumors has been hampered nevertheless we can try tofocus on whether there is a diï¬erence between these tumors froma new perspective the ct finding sc in the stomach have notbeen previously scientific reported or even detailed descriptionthere are only four simple descriptions chunchao reported that a patient with a giant sc presented a mass witha cm diameter in the antrum and body of stomach whichinfiltrated the gastric serosa the hepatic flexure of the colon andgallbladder were also involved on ct contrastenhanced ctimages showed obvious enhancement of the two lesions sato reported a patient with sc of the remnant stomachand the radiographic examination showed an elevated lesionwith a large ulcer at the gastric cardiac lesser curvature thatmeasured cm in diameter the other two reports only describeda soft tissue mass or a large tumor in the dilated stomach on the other hand within in the upper gastrointestinal tractalthough there are fewer reports of carcinosarcoma localized inthe stomach this type of tumor is still more common than sc gastric carcinosarcoma showed an elevated lesion or thickenedgastric walls in “ of all reviewed cases “ tomoaki reported a 79yearold man with gastric carcinosarcomaand his veins showed severe invasion enhanced abdominalct showed irregular thickening and slight enhancement of thegastric wall on the side of the lesser curvature with suspiciousbulky lymph nodes yoshiyuki reported a 70yearoldjapanese woman who presented with a soft tissue mass adjacentto the lesser curvature of the stomach that was lobulated andct revealed an ulcer on the lesion the contrastenhanced ctimages showed heterogeneous enhancement of the mass thefinal pathological diagnosis was gastric carcinosarcoma inthe present study we found that gsc showed local thickeningof the gastric wall and mass formation often accompaniedby ulcers the site of the disease was mostly in the proximalpart of the stomach but these tumors can also occur in theremnant stomach the signal of the tumor was homogeneousor heterogeneous on plain ct scans after contrast mediuminjection of tumors demonstrated heterogeneousenhancement on ap images due to cystic areas or necrosis inthe lesions in this study the enhancement degree of all tumorsreached a peak in the pp after contrast enhancement for thesetumors the enhancement degree in the delayed phase wasnot significantly reduced the overall enhancement mode wasdelayed enhancement in addition ct showed that four patientshad invasion into the gastric serosal region or gastric bare areatwo patients had the characteristics of enlarged perigastric orretroperitoneal lymph nodes and uneven enhancement and onepatient had invasion into the adjacent liver tissue these findingsreflect the metastatic and highly invasive characteristics of gscoverall ct and contrastenhanced ct can clearly show theprimary lesion infiltration range lymph node metastasis anddistant metastasis of gsctomographic diagnosis of gsc has not been attemptedbecause of the rarity of this entity according to the findingsof our study gsc needs to be diï¬erentiated from gac andgl on ct adenocarcinoma is the most common pathologicaltype of gastric tumor and is mainly distributed in the antrumseldomly in the body and fundus of the stomach the incidenceof gac is high in men and the median patient age is years the most common ct signs of gac are localor extensive thickening of the gastric wall mass formationincluding fungoidestype polypoidtype masses rough orsmooth serous surfaces and continuous interruption of themucosal layer tumors involving the mucosal surface can appearenhanced “ s after injecting a contrast agent the peakvalue for tumors invading the muscular layer usually appearsafter “ s and after the mucosal surface is strengthenedthe duration is longer primary gl accounts for “ ofmalignant gastric tumors and is predominantly situated in thegastric antrum gastric body and gastric fundus the incidenceof gl is high among males with a median patient age of yearsthe clinical symptoms included epigastric pain bleeding earlysatiety and fatigue the most common ct manifestations ofgl are diï¬use thickening of the gastric wall or a homogeneousfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatissue mass with slight attenuation or an appearancesoftsimilar to that of the normal gastric wall for gl becauseof hemorrhage necrosis submucosal edema or infarction thegastric wall may be heterogenous on ct gl originates froma submucosal process and gastric mucosa is commonly intactin the early stage but shows interruptions or ulceration in thelater stage after contrast medium injection most gl showedhomogeneous and slight enhancement in the delayed phase lymphoma is considered when distant structures the mesenteryretroperitoneum or other parts of the abdomen have lymphnode metastasis the ct findings may only reflect features of gsc but cannotaccurately diagnose gsc l one explore the origin of thesarcomatous portion immunohistochemistry ihc also failedto conclusively establish the origin of gsc rodrigues usedfluorescence in situ hybridization fish to confirm that sc andadenocarcinoma have a common origin that is the epithelium while primary gl originated from gastric submucosallymphoid tissuethe main treatment for localized lymphomas is eradicationof helicobacter pylori and surgical treatment whereas advanceddisease often requires radiation or chemotherapy alone surgery is the only treatment option for patients with gacadjuvant chemotherapy and chemoradiotherapy are also oftenused targeted therapy isin the exploration stage however there are currently no specific national comprehensivecancer network guidelines for the treatment of only gscbecause the tumor is relatively rare although complete surgicalresection is the most important treatment method for examplewhile chemotherapy is considered in clinical practice whetherchemotherapy can be applied for gsc and the efficacy ofchemotherapy remain controversial domblides firstevaluated the efficacy of immune checkpoint inhibitors icis forsc and found that lung sc patients exhibited high response ratesand prolonged overall survival os with icis this studyprovides a new idea for the treatment of gscbecause gl tends to be confined to the gastric wall forprolonged periods before tumor spread its prognosis is betterthan that of gac previous literature has found that scin the parotid gland lung hypopharynx liver and pancreashave poor prognoses due to metastasis or recurrence with asurvival period of a few months “ similarly gscpatients also died or developed metastasis or recurrence withina few months or it was already in the advanced stage at thefirst diagnosis all these clinical manifestations suggest that gschas a poorer prognosis than gac and gl in additiongsc can metastasize through the blood and lymph nodesand the most common sites of metastasis are the local lymphnodes and liver this conclusion is consistent with ourresearch resultsconclusionthe incidence rate of gsc is extremely low so clinicians andradiologists are not familiar with the features of this tumorbased on systematic research of this rare tumor and comparisonswith common gastric cancers we found that gsc is morecommon in men who are approximately years old and isoften accompanied by ulcers the disease is mostly located in theproximal part of the stomach and can also occur in the remnantstomach with delayed enhancement on contrastenhanced ctimages these characteristics can provide a reference for furtherresearch on gscs in the future however an accurate diagnosisof gsc depends on the combination of clinical imaging andhistopathological features due to the aggressive nature and poorprognosis of the tumor rapid clinical intervention and detailedfollowup with ct are essentialdata availability statementthe original contributions presented in the study are includedin the supplementary material further inquiries can bedirected to the corresponding authorethics statementthe studies involving human participants were reviewed andapproved by the medical ethical committee of the zhengzhouuniversity the patientsparticipants provided their writteninformed consent to participate in this study written informedconsent was obtained from the individuals for the publication ofany potentially identifiable images or data included in this author contributionsyl manuscript preparationliterature research and dataanalysis pl literature research and data analysis kf manuscriptreview and data collection kc guidance of pathologicalknowledge sy guidance of imaging knowledge jj imaging datacollection and analysis wl and xz manuscript editing jgstudy conception and design manuscript review and guarantor ofintegrity of the entire study all authors have read and approvedthe final manuscriptfundingthis work was supported by the national natural and sciencefund of china no references zhu cc li mr lin tl zhao g sarcomatoid carcinoma of the stomach acase report and literature review oncol lett “ doi ol20153460 snover dc levine gd rosaij thymic carcinoma five distinctivehistological variants am j surg pathol “ zhou dk gao bq zhang w qian xh ying lx wang wl sarcomatoidcarcinoma of the pancreas a case report world j clin cases “doi 1012998wjccv7i2236 xie y xiang y zhang d yao x sheng j yang y sarcomatoidthe “ doi 103892mmr2018and review ofthe pancreasreportcaseofcarcinomaliterature mol med repafrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinoma sato a oki e kohso h endo y uchida h hiroshige s sarcomatoidcarcinoma of the remnant stomach report of a case surg today “ doi 101007s0059501204027 nakayama y murayama h iwasaki h iwanaga s kikuchi m ikeda s gastric carcinosarcoma sarcomatoid carcinoma with rhabdomyoblastic andosteoblastic diï¬erentiation pathol int “ doi 101111j144018271997tb04540x robeycaï¬erty ss grignon dj ro jy cleary kr ayala ag ordonezng sarcomatoid carcinoma of the stomach a report of three caseswith immunohistochemical and ultrastructural observations cancer “ doi 101002109701421990040165730co2n ruess da kayser c neubauer j fichtnerfeigl s hopt ut wittel uacarcinosarcoma of the pancreas case report with comprehensive literaturereview pancreas “ doi 101097mpa0000000000000904 fujiie m yamamoto m taguchi k iwanaga a ohgaki k egashira a gastric carcinosarcoma with rhabdomyosarcomatous diï¬erentiation a casereport and review surg case rep doi 101186s407920160176z tanimura h furuta m carcinosarcoma of the stomach am j surg “ doi 101016000296106790325x kitamura s study on carcinosarcoma of stomach gan “ kumagai k kawai k kusano h matsuo k irie j tsuchiyama h a caseof socalled carcinosarcoma of the stomach gan no rinsho “ bekki t fujikuni n tanabe k yonehara s amano h noriyuki t thegastric carcinosarcoma with severe venous invasion a case report surg caserep doi 101186s4079201804218 ikeda y kosugi s nishikura k ohashi m kanda t kobayashi t gastriccarcinosarcoma presenting as a huge epigastric mass gastric cancer “ doi 101007s1012000604054 cid³n eu cuenca ij gastric adenocarcinoma is computed tomography ctuseful in preoperative staging clin med oncol “ doi cmos2641 hallinan jt venkatesh sk gastric carcinoma imaging diagnosis stagingand assessment of treatment response cancer imaging “doi gossios k katsimbri p tsianos e ct features of gastric lymphoma eurradiol “ doi 101007s003300050069 rodrigues dn hazell s miranda s crespo m fisher c de bono js sarcomatoid carcinoma of the prostate erg fluorescence insituhybridization confirms epithelial origin histopathology “doi 101111his12493 levine ms rubesin se pantongragbrown l buck jl herlinger h nonhodgkin™s lymphoma of the gastrointestinal tract radiographic findings ajram j roentgenol “ doi 102214ajr16818976941 russo ae strong ve gastric cancer etiology and management in asia and thewest annu rev med “ doi 101146annurevmed081117 domblides c leroy k monnet i mazi¨res j barlesi f gounant v efficacy of immune checkpoint inhibitors in lung sarcomatoid carcinoma jthor oncol “ doi 101016jjtho202001014 niu x sarcomatoid carcinoma in the parotid gland a review of years ofexperience laryngoscope “ doi 101002lary27474 li s jiang l he q wei w wang y zhang x the prognostic significanceof jmjd3 in primary sarcomatoid carcinoma of the lung a rare subtypeof lung cancer onco targets ther “ doi 102147otts22 dai l fang q li p liu f zhang x oncologic outcomes of patients withsarcomatoid carcinoma of the hypopharynx front oncol doi103389fonc201900950 seo n kim mj rhee h hepatic sarcomatoid carcinoma magnetic resonanceimaging evaluation by using the liver imaging reporting and data system eurradiol “ doi 101007s00330019060528 shi y chen j chen h hong x sarcomatoid carcinoma of the gallbladdera case report j int med res doi conflict of interest the authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestcopyright liu liang feng chen yue ji li zhao and gao this is anopenaccess distributed under the terms of the creative commons attributionlicense cc by the use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice no use distribution or reproduction is permitted which does not complywith these termsfrontiers in oncology wwwfrontiersinaugust volume 0c'

cancer7502

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: sarcomatoid carcinomas scs are extremely rare aggressivemalignant tumors characterized by distinct cellular morphology the features of this tumor were first described in by snover scs can occur in a wide variety of sitesincluding the respiratory tract digestive tract genitourinary tractbreast and thyroid glands however these tumors are rarein the digestive tract especially in the stomach as of april there are only six cases of gastric sarcomatoid carcinomagsc reported in the english medical literature these previousreports focused on the pathological and clinical manifestationsthem have not systematically described the radiologic appearanceof the tumor due to the more invasive nature and poorerprognosis of gsc than pure gastric adenocarcinoma gacand gastric lymphoma gl it is clinically beneficial to narrowdown the diï¬erential diagnoses by understanding the computedtomography ct characteristics of gsc the present studyanalyzed our experience in diagnosing five patients with gscin terms of the imaging findings and clinical features to thebest of our knowledge our study represents the largest seriesof gscs to datein addition due to the rarity of gsc the diï¬erential diagnosisbetween gsc and other types of malignant gastric tumors hasnot received much attention so we also initially explored thediï¬erential diagnosis of gsc from gac and glmaterials and methodsthe protocol was approved by the medical ethics committeeof zhengzhou universityinformed consent was obtainedfrom all patientspatient selectionfrom august to january we searched the pathologyrecords and the picture archiving and communication systemspacs of our hospital the search terms included stomachand sarcomatoid carcinomas a total of five patients wereidentified as having sc and were enrolled in the present study weretrospectively reviewed all clinical data demographic featureslaboratory findings clinical interventions and the histologicfindings of the five biopsy or operation specimensct evaluationfive gsc patients underwent ct examinations the ctscans were acquired with a 64row multidetector devicediscoveryct750hd ge healthcare waukesha wiunited states conventional axial scanning was performedbefore and after an intravenous iv injection of nonioniciohexol iopromide mgml ge medical systems mlkgand mls through a dualhead pump injector medradwarrendale pa united states the imaging parameters wereas follows tube voltage kv tube current ma fieldof view fov mm matrix — mm and sectionthickness mm finally a 20ml saline flush was performedat a rate of mlsgastric sarcomatoid carcinomacontrastenhanced ct scans were acquired with scanningdelays of s arterial phase ap and s portal venous phasepp after the iv injection of the contrast agent started the ctdose index volume for the three phases was msvimage analysistwo experienced radiologists and years of abdominal ctexperience performed a retrospective analysis of the ct imagesall analyses were performed with an aw47 workstation gehealthcare and the radiologists were blinded to the clinicalinformation of the patients the evaluated parameters includedthe tumor location gastric cardia gastric fundus gastric bodygastric angle and gastric antrum longaxis diameter shapegrowth pattern serosa condition attenuation and enhancementcharacteristics such as the enhancement pattern and degreeof enhancement the enhancement pattern of the tumor wasclassified as homogeneous or heterogeneous based on the apimage the degree of enhancement of the tumor was based ondynamic ct imaging using hu attenuation where œobviousenhancement was defined as hu œmoderate enhancementas hu and œmildly enhancement as huthe gscs were staged with the union for internationalcancer control uicc tnm staging standard all imagingfindings were compared with the postoperative pathologicalfindings the accuracy rate the number of cts coincidentwith the pathological diagnosisthe number of actual pathologicaldiagnoses — pathological evaluationthree patients underwent gastrectomy and two underwentendoscopic biopsy the three gastrectomy specimens measured cm — cm — cm cm — cm — cmcm — cm — cm respectively in two of theseand tumors the mucosal surface of the excised specimen showedulcerative masses of approximately cm — cm — cmand cm — cm the remaining specimen was a soft massmeasuring cm — cm — cm for biopsy multiple sampleswere acquired and the diameter of each sample was cmaccording to the relevant literature the diagnostic criteria fsc were generally as follows the tumor originated fromthe stomach and the tumor consisted of both carcinomatousand sarcomatoid components and the sarcomatoid componentaccounted for more than of the tissue in addition if biopsywas performed the sarcomatoid component can be seen in everysample furthermore sarcomatoid regions express epithelialmarkers such as ck or emathe specimens were fully stretched fixed and soaked in formaldehyde solution for h all biopsy specimenswere analyzed the specimens underwent routine dehydrationparaffin embedding sectioning into µm thick sectionsand hematoxylin eosin he staining immunohistochemicalstaining was performed using a roche benchmark xt automaticimmunohistochemical detector the antibodies used in thisstudy included ae1ae3 ckl ck818 epithelial membraneantigen ema vimentin p40 p63 and antigen ki67 ki67all antibodies listed above were purchased from dako dakoglostrup denmarkfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable comparison between gsc and gac glage median age rangemain symptomsepigastric discomfortpainintermittent vomitingacute hematemesisbloody stooldysphagialocationcardia and fundusbodyantrumthe longaxis diameter median size rangeshapefocal thickeningdiffuse thickeningmassserosal surfacebare areaclearunclearulcersyesnodensity characteristicsheterogeneoushomogeneousenhancement patterheterogeneoushomogeneouslymph node involvementyesnoliver involvementyesnotherapyresectionchemotherapyresection and chemotherapyneoadjuvant chemotherapyradiation therapygscgacgl “ years “ years “ years “ cm “ cm “ cm comprehensive comparative analysiseach patient with gsc was matched by age ± years year ofdiagnosis and sex to four patients with gac gl patients witheach disease were retrieved from pacs patients with gsc werecompared with those with gac gl in terms of demographicclinical and ct characteristics table resultspatient characteristicsthe patients included four men and one woman ranging inage from to years with a median age of years theclinical and ct features of these patients are summarized intables all patients had nonspecific symptoms includingabdominal discomfort epigastric discomfort nausea or vomitingthe other presenting symptoms included hematemesis or weightloss three patients underwent radical resectionin whichonly one patient was treated with adjuvant chemotherapyafter surgery and two patients chose to deny treatment inaddition we also reviewed the upper gastrointestinal radiographyresults figure the laboratory findings revealed that patient was positivefor tumor abnormal protein tap and patient was positivefor carbohydrate antigen ca125 before treatmenthemoglobin and erythrocyte count decreased in three patientsfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and pathological factors of the five gsc patientscasesexage yearscomplaintlocationmaximumdiametertumor markercmanemiatherapymetastasismmfmmsuddenhematemesisepigastricdiscomfortintermittentvomitingepigastric painepigastric painepigastric painlesser curvatureremnant stomachcardia andfunduscardia and fundusfunduscardia and fundusnormaltap ca125 normalna““rrnnonenonerc“““œ yespresentpositive œ“ noabsentnegative f female m male age in years r radical gastrectomy rn remnant gastrectomy c chemotherapyna not availabletable computed tomography features of the five gsc patientscasegross features of the tumorulcersgrowth modedensity characteristicsenhancement patterfocal thickeningfocal thickeningmassfocal thickeningfocal thickening“intracavityintracavityintracavityintracavityintracavityheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousmarginunclearunclearunclearclearunclearœ yespresentpositive œ“ noabsentnegativefigure characteristics of xray examinations of a 65yearold male patient with gsc ab reveals that there is a huge niche with irregular shapes at the smallcurvature of the stomach the niche is located inside the outline of the stomach the niche is surrounded by transparent bands with different widths that is ringembankments with irregular outlines the surrounding mucosa is thickened interrupted and the local gastric cavity is narrowedpatients and and platelet count was elevated in fourpatients patients and pathological featuresmicropathologically the gastric tumor cells showed infiltrativegrowth the cytological characteristics ofthe tumor cellsshowed obvious malignant characteristics microscopicallythe spindle cell structure and the nucleus were obviouslyatypical pleomorphic and enlarged mitotic figures were visiblefigures 2ab on immunohistochemical examination thetumor cells showed positive staining for ae1ae3 cklck818 ema p40 vimentin the ki67 index was higher than figures 2c“i all five tumors were diagnosed as gscin addition the sarcomatoid component showed spindle cellsarcomatoid morphologyct findingsof the cases of gsc were in the gastric fundus and cardiafigure was in the gastric body and was in the gastricfundus of these tumors one was a recurrence in the remnantstomach the ct manifestations of this tumor included localthickening n mass formation n the longaxisfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure histological and immunohistochemical features of gsc ab hematoxylineosin he staining showing tumor cells demonstrated spindleshapedstructures significant atypical nuclei pleomorphic nuclei and giant nuclei mitotic figures visible tumor cells showed infiltrative growth cells were stained withhematoxylin and eosin stain magnification a — b — by immunohistochemistry the tumor cells were positive for ae1ae3 c ckl d ck818 e emaf p40 g and vimentin h moreover of them were positive for ki67 i the final diagnosis was sc [magnification c“i —]diameter of the lesions ranged from to cm mean size cm in addition ulcers with an irregular base and slightlyraised borders were observed in of cases among the threepatients who underwent surgery two lesions invaded the gastricserosa and the remaining lesion invaded the gastric bare areaamong the two patients with biopsyproven gsc one patientexhibited tumor invasion of the gastric bare area the majorchanges in the ct imaging characteristics were an irregularouter layer of the gastric wall and obscuration of the perigastricfat initially the ct findings were interpreted as gac in fourcases and gl in the tumor showed predominantly inhomogeneous densityand the radiodensity values were “ hu in the noncontrastphase heterogeneous enhancement was seen in four casesdue to necrotic or cystic areas and the other tumor revealedhomogeneous enhancement the radiodensity values on the apimages ranged from to hu and to hu in thevenous phase after contrast medium injection two tumorsshowed obvious enhancement and moderate enhancementwas seen in the other three tumors the peak tumor valuewas observed in the portal phase one of the three patientswho underwentlymphsurgery demonstrated evidence ofin one patientthe boundary betweennode involvementthe lesion and the left lobe of the liver was unclear andthe area with low attenuation was confirmed by pathologythe liver withas a metastatic lesion in the rightcircular enhancement the remaining patientshowed noevidence of metastasis among the two patients with biopsyspecimens one patient was identified as having lymph nodemetastasis on ctlobe ofct staging versus pathological stagingof gscnone of the gscs were staged as t1t2 by ct or pathologythe accuracy of ct staging t3 and t4 gsc was and respectively the overall diagnostic accuracy of ctfor determining the t stage of gsc was none of the gscs were staged as n2n3 by ct or pathologythe accuracy of ct in staging n3 and n4 gsc was and respectively the overall diagnostic accuracy of ct fordetermining the n stage of gsc was the comparison of tn staging based on ct and pathology isshown in table frontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure sarcomatoid carcinoma of the stomach in 62yearold women a unenhanced ct image of stomach reveals an intraluminal mass of homogeneousattenuation with an irregular surface at the gastric fundus and cardiac region b“d contrastenhanced ct image shows obvious homogeneous enhancement ofmass with the peak value of the tumor on the portal phase in perigastric lymph nodes an enlarged and significantly enhancement lymph node can be seenb arterial phase of contrast enhancement image c portal phase of contrast enhancement image d portal phase of contrast enhancement coronal imagediscussiontable ct and pathological tn staging for comparisonsarcomatoid carcinoma is an extremely rare and complicatedmalignant tumor composed of malignant epithelial componentsand atypical spindle cells however the spindle cells of scsappear to show evidence of epithelial diï¬erentiationforexample showing epithelial markers or epithelial ultrastructuralinstead of a specific line of mesenchymalcharacteristicsdiï¬erentiation moreoverliteratureemphasizes that the sarcomatous components occupy ofthe elements involved in the present study our patients™tumor cells displayed atypical spindle shapes that expressed theepithelial phenotypethe currentsome ofsarcomatoid carcinomas can occur in almost any an wherecarcinoma can occur in the digestive system the incidencesof scs in the esophagus and liver are relatively high but scsare exceedingly rare in the stomach we could find only sixprevious reports in the english literature table between and patients with sc confirmed by pathologywere retrospectively analyzed with only five tumors occurringin the stomach the average age of the reported patientswas years range “ and that in our series was years range “ a previous study reported that the sexcaseno no no no no ctt4an0t3n0t3n1t3n0t4an1na not available t tumor n nodepathological staget4an1t3n0nanat4an0distribution of male to female gsc patients was and thecorresponding proportion in our patients was “ ithas been noticed that scs are more common in male patientsand sex is a probable risk factor gsc patients may present withepigastric pain or discomfort dysphagia nausea and vomitinghematemesis and emaciation due to thickening of the gastricwall pain or discomfort in the upper abdomen is common thesymptoms can last from a few days to several years withoutobvious specificityin the present study of the cases of gsc were recognizedin the proximal stomach and the remaining tumor was founddistal to the stomach four cases of gsc in the present study hadfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and imaging features of six previously reported cases of gcscasegenderageyearslocationsize cmshapeulcersenhanceappearancerecurrencemetastasistherapyprognosis mmffmmremnant stomachgreater curvaturelesser curvaturegastroesophageal junctionremnant stomachdistal stomachpolypoidpolypoidpolypoidulceratedpolypoidmass“““nenenenenehyperne“““““nonesurgerysurgerysurgeryendoscopysurgeryna mo d mo d mo d mo d mo dthe patient succumbed to heart failure before the surgical treatment an autopsy was performed œ yespresentpositive œ“ noabsentnegative hyper hyperdensene no evaluate mo month d dieareregarded asa longaxis diameter less than cm and the remaining tumorhad the largest longaxis diameter among our patients cmthe location distribution and longaxis diameters of the gscs inour patients were similar to those in previous reports “in the actual diagnosis processthe diagnosis of sc has always been difficult for cliniciansand pathologists especially the diï¬erential diagnosis fromcarcinosarcoma carcinosarcomastrulybiphasic neoplasms composed of distinct malignant epithelialcarcinomatous and mesenchymal sarcomatous componentsthe sarcoma components show typical specialized diï¬erentiation howeverthe termsœsarcomatoid carcinoma and œcarcinosarcoma have been usedinterchangeably in some cases therefore the understanding ofthese tumors has been hampered nevertheless we can try tofocus on whether there is a diï¬erence between these tumors froma new perspective the ct finding sc in the stomach have notbeen previously scientific reported or even detailed descriptionthere are only four simple descriptions chunchao reported that a patient with a giant sc presented a mass witha cm diameter in the antrum and body of stomach whichinfiltrated the gastric serosa the hepatic flexure of the colon andgallbladder were also involved on ct contrastenhanced ctimages showed obvious enhancement of the two lesions sato reported a patient with sc of the remnant stomachand the radiographic examination showed an elevated lesionwith a large ulcer at the gastric cardiac lesser curvature thatmeasured cm in diameter the other two reports only describeda soft tissue mass or a large tumor in the dilated stomach on the other hand within in the upper gastrointestinal tractalthough there are fewer reports of carcinosarcoma localized inthe stomach this type of tumor is still more common than sc gastric carcinosarcoma showed an elevated lesion or thickenedgastric walls in “ of all reviewed cases “ tomoaki reported a 79yearold man with gastric carcinosarcomaand his veins showed severe invasion enhanced abdominalct showed irregular thickening and slight enhancement of thegastric wall on the side of the lesser curvature with suspiciousbulky lymph nodes yoshiyuki reported a 70yearoldjapanese woman who presented with a soft tissue mass adjacentto the lesser curvature of the stomach that was lobulated andct revealed an ulcer on the lesion the contrastenhanced ctimages showed heterogeneous enhancement of the mass thefinal pathological diagnosis was gastric carcinosarcoma inthe present study we found that gsc showed local thickeningof the gastric wall and mass formation often accompaniedby ulcers the site of the disease was mostly in the proximalpart of the stomach but these tumors can also occur in theremnant stomach the signal of the tumor was homogeneousor heterogeneous on plain ct scans after contrast mediuminjection of tumors demonstrated heterogeneousenhancement on ap images due to cystic areas or necrosis inthe lesions in this study the enhancement degree of all tumorsreached a peak in the pp after contrast enhancement for thesetumors the enhancement degree in the delayed phase wasnot significantly reduced the overall enhancement mode wasdelayed enhancement in addition ct showed that four patientshad invasion into the gastric serosal region or gastric bare areatwo patients had the characteristics of enlarged perigastric orretroperitoneal lymph nodes and uneven enhancement and onepatient had invasion into the adjacent liver tissue these findingsreflect the metastatic and highly invasive characteristics of gscoverall ct and contrastenhanced ct can clearly show theprimary lesion infiltration range lymph node metastasis anddistant metastasis of gsctomographic diagnosis of gsc has not been attemptedbecause of the rarity of this entity according to the findingsof our study gsc needs to be diï¬erentiated from gac andgl on ct adenocarcinoma is the most common pathologicaltype of gastric tumor and is mainly distributed in the antrumseldomly in the body and fundus of the stomach the incidenceof gac is high in men and the median patient age is years the most common ct signs of gac are localor extensive thickening of the gastric wall mass formationincluding fungoidestype polypoidtype masses rough orsmooth serous surfaces and continuous interruption of themucosal layer tumors involving the mucosal surface can appearenhanced “ s after injecting a contrast agent the peakvalue for tumors invading the muscular layer usually appearsafter “ s and after the mucosal surface is strengthenedthe duration is longer primary gl accounts for “ ofmalignant gastric tumors and is predominantly situated in thegastric antrum gastric body and gastric fundus the incidenceof gl is high among males with a median patient age of yearsthe clinical symptoms included epigastric pain bleeding earlysatiety and fatigue the most common ct manifestations ofgl are diï¬use thickening of the gastric wall or a homogeneousfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatissue mass with slight attenuation or an appearancesoftsimilar to that of the normal gastric wall for gl becauseof hemorrhage necrosis submucosal edema or infarction thegastric wall may be heterogenous on ct gl originates froma submucosal process and gastric mucosa is commonly intactin the early stage but shows interruptions or ulceration in thelater stage after contrast medium injection most gl showedhomogeneous and slight enhancement in the delayed phase lymphoma is considered when distant structures the mesenteryretroperitoneum or other parts of the abdomen have lymphnode metastasis the ct findings may only reflect features of gsc but cannotaccurately diagnose gsc l one explore the origin of thesarcomatous portion immunohistochemistry ihc also failedto conclusively establish the origin of gsc rodrigues usedfluorescence in situ hybridization fish to confirm that sc andadenocarcinoma have a common origin that is the epithelium while primary gl originated from gastric submucosallymphoid tissuethe main treatment for localized lymphomas is eradicationof helicobacter pylori and surgical treatment whereas advanceddisease often requires radiation or chemotherapy alone surgery is the only treatment option for patients with gacadjuvant chemotherapy and chemoradiotherapy are also oftenused targeted therapy isin the exploration stage however there are currently no specific national comprehensivecancer network guidelines for the treatment of only gscbecause the tumor is relatively rare although complete surgicalresection is the most important treatment method for examplewhile chemotherapy is considered in clinical practice whetherchemotherapy can be applied for gsc and the efficacy ofchemotherapy remain controversial domblides firstevaluated the efficacy of immune checkpoint inhibitors icis forsc and found that lung sc patients exhibited high response ratesand prolonged overall survival os with icis this studyprovides a new idea for the treatment of gscbecause gl tends to be confined to the gastric wall forprolonged periods before tumor spread its prognosis is betterthan that of gac previous literature has found that scin the parotid gland lung hypopharynx liver and pancreashave poor prognoses due to metastasis or recurrence with asurvival period of a few months “ similarly gscpatients also died or developed metastasis or recurrence withina few months or it was already in the advanced stage at thefirst diagnosis all these clinical manifestations suggest that gschas a poorer prognosis than gac and gl in additiongsc can metastasize through the blood and lymph nodesand the most common sites of metastasis are the local lymphnodes and liver this conclusion is consistent with ourresearch resultsconclusionthe incidence rate of gsc is extremely low so clinicians andradiologists are not familiar with the features of this tumorbased on systematic research of this rare tumor and comparisonswith common gastric cancers we found that gsc is morecommon in men who are approximately years old and isoften accompanied by ulcers the disease is mostly located in theproximal part of the stomach and can also occur in the remnantstomach with delayed enhancement on contrastenhanced ctimages these characteristics can provide a reference for furtherresearch on gscs in the future however an accurate diagnosisof gsc depends on the combination of clinical imaging andhistopathological features due to the aggressive nature and poorprognosis of the tumor rapid clinical intervention and detailedfollowup with ct are essentialdata availability statementthe original contributions presented in the study are includedin the supplementary material further inquiries can bedirected to the corresponding authorethics statementthe studies involving human participants were reviewed andapproved by the medical ethical committee of the zhengzhouuniversity the patientsparticipants provided their writteninformed consent to participate in this study written informedconsent was obtained from the individuals for the publication ofany potentially identifiable images or data included in this author contributionsyl manuscript preparationliterature research and dataanalysis pl literature research and data analysis kf manuscriptreview and data collection kc guidance of pathologicalknowledge sy guidance of imaging knowledge jj imaging datacollection and analysis wl and xz manuscript editing jgstudy conception and design manuscript review and guarantor ofintegrity of the entire study all authors have read and approvedthe final manuscriptfundingthis work was supported by the national natural and sciencefund of china no references zhu cc li mr lin tl zhao g sarcomatoid carcinoma of the stomach acase report and literature review oncol lett “ doi ol20153460 snover dc levine gd rosaij thymic carcinoma five distinctivehistological variants am j surg pathol “ zhou dk gao bq zhang w qian xh ying lx wang wl sarcomatoidcarcinoma of the pancreas a case report world j clin cases “doi 1012998wjccv7i2236 xie y xiang y zhang d yao x sheng j yang y sarcomatoidthe “ doi 103892mmr2018and review ofthe pancreasreportcaseofcarcinomaliterature mol med repafrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinoma sato a oki e kohso h endo y uchida h hiroshige s sarcomatoidcarcinoma of the remnant stomach report of a case surg today “ doi 101007s0059501204027 nakayama y murayama h iwasaki h iwanaga s kikuchi m ikeda s gastric carcinosarcoma sarcomatoid carcinoma with rhabdomyoblastic andosteoblastic diï¬erentiation pathol int “ doi 101111j144018271997tb04540x robeycaï¬erty ss grignon dj ro jy cleary kr ayala ag ordonezng sarcomatoid carcinoma of the stomach a report of three caseswith immunohistochemical and ultrastructural observations cancer “ doi 101002109701421990040165730co2n ruess da kayser c neubauer j fichtnerfeigl s hopt ut wittel uacarcinosarcoma of the pancreas case report with comprehensive literaturereview pancreas “ doi 101097mpa0000000000000904 fujiie m yamamoto m taguchi k iwanaga a ohgaki k egashira a gastric carcinosarcoma with rhabdomyosarcomatous diï¬erentiation a casereport and review surg case rep doi 101186s407920160176z tanimura h furuta m carcinosarcoma of the stomach am j surg “ doi 101016000296106790325x kitamura s study on carcinosarcoma of stomach gan “ kumagai k kawai k kusano h matsuo k irie j tsuchiyama h a caseof socalled carcinosarcoma of the stomach gan no rinsho “ bekki t fujikuni n tanabe k yonehara s amano h noriyuki t thegastric carcinosarcoma with severe venous invasion a case report surg caserep doi 101186s4079201804218 ikeda y kosugi s nishikura k ohashi m kanda t kobayashi t gastriccarcinosarcoma presenting as a huge epigastric mass gastric cancer “ doi 101007s1012000604054 cid³n eu cuenca ij gastric adenocarcinoma is computed tomography ctuseful in preoperative staging clin med oncol “ doi cmos2641 hallinan jt venkatesh sk gastric carcinoma imaging diagnosis stagingand assessment of treatment response cancer imaging “doi gossios k katsimbri p tsianos e ct features of gastric lymphoma eurradiol “ doi 101007s003300050069 rodrigues dn hazell s miranda s crespo m fisher c de bono js sarcomatoid carcinoma of the prostate erg fluorescence insituhybridization confirms epithelial origin histopathology “doi 101111his12493 levine ms rubesin se pantongragbrown l buck jl herlinger h nonhodgkin™s lymphoma of the gastrointestinal tract radiographic findings ajram j roentgenol “ doi 102214ajr16818976941 russo ae strong ve gastric cancer etiology and management in asia and thewest annu rev med “ doi 101146annurevmed081117 domblides c leroy k monnet i mazi¨res j barlesi f gounant v efficacy of immune checkpoint inhibitors in lung sarcomatoid carcinoma jthor oncol “ doi 101016jjtho202001014 niu x sarcomatoid carcinoma in the parotid gland a review of years ofexperience laryngoscope “ doi 101002lary27474 li s jiang l he q wei w wang y zhang x the prognostic significanceof jmjd3 in primary sarcomatoid carcinoma of the lung a rare subtypeof lung cancer onco targets ther “ doi 102147otts22 dai l fang q li p liu f zhang x oncologic outcomes of patients withsarcomatoid carcinoma of the hypopharynx front oncol doi103389fonc201900950 seo n kim mj rhee h hepatic sarcomatoid carcinoma magnetic resonanceimaging evaluation by using the liver imaging reporting and data system eurradiol “ doi 101007s00330019060528 shi y chen j chen h hong x sarcomatoid carcinoma of the gallbladdera case report j int med res doi conflict of interest the authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestcopyright liu liang feng chen yue ji li zhao and gao this is anopenaccess distributed under the terms of the creative commons attributionlicense cc by the use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice no use distribution or reproduction is permitted which does not complywith these termsfrontiers in oncology wwwfrontiersinaugust volume 0c' Answer:

Colon_Cancer

laparoscopic surgery for rectal cancer is commonly performed in china however compared with open surgerythe effectiveness of laparoscopic surgery especially the longterm survival has not been sufficiently provedmethods data of eligible patients with nonmetastatic rectal cancer at nanfang hospital of southern medical university andguangdong provincial hospital of chinese medicine between and were retrospectively reviewed longterm survival outcomes and shortterm surgical safety were analysed with propensity score matching between groupsresults of cases collated from two institutes matched pairs were analysed after propensity score matching the estimated blood loss during laparoscopic surgery was significantly less than that during open surgery p¼ and the operativetime and hospital stay were shorter in the laparoscopic group both p the postoperative complications rate was inthe laparoscopic group and in the open group p¼ no significant difference was observed between the laparoscopicgroup and the open group in the 5year overall survival rate vs p¼ 5year relapsefree survival rate vs p¼ or 5year cancerspecific survival rate vs p¼ an elevated carcinoembryonic antigen harvested lymph nodes and perineural invasion were independent prognostic factors affecting overall survival and relapsefreesurvivals our findings suggest that open surgery should still be the priority recommendation but laparoscopic surgery isalso an acceptable treatment for nonmetastatic rectal cancerkey words laparoscopic surgery open surgery propensity score matching rectal cancersubmitted february revised april accepted june vc the authors published by oxford university press and sixth affiliated hospital of sun yatsen universitythis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0c kl tan introductioncolorectal cancer is the second most commonly diagnosedcancer and the fifth leading cause of cancerrelated death forboth sexes in china in a multidisciplinary approach thatcombines chemotherapy with radiotherapy for the treatmentof colorectal cancer surgery remains the major approach thefirst successful use of laparoscopy in colorectal surgery waspublished in by jacobs laparoscopic surgery hasbeen performed widely in colon cancer all over the world andseveral randomized“controlled trials have demonstrated thatlaparoscopic surgery for colon cancer is safe and feasible withbetter shortterm outcomes including a decrease in postoperative pain a shorter hospital stay and earlier recoveryand equivalent longterm results compared to open surgery[“] however laparoscopic surgery for rectal cancer is morearduous than that for colon cancer so the early clinical trialsexcluded rectal cancer [“] although a few clinical trialshave shown the advantages of laparoscopic rectalcancer resection compared with open surgery [“] both the acosogz6051 and alacart trials did not support the use of laparoscopic surgery for rectal cancer [ ] it is still controversialwhether laparoscopic surgery is suitable for rectal cancer especially for low rectal cancer therefore we conducted thisretrospective cohort study to compare longterm survival outcomes and shortterm surgical safety between laparoscopicand open surgery for nonmetastatic rectal cancer in thechinese population propensity score matching psm was performed for the study designpatients and methodsstudy designall consecutive eligible patients with rectal cancer were confirmed from the department of general surgery of nanfanghospital of southern medical university and the department ofproctology of guangdong provincial hospital of chinesemedicine between january and december these twocenters were members of the southern chinese laparoscopiccolorectal surgery study group demographic clinical pathologic and imaging features together with the management andoutcomes were carefully reviewed written informed consentwas acquired from patients preceding the surgical proceduresthis study was approved by the ethical committee of nanfanghospital and guangdong provincial hospital of chinesemedicine no ze2019052“study subjectsinclusion criteria were patients with clinical stage i“iii rectalcancer who underwentrectal cancerexclusion criteria were patients with i combined operationsextending to the surrounding an ii multiple cancers iiiemergency operation iv conversion to open surgery or vpatients who received neoadjuvant therapyradical surgery forall included cases were classified into two groups based onthe surgical approach which was either laparoscopic or opensurgery the surgical approach was decided by the individualcolorectal surgeon based on a combined assessment of clinicalendoscopic and imaging featuresdata collectiondata were collected in a prospectively maintained databasefrom clinical report forms the demographic and clinicopathological data included age gender body mass index bmi preoperative carcinoembryonic antigen cealocationoperative time estimated blood loss surgical procedure protective ileostomy tumor grade tumor stage and hospital staypreoperative cea was defined as cea measured closest to theoperation time tumor location was divided into the followingthree sections upper rectum above cm from the anal vergemiddle rectum “ cm from the anal verge and lower rectumbelow cm from the anal verge surgical procedures consistedof three categories low anterior resection abdominoperinealtumorfigure flow diagram of patient disposition 0claparoscopic vs open surgery for rectal cancer table baseline characteristics of the study populationcharacteristictotal cohortmatched cohortlaparoscopic groupn¼ open groupn¼ pvaluelaparoscopic groupn¼ open groupn ¼ pvalueage years mean sdgender n malefemalebmi kgm2 mean sdpreoperative cea n 14 ngml ngmltumor location n upper rectummiddle rectumlower rectumtumor stage n iiiiii sd standard deviation bmi body mass index cea carcinoembryonic antigentable operative and pathological results in matched cohorts variablesurgical procedure n low anterior resectionabdominoperineal resectionhartmann™s procedureprotective ileostomy n operative time min median iqrintraoperative blood loss ml median iqrhospital stay day median iqrtumor grade n wellmoderatepoorothersharvested lymph nodes n 15lymphovascular invasion n perineural invasion n tumor deposits n postoperative complications n wound infectionileusurinary dysfunctionanastomosis leakageintraabdominal bleedingpneumoniacardiac eventreoperation n mortality n iqr interquartile rangelaparoscopic group n¼ open group n ¼ “ “ “ “ “ “ pvalueresection and hartmann™s procedure tumor grade was dividedinto three types well differentiated moderately differentiatedand poorly differentiated including signet or mucinous adenocarcinoma tumor stage was based on the final pathologic report and preoperative imaging examinationoutcome measurementsthe primary endpoint of this study was overall survival osrelapsefree survival rfs and cancerspecific survival cssos was defined as the time from operation to death from any 0c kl tan figure survival curve after laparoscopic surgery vs open surgery in matchedcohortscause or the last followup rfs was defined as the time fromoperation to identified recurrence or any cause of death csswas defined as the time from operation to death due to rectalcancer the last followup was january ileus urinary dysfunctionthe secondary endpoints were operative time estimated bloodloss hospital stay reoperation postoperative complications andmortality postoperative complications were defined as woundinfectionleakageintraabdominal bleeding pneumonia and cardiac eventsintraabdominal bleeding was defined in this study as bleeding requiring transfusion or reoperation all complications within daysafter surgery were recorded postoperative mortality was traditionally defined as any death occurring within days after surgeryanastomoticstatistical analysisdata are presented as mean standard deviation or medianwith interquartile range iqr for quantitative variables withparanormal distribution and numbers with percentages for categorical variables quantitative variables were compared usingthe student™s ttest or mann“whitney u test categorical variables were analysed using the chisquare test or fisher™s exacttest the estimates of the differences in age gender bmi preoperative cea level tumor location and tumor stage between thetwo groups were performed using psm [ ]survival rates were calculated by using the kaplan“meiermethod and comparisons between groups were performed withthe logrank test to identify the prognostic factors univariateand multivariate analyses were performed using the cox proportional hazards regression model and the results were presented as hazard ratios hrs with confidence intervalscis only factors with p in the univariate analysis wereevaluated in subsequent multivariate analysis using forwardstepwise selection for os and rfs a p was regarded asstatistically significant all statistical analyses were carried outwith ibmvr spssvr statistics version resultsbaseline characteristicsbetween january and december eligiblepatients were collected from hospitals in china of patients cases were excluded among the remaining cases underwent laparoscopic surgery and underwent open surgery after psm of pairs ofpatients were successfully matched figure baseline characteristics are outlined in table before psm there were differences in age and preoperative cea between the two groups afterpsm all variables were well balancedshortterm surgical outcomesthe perioperative and pathological results in matched cohortsare presented in table the estimated blood loss during laparoscopic surgery was significantly less than that during opensurgery p¼ in the laparoscopic group the operative timeand hospital stay were shorter than in the open groupp the incidence of postoperative complications was in the laparoscopic group and in the open groupp¼ in the open group the most common complicationswere wound infection and pneumonia followed byanastomosis leakage whereas in the laparoscopic groupthe most common complication was anastomosis leakage followed by pneumonia longterm survival outcomesin the matched cohorts the median followup period was months in the laparoscopic group iqr “ monthsand months in the open group iqr “ monthsduring the followup patients died among whom diedfrom rectal cancer and had locoregional recurrence or distantmetastasis no significant difference was observed between thelaparoscopic group and the open group in 5year os vs p¼ 5year rfs vs p¼ or 5yearcss vs p¼ figure subgroup analyses for os were conducted for gender agebmi tumor location and tumor stage compared with open surgery male patients or those with an intermediate bmi to who underwent laparoscopic surgery tended to show worseos figure 0claparoscopic vs open surgery for rectal cancer figure subgroup analysis of overall survival in matched cohortstable multivariate analysis for os and rfs in matched cohortsvariableosrfspreoperative cea vs 14 ngmlnumber of harvested lymph nodes 15 vs perineural invasion yes vs nohr cipvalue“““hr cipvalue“““os overall survival rfs relapsefree survival cea carcinoembryonic antigen hr hazard ratio ci confidence intervalprognostic factors for longterm survivalprognostic factors affecting survival are presented in table univariate analyses revealed that an elevated cea ngml harvested lymph nodes perineural invasion and tumordeposits were associated with poor os and that an elevatedcea harvested lymph nodes perineural invasion and lymphovascular invasion were associated with poor rfs data notshown the surgical approach laparoscopic vs open was notassociated with os hr ci “ and rfs hr ci “ multivariate analyses testified that an elevated cea harvested lymph nodes and perineural invasionwere independent factors affecting os and rfs table discussionlaparoscopic surgery for rectal cancer is commonly performedin many countries nevertheless the evidence for laparoscopicsurgery for rectal cancer is insufficient this study focused onthe longterm survival outcomes and surgical safety of patientswho underwentlaparoscopic or open surgery for nonmetastatic rectal cancer in the chinese population in this twocenter study psm was performed to make selection balance between patients treated with laparoscopic and open surgery thesix factors of age gender bmi preoperative cea level tumor location and tumor stage were used as described in the protocolthe baseline characteristics were ideally balanced between thelaparoscopic and open groupssome studies have reported similar postoperative complications and mortality between laparoscopic surgery and opensurgery for rectal cancer [ ] and other studies have reportedfewer postoperative complications after laparoscopic surgerythan after open surgery [ ] in our study there were no significant differences in postoperative complications includingwound infectionileus urinarytract infection anastomosisleakageintraabdominal bleeding pneumonia and cardiacevent between the two groups the longer operative time is often considered a disadvantage of laparoscopic surgery according to some previous reports [ ] in contrast our studyshowed that the operative time of laparoscopic surgery wasshorter than that of open surgery the clasicc trial and colorii trial both showed that hospital stay was significantly shorterin the laparoscopic group [ ] similarly our study alsoshowed that the hospital stay for laparoscopic surgery wasshorter than for open surgerywith regard to longterm survival no largescale clinical trials have demonstrated a statistically significant difference between laparoscopic and open surgery for rectal cancer thecolor ii trial indicated no statistically significant differences indfs and os between laparoscopic and open surgeries inthe corean study dfs in laparoscopic surgery is noninferiorcompared to that in open surgery for mid or low rectal cancer consistently with previous studies os rfs and css didnot differ in both groups in our study interestingly subgroupanalyses for os showed that male and intermediate bmi to kgm2 subgroups were associated with unfavorable outcomes in the laparoscopicsurgery group vs the opensurgerygroup chinese male populations have a narrow pelvis whichmight affect the visualization of and access to the deep pelvic 0c kl tan anatomy during laparoscopic surgery kitano foundthat laparoscopic surgery might affect longterm outcomes inthe highbmi kgm2 subgroup in the current study thebmi subgroup unfavorable for laparoscopic surgery that weidentified was intermediate bmi not high bmi it might be dueto lower bmi in the chinese population compared to that in thewestern population and the small proportion of patientswith high bmi in our cohort further evaluation will be neededto determine which subgroups of patients require additional attention when undergoing laparoscopic surgerylymphovascularwe evaluated several possible prognostic factors that mayinfluence survival in patients with rectal cancer including tumor location tumor stage tumor grade surgical approach preoperative cea levelinvasion perineuralinvasion and tumor deposits [“] as expected our studyshowed that perineural invasion was the significant prognosticfactor affecting os and rfs perineural invasion refers to the invasion of cancer cells into any of the layers of the nerve sheatha higher grade of perineural invasion was related to local recurrence and metastasis in distant ans such as the liver lungand peritoneum all patients in this study underwent radical surgery with lymphnode dissection a minimum of harvested lymph nodes is recommended to ensure adequatestaging and oncologic resection for colorectal cancer themore lymph nodes harvested the better the prognosis [ ]in this study the average number of harvested lymph nodeswas we found that patients with 15 harvested lymphnodes had better os and rfs than those with harvestedlymph nodes several studies have shown that elevated preoperative cea was a poor prognostic factor in colorectal cancer[“] in our study we also found that patients with an elevated preoperative cea had poorer os and rfsour study has several limitations first a selection biasexisted due to its retrospective design to reduce this the twogroups were matched carefully using psm second the statistical power is insufficient because the number of patients enrolled may not be sufficient after matching third data aboutadjuvant therapy after surgery were not collected which mightbe different between both groups and thus have influenced survival outcomes fourth the exclusion of converted cases mayintroduce a bias in favor of laparoscopic surgery finally thebowelrecovery data could not be exactly assessed due to thelack of records in this retrospective study therefore further research with a large population is still awaitedanydifferencesinin our study revealed the benefit of laparoscopicsurgery on shortterm outcomes including less blood lossshorter operative time and shorter hospital stay we did notfindcomplicationslaparoscopic surgery was similar to open surgery in terms ofos rfs and css for patients however male patients and thosewith an intermediate bmi in the laparoscopic group tended toshow worse os than those in the open group findings fromthis study suggest that open surgery should still be the priorityrecommendation but laparoscopic surgery is also an acceptabletreatment for nonmetastatic rectal cancer in the chinesepopulationpostoperativeauthors™ contributionsklt hjd zqc and tym collected and analysed the dataklt hl and rsx performed statistical analysis klt andhjd drafted the manuscript gxl and xhf performed theprocedure conceived of and designed the study and criticallyrevised all the intellectual content of the manuscript allauthors read and approved the final manuscriptfundingthis work was supported by clinical research of guangdongprovincial hospital of chinese medicine [no yn10101902]and a scientific research project of guangdong provincialacademy of chinese medical sciences [no yn2018ml11]acknowledgementsthe authors thank the patients and their families for making this retrospective study possible we also thank all theinvestigators and staff who contributed to the patientfollowup and data collection in nanfang hospital ofsouthern medical university and guangdong provincialhospital of chinese medicineconflicts of interestthe authors declare that there is no conflict of interests inthis studyreferences feng rm zong yn cao sm current cancer situation inchina good or bad news from the global cancerstatistics cancer commun “jacobs m verdeja jc goldstein hs minimally invasive colonresection laparoscopic colectomy surg laparosc endosc “ hewett pj allardyce ra bagshaw pf shortterm outcomes of the australasian randomized clinical study comparing laparoscopic and conventional open surgical treatmentsfor colon cancer the alccas trial ann surg “ buunen m veldkamp r hop wc survival after laparoscopic surgery versus open surgery for colon cancer longterm outcome of a randomised clinical trial lancet oncol “ clinical outcomes of surgical therapy study group a comparison of laparoscopically assisted and open colectomy forcolon cancer n engl j med “ bonjer hj haglind e jeekel i laparoscopic surgery versusopen surgery for colon cancer shortterm outcomes of arandomised trial lancet oncol “ fleshman j sargent dj green e laparoscopic colectomyfor cancer is not inferior to open surgery based on 5year datafrom the cost study group trial ann surg “ van der pas mh haglind e cuesta ma laparoscopic versus open surgery for rectal cancer color ii shortterm outcomes of a randomised phase trial lancet oncol “ kang sb park jw jeong sy open versus laparoscopicsurgery for mid or low rectal cancer after neoadjuvant chemoradiotherapy corean trial shortterm outcomes of anopenlabel randomised controlled trial lancet oncol “ zhou zx zhao ly lin t longterm oncologic outcomesof laparoscopic vs open surgery for stages ii and iii rectal 0ccancer a retrospective cohort study world j gastroenterol“iii colon cancer jcog0404 a phase randomised controlledtrial lancet gastroenterol hepatol “laparoscopic vs open surgery for rectal cancer fleshman j branda m sargent dj effect of laparoscopicassisted resection vs open resection of stage ii or iii rectalcancer on pathologic outcomes the acosog z6051 randomized clinical trial jama “ stevenson ar solomon mj lumley jw effect oflaparoscopicassisted resection vs open resection on pathological outcomes in rectal cancer the alacart randomizedclinical trial jama “ austin pc an introduction to propensity score methods forreducing the effects of confounding in observational studiesmultivariate behav res “ wang h chen x liu h laparoscopyassisted colectomyas an oncologically safe alternative for patients with stage t4colon cancer a propensitymatched cohort study bmc cancer bonjer hj deijen cl abis ga a randomized trial of laparoscopic versus open surgery for rectal cancer n engl j med“ lujan j valero g biondo s laparoscopic versus opensurgery for rectal cancer results of a prospective multicentreanalysis of patients surg endosc “ landi f vallribera f rivera jp morbidity after laparoscopic and open rectal cancer surgery a comparative analysisof morbidity in octogenarians and younger patientscolorectal dis “ toda s kuroyanagi h laparoscopic surgery for rectal cancercurrent status and future perspective asian j endosc surg“ guillou pj quirke p thorpe h shortterm endpoints ofconventionalinpatients with colorectal cancer mrc clasicc trial multicentre randomised controlled trial lancet “laparoscopicassisted surgeryversus jeong sy park jw nam bh open versus laparoscopicsurgery for midrectal or lowrectal cancer after neoadjuvantchemoradiotherapy corean trial survival outcomes of anopenlabel noninferiorityrandomised controlled triallancet oncol “ kitano s inomata m mizusawa j survival outcomes following laparoscopic versus open d3 dissection for stage ii or mehrkhani f nasiri s donboli k prognostic factors insurvival of colorectal cancer patients after surgery colorectaldis “ wiratkapun s kraemer m seowchoen f high preoperative serum carcinoembryonic antigen predicts metastatic recurrence in potentially curative colonic cancer results of afiveyear study dis colon rectum “ huang qs qin hb xiao j association of tumor differentiation and prognosis in patients with rectal cancer undergoingneoadjuvant chemoradiation therapy gastroenterol rep “ liebig c ayala g wilks ja perineural invasion is an independent predictor of outcome in colorectal cancer j clin oncol“ ueno h shirouzu k eishi y study group for perineuralinvasion projected by the japanese society for cancer of thecolon and rectum jsccr characterization of perineural invasion as a component of colorectal cancer staging am j surgpathol “ amin mb edge sb greene fl eds ajcc cancer stagingmanual 8th edn new york springer “ rosenberg r engel j bruns c the prognostic value oflymph node ratio in a populationbased collective of colorectal cancer patients ann surg “ sjo oh merok ma svindland a prognostic impact oflymph node harvest and lymph node ratio in patients withcolon cancer dis colon rectum “ tarantino i warschkow r worni m elevated preoperative cea is associated with worse survival in stage iiii rectalcancer patients br j cancer “ huang sh tsai ws you jf preoperative carcinoembryonic antigen as a poor prognostic factor in stage iiii colorectal cancer after curativeintent resection a propensity scorematching analysis ann surg oncol “ konishi t shimada y hsu m association of preoperativeand postoperative serum carcinoembryonic antigen and colon cancer outcome jama oncol “ 0c'

cancer7503

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: laparoscopic surgery for rectal cancer is commonly performed in china however compared with open surgerythe effectiveness of laparoscopic surgery especially the longterm survival has not been sufficiently provedmethods data of eligible patients with nonmetastatic rectal cancer at nanfang hospital of southern medical university andguangdong provincial hospital of chinese medicine between and were retrospectively reviewed longterm survival outcomes and shortterm surgical safety were analysed with propensity score matching between groupsresults of cases collated from two institutes matched pairs were analysed after propensity score matching the estimated blood loss during laparoscopic surgery was significantly less than that during open surgery p¼ and the operativetime and hospital stay were shorter in the laparoscopic group both p the postoperative complications rate was inthe laparoscopic group and in the open group p¼ no significant difference was observed between the laparoscopicgroup and the open group in the 5year overall survival rate vs p¼ 5year relapsefree survival rate vs p¼ or 5year cancerspecific survival rate vs p¼ an elevated carcinoembryonic antigen harvested lymph nodes and perineural invasion were independent prognostic factors affecting overall survival and relapsefreesurvivals our findings suggest that open surgery should still be the priority recommendation but laparoscopic surgery isalso an acceptable treatment for nonmetastatic rectal cancerkey words laparoscopic surgery open surgery propensity score matching rectal cancersubmitted february revised april accepted june vc the authors published by oxford university press and sixth affiliated hospital of sun yatsen universitythis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0c kl tan introductioncolorectal cancer is the second most commonly diagnosedcancer and the fifth leading cause of cancerrelated death forboth sexes in china in a multidisciplinary approach thatcombines chemotherapy with radiotherapy for the treatmentof colorectal cancer surgery remains the major approach thefirst successful use of laparoscopy in colorectal surgery waspublished in by jacobs laparoscopic surgery hasbeen performed widely in colon cancer all over the world andseveral randomized“controlled trials have demonstrated thatlaparoscopic surgery for colon cancer is safe and feasible withbetter shortterm outcomes including a decrease in postoperative pain a shorter hospital stay and earlier recoveryand equivalent longterm results compared to open surgery[“] however laparoscopic surgery for rectal cancer is morearduous than that for colon cancer so the early clinical trialsexcluded rectal cancer [“] although a few clinical trialshave shown the advantages of laparoscopic rectalcancer resection compared with open surgery [“] both the acosogz6051 and alacart trials did not support the use of laparoscopic surgery for rectal cancer [ ] it is still controversialwhether laparoscopic surgery is suitable for rectal cancer especially for low rectal cancer therefore we conducted thisretrospective cohort study to compare longterm survival outcomes and shortterm surgical safety between laparoscopicand open surgery for nonmetastatic rectal cancer in thechinese population propensity score matching psm was performed for the study designpatients and methodsstudy designall consecutive eligible patients with rectal cancer were confirmed from the department of general surgery of nanfanghospital of southern medical university and the department ofproctology of guangdong provincial hospital of chinesemedicine between january and december these twocenters were members of the southern chinese laparoscopiccolorectal surgery study group demographic clinical pathologic and imaging features together with the management andoutcomes were carefully reviewed written informed consentwas acquired from patients preceding the surgical proceduresthis study was approved by the ethical committee of nanfanghospital and guangdong provincial hospital of chinesemedicine no ze2019052“study subjectsinclusion criteria were patients with clinical stage i“iii rectalcancer who underwentrectal cancerexclusion criteria were patients with i combined operationsextending to the surrounding an ii multiple cancers iiiemergency operation iv conversion to open surgery or vpatients who received neoadjuvant therapyradical surgery forall included cases were classified into two groups based onthe surgical approach which was either laparoscopic or opensurgery the surgical approach was decided by the individualcolorectal surgeon based on a combined assessment of clinicalendoscopic and imaging featuresdata collectiondata were collected in a prospectively maintained databasefrom clinical report forms the demographic and clinicopathological data included age gender body mass index bmi preoperative carcinoembryonic antigen cealocationoperative time estimated blood loss surgical procedure protective ileostomy tumor grade tumor stage and hospital staypreoperative cea was defined as cea measured closest to theoperation time tumor location was divided into the followingthree sections upper rectum above cm from the anal vergemiddle rectum “ cm from the anal verge and lower rectumbelow cm from the anal verge surgical procedures consistedof three categories low anterior resection abdominoperinealtumorfigure flow diagram of patient disposition 0claparoscopic vs open surgery for rectal cancer table baseline characteristics of the study populationcharacteristictotal cohortmatched cohortlaparoscopic groupn¼ open groupn¼ pvaluelaparoscopic groupn¼ open groupn ¼ pvalueage years mean sdgender n malefemalebmi kgm2 mean sdpreoperative cea n 14 ngml ngmltumor location n upper rectummiddle rectumlower rectumtumor stage n iiiiii sd standard deviation bmi body mass index cea carcinoembryonic antigentable operative and pathological results in matched cohorts variablesurgical procedure n low anterior resectionabdominoperineal resectionhartmann™s procedureprotective ileostomy n operative time min median iqrintraoperative blood loss ml median iqrhospital stay day median iqrtumor grade n wellmoderatepoorothersharvested lymph nodes n 15lymphovascular invasion n perineural invasion n tumor deposits n postoperative complications n wound infectionileusurinary dysfunctionanastomosis leakageintraabdominal bleedingpneumoniacardiac eventreoperation n mortality n iqr interquartile rangelaparoscopic group n¼ open group n ¼ “ “ “ “ “ “ pvalueresection and hartmann™s procedure tumor grade was dividedinto three types well differentiated moderately differentiatedand poorly differentiated including signet or mucinous adenocarcinoma tumor stage was based on the final pathologic report and preoperative imaging examinationoutcome measurementsthe primary endpoint of this study was overall survival osrelapsefree survival rfs and cancerspecific survival cssos was defined as the time from operation to death from any 0c kl tan figure survival curve after laparoscopic surgery vs open surgery in matchedcohortscause or the last followup rfs was defined as the time fromoperation to identified recurrence or any cause of death csswas defined as the time from operation to death due to rectalcancer the last followup was january ileus urinary dysfunctionthe secondary endpoints were operative time estimated bloodloss hospital stay reoperation postoperative complications andmortality postoperative complications were defined as woundinfectionleakageintraabdominal bleeding pneumonia and cardiac eventsintraabdominal bleeding was defined in this study as bleeding requiring transfusion or reoperation all complications within daysafter surgery were recorded postoperative mortality was traditionally defined as any death occurring within days after surgeryanastomoticstatistical analysisdata are presented as mean standard deviation or medianwith interquartile range iqr for quantitative variables withparanormal distribution and numbers with percentages for categorical variables quantitative variables were compared usingthe student™s ttest or mann“whitney u test categorical variables were analysed using the chisquare test or fisher™s exacttest the estimates of the differences in age gender bmi preoperative cea level tumor location and tumor stage between thetwo groups were performed using psm [ ]survival rates were calculated by using the kaplan“meiermethod and comparisons between groups were performed withthe logrank test to identify the prognostic factors univariateand multivariate analyses were performed using the cox proportional hazards regression model and the results were presented as hazard ratios hrs with confidence intervalscis only factors with p in the univariate analysis wereevaluated in subsequent multivariate analysis using forwardstepwise selection for os and rfs a p was regarded asstatistically significant all statistical analyses were carried outwith ibmvr spssvr statistics version resultsbaseline characteristicsbetween january and december eligiblepatients were collected from hospitals in china of patients cases were excluded among the remaining cases underwent laparoscopic surgery and underwent open surgery after psm of pairs ofpatients were successfully matched figure baseline characteristics are outlined in table before psm there were differences in age and preoperative cea between the two groups afterpsm all variables were well balancedshortterm surgical outcomesthe perioperative and pathological results in matched cohortsare presented in table the estimated blood loss during laparoscopic surgery was significantly less than that during opensurgery p¼ in the laparoscopic group the operative timeand hospital stay were shorter than in the open groupp the incidence of postoperative complications was in the laparoscopic group and in the open groupp¼ in the open group the most common complicationswere wound infection and pneumonia followed byanastomosis leakage whereas in the laparoscopic groupthe most common complication was anastomosis leakage followed by pneumonia longterm survival outcomesin the matched cohorts the median followup period was months in the laparoscopic group iqr “ monthsand months in the open group iqr “ monthsduring the followup patients died among whom diedfrom rectal cancer and had locoregional recurrence or distantmetastasis no significant difference was observed between thelaparoscopic group and the open group in 5year os vs p¼ 5year rfs vs p¼ or 5yearcss vs p¼ figure subgroup analyses for os were conducted for gender agebmi tumor location and tumor stage compared with open surgery male patients or those with an intermediate bmi to who underwent laparoscopic surgery tended to show worseos figure 0claparoscopic vs open surgery for rectal cancer figure subgroup analysis of overall survival in matched cohortstable multivariate analysis for os and rfs in matched cohortsvariableosrfspreoperative cea vs 14 ngmlnumber of harvested lymph nodes 15 vs perineural invasion yes vs nohr cipvalue“““hr cipvalue“““os overall survival rfs relapsefree survival cea carcinoembryonic antigen hr hazard ratio ci confidence intervalprognostic factors for longterm survivalprognostic factors affecting survival are presented in table univariate analyses revealed that an elevated cea ngml harvested lymph nodes perineural invasion and tumordeposits were associated with poor os and that an elevatedcea harvested lymph nodes perineural invasion and lymphovascular invasion were associated with poor rfs data notshown the surgical approach laparoscopic vs open was notassociated with os hr ci “ and rfs hr ci “ multivariate analyses testified that an elevated cea harvested lymph nodes and perineural invasionwere independent factors affecting os and rfs table discussionlaparoscopic surgery for rectal cancer is commonly performedin many countries nevertheless the evidence for laparoscopicsurgery for rectal cancer is insufficient this study focused onthe longterm survival outcomes and surgical safety of patientswho underwentlaparoscopic or open surgery for nonmetastatic rectal cancer in the chinese population in this twocenter study psm was performed to make selection balance between patients treated with laparoscopic and open surgery thesix factors of age gender bmi preoperative cea level tumor location and tumor stage were used as described in the protocolthe baseline characteristics were ideally balanced between thelaparoscopic and open groupssome studies have reported similar postoperative complications and mortality between laparoscopic surgery and opensurgery for rectal cancer [ ] and other studies have reportedfewer postoperative complications after laparoscopic surgerythan after open surgery [ ] in our study there were no significant differences in postoperative complications includingwound infectionileus urinarytract infection anastomosisleakageintraabdominal bleeding pneumonia and cardiacevent between the two groups the longer operative time is often considered a disadvantage of laparoscopic surgery according to some previous reports [ ] in contrast our studyshowed that the operative time of laparoscopic surgery wasshorter than that of open surgery the clasicc trial and colorii trial both showed that hospital stay was significantly shorterin the laparoscopic group [ ] similarly our study alsoshowed that the hospital stay for laparoscopic surgery wasshorter than for open surgerywith regard to longterm survival no largescale clinical trials have demonstrated a statistically significant difference between laparoscopic and open surgery for rectal cancer thecolor ii trial indicated no statistically significant differences indfs and os between laparoscopic and open surgeries inthe corean study dfs in laparoscopic surgery is noninferiorcompared to that in open surgery for mid or low rectal cancer consistently with previous studies os rfs and css didnot differ in both groups in our study interestingly subgroupanalyses for os showed that male and intermediate bmi to kgm2 subgroups were associated with unfavorable outcomes in the laparoscopicsurgery group vs the opensurgerygroup chinese male populations have a narrow pelvis whichmight affect the visualization of and access to the deep pelvic 0c kl tan anatomy during laparoscopic surgery kitano foundthat laparoscopic surgery might affect longterm outcomes inthe highbmi kgm2 subgroup in the current study thebmi subgroup unfavorable for laparoscopic surgery that weidentified was intermediate bmi not high bmi it might be dueto lower bmi in the chinese population compared to that in thewestern population and the small proportion of patientswith high bmi in our cohort further evaluation will be neededto determine which subgroups of patients require additional attention when undergoing laparoscopic surgerylymphovascularwe evaluated several possible prognostic factors that mayinfluence survival in patients with rectal cancer including tumor location tumor stage tumor grade surgical approach preoperative cea levelinvasion perineuralinvasion and tumor deposits [“] as expected our studyshowed that perineural invasion was the significant prognosticfactor affecting os and rfs perineural invasion refers to the invasion of cancer cells into any of the layers of the nerve sheatha higher grade of perineural invasion was related to local recurrence and metastasis in distant ans such as the liver lungand peritoneum all patients in this study underwent radical surgery with lymphnode dissection a minimum of harvested lymph nodes is recommended to ensure adequatestaging and oncologic resection for colorectal cancer themore lymph nodes harvested the better the prognosis [ ]in this study the average number of harvested lymph nodeswas we found that patients with 15 harvested lymphnodes had better os and rfs than those with harvestedlymph nodes several studies have shown that elevated preoperative cea was a poor prognostic factor in colorectal cancer[“] in our study we also found that patients with an elevated preoperative cea had poorer os and rfsour study has several limitations first a selection biasexisted due to its retrospective design to reduce this the twogroups were matched carefully using psm second the statistical power is insufficient because the number of patients enrolled may not be sufficient after matching third data aboutadjuvant therapy after surgery were not collected which mightbe different between both groups and thus have influenced survival outcomes fourth the exclusion of converted cases mayintroduce a bias in favor of laparoscopic surgery finally thebowelrecovery data could not be exactly assessed due to thelack of records in this retrospective study therefore further research with a large population is still awaitedanydifferencesinin our study revealed the benefit of laparoscopicsurgery on shortterm outcomes including less blood lossshorter operative time and shorter hospital stay we did notfindcomplicationslaparoscopic surgery was similar to open surgery in terms ofos rfs and css for patients however male patients and thosewith an intermediate bmi in the laparoscopic group tended toshow worse os than those in the open group findings fromthis study suggest that open surgery should still be the priorityrecommendation but laparoscopic surgery is also an acceptabletreatment for nonmetastatic rectal cancer in the chinesepopulationpostoperativeauthors™ contributionsklt hjd zqc and tym collected and analysed the dataklt hl and rsx performed statistical analysis klt andhjd drafted the manuscript gxl and xhf performed theprocedure conceived of and designed the study and criticallyrevised all the intellectual content of the manuscript allauthors read and approved the final manuscriptfundingthis work was supported by clinical research of guangdongprovincial hospital of chinese medicine [no yn10101902]and a scientific research project of guangdong provincialacademy of chinese medical sciences [no yn2018ml11]acknowledgementsthe authors thank the patients and their families for making this retrospective study possible we also thank all theinvestigators and staff who contributed to the patientfollowup and data collection in nanfang hospital ofsouthern medical university and guangdong provincialhospital of chinese medicineconflicts of interestthe authors declare that there is no conflict of interests inthis studyreferences feng rm zong yn cao sm current cancer situation inchina good or bad news from the global cancerstatistics cancer commun “jacobs m verdeja jc goldstein hs minimally invasive colonresection laparoscopic colectomy surg laparosc endosc “ hewett pj allardyce ra bagshaw pf shortterm outcomes of the australasian randomized clinical study comparing laparoscopic and conventional open surgical treatmentsfor colon cancer the alccas trial ann surg “ buunen m veldkamp r hop wc survival after laparoscopic surgery versus open surgery for colon cancer longterm outcome of a randomised clinical trial lancet oncol “ clinical outcomes of surgical therapy study group a comparison of laparoscopically assisted and open colectomy forcolon cancer n engl j med “ bonjer hj haglind e jeekel i laparoscopic surgery versusopen surgery for colon cancer shortterm outcomes of arandomised trial lancet oncol “ fleshman j sargent dj green e laparoscopic colectomyfor cancer is not inferior to open surgery based on 5year datafrom the cost study group trial ann surg “ van der pas mh haglind e cuesta ma laparoscopic versus open surgery for rectal cancer color ii shortterm outcomes of a randomised phase trial lancet oncol “ kang sb park jw jeong sy open versus laparoscopicsurgery for mid or low rectal cancer after neoadjuvant chemoradiotherapy corean trial shortterm outcomes of anopenlabel randomised controlled trial lancet oncol “ zhou zx zhao ly lin t longterm oncologic outcomesof laparoscopic vs open surgery for stages ii and iii rectal 0ccancer a retrospective cohort study world j gastroenterol“iii colon cancer jcog0404 a phase randomised controlledtrial lancet gastroenterol hepatol “laparoscopic vs open surgery for rectal cancer fleshman j branda m sargent dj effect of laparoscopicassisted resection vs open resection of stage ii or iii rectalcancer on pathologic outcomes the acosog z6051 randomized clinical trial jama “ stevenson ar solomon mj lumley jw effect oflaparoscopicassisted resection vs open resection on pathological outcomes in rectal cancer the alacart randomizedclinical trial jama “ austin pc an introduction to propensity score methods forreducing the effects of confounding in observational studiesmultivariate behav res “ wang h chen x liu h laparoscopyassisted colectomyas an oncologically safe alternative for patients with stage t4colon cancer a propensitymatched cohort study bmc cancer bonjer hj deijen cl abis ga a randomized trial of laparoscopic versus open surgery for rectal cancer n engl j med“ lujan j valero g biondo s laparoscopic versus opensurgery for rectal cancer results of a prospective multicentreanalysis of patients surg endosc “ landi f vallribera f rivera jp morbidity after laparoscopic and open rectal cancer surgery a comparative analysisof morbidity in octogenarians and younger patientscolorectal dis “ toda s kuroyanagi h laparoscopic surgery for rectal cancercurrent status and future perspective asian j endosc surg“ guillou pj quirke p thorpe h shortterm endpoints ofconventionalinpatients with colorectal cancer mrc clasicc trial multicentre randomised controlled trial lancet “laparoscopicassisted surgeryversus jeong sy park jw nam bh open versus laparoscopicsurgery for midrectal or lowrectal cancer after neoadjuvantchemoradiotherapy corean trial survival outcomes of anopenlabel noninferiorityrandomised controlled triallancet oncol “ kitano s inomata m mizusawa j survival outcomes following laparoscopic versus open d3 dissection for stage ii or mehrkhani f nasiri s donboli k prognostic factors insurvival of colorectal cancer patients after surgery colorectaldis “ wiratkapun s kraemer m seowchoen f high preoperative serum carcinoembryonic antigen predicts metastatic recurrence in potentially curative colonic cancer results of afiveyear study dis colon rectum “ huang qs qin hb xiao j association of tumor differentiation and prognosis in patients with rectal cancer undergoingneoadjuvant chemoradiation therapy gastroenterol rep “ liebig c ayala g wilks ja perineural invasion is an independent predictor of outcome in colorectal cancer j clin oncol“ ueno h shirouzu k eishi y study group for perineuralinvasion projected by the japanese society for cancer of thecolon and rectum jsccr characterization of perineural invasion as a component of colorectal cancer staging am j surgpathol “ amin mb edge sb greene fl eds ajcc cancer stagingmanual 8th edn new york springer “ rosenberg r engel j bruns c the prognostic value oflymph node ratio in a populationbased collective of colorectal cancer patients ann surg “ sjo oh merok ma svindland a prognostic impact oflymph node harvest and lymph node ratio in patients withcolon cancer dis colon rectum “ tarantino i warschkow r worni m elevated preoperative cea is associated with worse survival in stage iiii rectalcancer patients br j cancer “ huang sh tsai ws you jf preoperative carcinoembryonic antigen as a poor prognostic factor in stage iiii colorectal cancer after curativeintent resection a propensity scorematching analysis ann surg oncol “ konishi t shimada y hsu m association of preoperativeand postoperative serum carcinoembryonic antigen and colon cancer outcome jama oncol “ 0c' Answer:

Colon_Cancer

since leung reported a single case of lowdose aspirin lda induced multiple smallintestinal ulcers in many investigators have described ldainducedsmall intestinal mucosal injuries watanabe reportedthat all of their patients who used lda had small intestinallesions that were detected using capsule endoscopy 0cgastroenterology research and practicece iwamoto investigated patients whounderwent ce for occult bleeding and erosions wereobserved in cases of whom were taking lda ornonlda nonsteroidal anti‚ammatory drugs endo described small intestinal lesions in of subjects who took aspirin mg for weeks shiotani performed ce on young healthy individuals beforeand after medium doses of enteric aspirin were administered for days and rabeprazole mg was administered for week and found large erosions that included small intestinal ulcers in of the subjects recently direct oral anticoagulants doacs have beenadministered as alternatives to warfarin dabigatran is adirect thrombin inhibitor and rivaroxaban apixaban andedoxaban are factor xa inhibitors doacs have significantlyfewer side eï¬ects than warfarin including intracranial hemorrhage hence the number of patients taking doacs isgradually increasing [“] howeverthe findings frommetaanalyses have shown that compared with warfarinthe incidence of gastrointestinal gi bleeding is higher inassociation with doacs [ ] the causes of gi bleedingin association with doacs were bleeding from colon astric cancers and diverticular hemorrhages comparedwith warfarin dabigatran and rivaroxaban are associatedwith higher risks of gi bleeding depending on their dosesand the risk of gi bleeding associated with apixaban iscomparable edoxaban is associated with significantlyless gi bleeding at low doses mg once daily comparedwith warfarin but the risk is significantly greater at highdoses mg once daily but unlike western countries the rate of gi bleeding for both dabigatran and rivaroxaban is equivalent to warfarin in asian countries furthermore edoxaban tends to cause less digestive tractbleeding than warfarin esophageal ulcers caused bydabigatran have been described by toya kasai and okada and okada the tartaric acidcoating on dabigatran causes esophageal mucosal disorders because dabigatran persists in the midesophagus ifit is consumed without water however there havebeen no reports of smalllesions in patientswho receive doacs here we aimed to evaluate smallintestinal mucosal injuries in patients taking doacs usingvideo capsule endoscopy vceintestinal methodsthis study was a prospective openlabel nonblinded multicenter and observational study from september tomarch pat5ents taking doacs namely dabigatranrivaroxaban and apixaban for atrial fibrillation at saitamamedical university hospital keio university hospital saitama medical center and yokohama municipal citizen™shospital were enrolled patients with severe comorbiditiesincluding severe anemia and exacerbations of heart failurethat required blood transfusion crohn™s disease and ileuswere excludedthe hemoglobin hb and serum ferritin levels the esophagogastroduodenoscopy egd findings and colonoscopicfindings were examined vce pillcam sb2 given imagingfigure rednessfigure erosionltd yoqneam israel was performed to examine small intestinal lesions according to the doac used redness erosionulcer and angioectasia were checked redness was a red spotfigure and erosions were defined as small and superficialmucosal disruptions denuded of villi figure ulcers weredefined as large submucosal disruptions with a central areacovered with exudate and angioectasia was a patchy erythematous lesion figure the images were analyzed using theproprietary rapid software by an expert n h whohad performed more than vce examinations blindlythe type and location of smallbowel lesions were registeredalso the proportion of lesions detected between types ofdoac was evaluated and the hemoglobin hb and serumferritin levels were compared between patients with and without smallbowel lesionsthe study protocol accorded with the tenets of therevised declaration of helsinkiand it wasapproved by the institutional review boards at our institutions written informed consent was obtained from all ofthe patients this study was registered with the universityhospital medicalinformation network clinical trialsregistry umin000011527 october 0cgastroenterology research and practicetable patients™ characteristicsparametermean age years rangesex n malefemalecomorbid disease natrial fibrillationparoxysmal atrial fibrillationhypertensionhyperlipidemiacerebral infarctiondoac n years “dabigatranrivaroxabanapixabanbayaspirin ncelecoxib nppi nh2 blocker n “mean hb gdl rangedoac direct oral anticoagulant ppi proton pump inhibitor hbhemoglobinlower portion in patients table erosions wereobserved in patients and they were present in theupper portion in in the middle portion in and in the lower portion in patientstable erosions tended to occur less frequently in themiddle portion however the diï¬erence was not significantp compared with the upper portion p compared with the lower portion angioectasia was observedin patients and was present in the upper portionin patients and in the middle portion in patient and was absent from the lower portion table there were no ulcers in any patients erosions tended to bemore frequent in patients taking dabigatran or apixaban thanin patients taking rivaroxaban this diï¬erence was not significant p table no significant diï¬erences wereobserved regarding angioectasia among the patients takingthe diï¬erent doacs none of these patients had activebleeding from small intestinal lesionsthe mean hb concentrations in the patients with andlesions were gdl and gdlwithout smallbowelrespectively a diï¬erence which was not significant p the mean ferritin levels in the patients with and withoutsmallbowel lesions were mgdl and mgdl respectively a diï¬erence which was not significant p discussionthis study™s findings showed that of the patients who tookdoacs had redness had erosionsor small ulcers had angioectasia and had no abnormalities in their small bowel smallbowellesions were observed in of patients thereforethere was a high incidence of smallbowel lesions in patientsfigure angioectasiaibm®spss® statistical software version ibm corporation armonk ny usa was used for the statisticalanalyses the data were analyzed using ttests and fisher™sexact test resultsthirtythree patients were enrolled to participate in thisstudy but patients withdrew their consent and vce wasperformed on patients the patients™ mean age was years range “ years and males and females participated in this study twenty patients had atrial fibrillation had paroxysmal atrial fibrillation had hypertension hadhyperlipidemia and had cerebral infarction eight patientstook dabigatran took rivaroxaban and took apixabanthe mean duration of doac use was months months additionally patient took bayaspirin patienttook celecoxib patients took proton pump inhibitorsppis and patients took h2 receptor antagonists theaverage hb concentration was gdl range “ gdl table twentytwo patients underwent egdand atrophic gastritis was present in patients hiatal hernias in patients gastric polyps in patients erosive gastritisin patients gastric ulcer or ulcer scar in patients refluxesophagitis in patients endoscopic submucosal dissectionscar for early gastric cancer in patients and an esophagealulcer in patient nineteen patients underwent colonoscopyand colonic polyps were present in patients colonic diverticulum were present in patients and a rectal ulcer waspresent in patient none of these lesions detected by egdand colonoscopy had active bleedingthe patients evaluated with vce was redness in thelower esophagus was present in patient gastric erosionswere present in patients and gastric redness was presentin patient table the patient who had redness in thelower esophagus was taking apixaban smallbowel transitwas complete in of patients smallbowellesions were observed in of patients redness was observed throughout the small intestine in patients and it was present in the upper portionin in the middle portion in and in the 0cgastroenterology research and practicetable capsule endoscopy findingsentire small intestine observation rate n detection rate n esophagusstomachupper small intestinemiddle small intestinelower small intestineentire small intestineredness lower n erosions n redness n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions nangioectasia nno abnormalities n table findings at each small intestinal site according to the directoral anticoagulant useddabigatranrivaroxabanapixabansitefindingupper small intestineredness nerosions nangioectasia nmiddle small intestineredness nerosions nangioectasialower small intestineredness nerosions nangioectasia nentire small intestineredness nerosions nangioectasia ntaking doacs however none of these patients had activebleeding and most of the lesions were mild patients withsevere anemia or active bleeding were excluded from thisstudy hence only patients with mild symptoms wereincluded ldainduced lesions cause redness erosions andulcers [“] previous studies™ findings that describe the characteristics of smallbowel injuries associated with chroniclda use suggest that ulcers are observed mainly in the distalpart of the small bowel [“] in this study erosionstended to be observed less frequently in the middle portionof the small bowel in the patients taking doacs howeverthere were no significant diï¬erences regarding the distributions of the lesions there were no ulcers in any patientstherefore the intake of doac might not be related withsevere ulcers in the small intestinein this study angioectasia was observed in the upper andmiddle portions but not in the lower portion of the smallbowel kaufman used a transit timebased quartilemethod to evaluate patients with angioectasia whounderwent ce and found that most lesions were inthe first quartile igawa reported that while therewere no diï¬erences regarding the location of type 1a angioectasia among patients with occult gastrointestinal bleedingtype 1b angioectasia was relatively less frequent in the lowerportion compared with that in the upper and middle portionsof the small bowel the data reported before thereforeangioectasia might not be aï¬ected by the intake of doacscomparison of vce findings before and after the administration of d719acs is neededno significant relationships were determined in relationto the presence of the hb level or the serum ferritin levelbetween the patients with and without smallbowel lesionsin this study despite detecting abnormal findings in thesmall bowel no active bleeding was seen by vce and therewas no severe anemia in any patients in this study furthermore compared with warfarin the incidence of gi bleedingis higher in association with doacs the causes of gi bleeding in association with doacs are bleeding from colon astric cancers and diverticular hemorrhages however noneof these lesions detected by egd and colonoscopy had activebleeding doacs did not aï¬ect bleeding from the upper gitract and the colon in this studyno significant diï¬erences were observed among thedoacs in relation to smallbowel lesions the findings fromthe randomized evaluation of longterm anticoagulationtherapy trial of dabigatran showed that in the warfaringroup patients with gi tract bleeding had gastric canceror colonic cancer and that in the dabigatran group patients with gi tract bleeding had colonic cancer and patient had gastric cancer hence dabigatran mightinduce gi tract bleeding from colon cancer rivaroxaban apixaban and edoxaban compete directly with the s1pocket of factor xa and inhibit factor xa activity whereasdabigatran is a prodrug that is activated in the presence ofesterase in the gi tract plasma and liver the causes of themucosal damage by dabigatran were thought to be due todirect acting at the local area where it is absorbed in addition tartaric acid coats dabigatran tablets and the tabletscan cause mucosal damage if they are retained within theesophagus while we expected an increase in the frequencyof intestinal mucosal injury among the patients who tookdabigatran as a consequence of the tartaric acid coating this 0cgastroenterology research and practicestudy™s findings did not demonstrate a higher rate of smallbowel lesions associated with the use of this doac themechanisms underlying mucosal injuries caused by doacsother than dabigatran remain unclear smallbowel lesionsincluding redness erosions and angioectasia might be moreeasily detected by performing ce on patients who takedoacs because the doacs might cause bleeding thatcould facilitate the detection of the lesionsthis study has several limitations while this was a multicenter study the sample size was small hence more patientsshould be accrued and investigated in the near future moreover this study only included data that described the patients™ï¬ndings after the administration of the doacs and datadescribing the findings before their administration wereabsent therefore it remains unclear whether small intestinallesions are directly induced by doacs studies of patients™ï¬ndings before and after the administration of doacs areneeded in the near future furthermore patients with severeanemia and overt bleeding were excluded from this studyand most of the enrolled patients did not have bleeding orhad minor bleeding patients taking edoxaban were notincluded in this study because edoxaban was not available injapan when this study began conclusionlesionssmallbowelincluding redness erosions smallulcers and angioectasia were detected in of patientswho took doacs more patients using doacs should beinvestigated using ce in the near futureabbreviationsdoac direct oral anticoagulantvideo capsule endoscopyvceldalowdose aspirincapsule endoscopycegastrointestinalgihemoglobinhbegdesophagogastroduodenoscopyproton pump inhibitorppidata availabilitythis manuscript describes a study that was aimed at evaluating direct oral anticoagulant doac related to smallbowellesions using video capsule endoscopy we believe that ourstudy makes a significant contribution to the literaturebecause its findings showed that many patients takingdoacs had smallbowel lesions however most lesions wererelatively mild and they did not cause bleedingconflicts of interestthe authors have no conflicts of interest to disclose that arerelevant to this studyauthors™ contributionshiroyuki imaeda was responsible for the conception anddesign and final approval of the article minoru yamaokahiroyuki imaeda naoki hosoe kazuaki yoneno ryukanno hisashi mitsufuji takahiro sasaki and keijiyamamoto were responsible for enrollment of patientshiroyuki imaeda and naoki hosoe were responsible foranalysis and interpretation of the data minoru yamaokaand hiroyuki imaeda were responsible for drafting of thearticle naoki hosoe was responsible for the critical revision of the article for important intellectual content takanori kanai toshimasa yamamoto toshihide mimuraharuhiko ogata nobuo araki and hidetomo nakamotowere the supervisorsreferences w k leung i bjarnason v w s wong j j y sung andf k l chan œsmall bowel enteropathy associated withchronic lowdose aspirin therapy lancet vol no t watanabe s sugimori n kameda œsmall bowelinjury by lowdose entericcoated aspirin and treatment withmisoprostol a pilot study clinical gastroenterology andhepatology vol no pp “ j iwamoto y mizokami y saito œsmallbowel mucosalinjuries in lowdose aspirin users with obscure gastrointestinalbleeding world journal of gastroenterology vol no pp “ h endo k hosono m inamori œincidence of smallbowel injury induced by lowdose aspirin a crossover studyusing capsule endoscopy in healthy volunteers digestionvol no pp “ a shiotani k haruma r nishi œrandomized doubleblind pilot study of geranylgeranylacetone versus placebo inpatients taking lowdose entericcoated aspirin lowdoseaspirininduced small bowel damage scandinavian journalof gastroenterology vol no pp “ s j connolly m d ezekowitz s yusuf œdabigatranversus warfarin in patients with atrial fibrillation the newengland journal of medicine vol no pp “ m r patel k w mahaï¬ey j garg œrivaroxaban versuswarfarin in nonvalvular atrial fibrillation new england journal of medicine vol pp “ c b granger j h alexander j j v mcmurray œapixaban versus warfarin in patients with atrial fibrillation thenew england journal of medicine vol no pp “ c t ruï¬ r p giugliano e braunwald œcomparison ofthe efficacy and safety of new oral anticoagulants with warfarinin patients with atrial fibrillation a metaanalysis of randomised trials lancet vol no pp “ c s miller a dorreen m martel t huynh and a n barkun œrisk of gastrointestinal bleeding in patients taking nonvitamin k antagonist oral anticoagulants a systematic reviewand metaanalysis clinical gastroenterology and hepatologyvol no pp “1683e3 d caldeira m barra a ferreira œsystematic reviewwith metaanalysis the risk of major gastrointestinal bleeding 0cgastroenterology research and practicewith nonvitamin k antagonist oral anticoagulants alimentary pharmacology therapeutics vol no pp “ t yamashita y koretsune y yang œedoxaban vs warfarin in east asian patients with atrial fibrillationan engageaftimi subanalysis circulation journal vol no pp “ m hori m matsumoto n tanahashi œrivaroxaban vswarfarin in japanese patients with atrial fibrillation circulation journal vol no pp “ y toya s nakamura k tomita œdabigatraninducedesophagitis the prevalence and endoscopic characteristicsjournal of gastroenterology and hepatology vol no pp “ k kasai e ishida and y kobayashi œtwo cases of esophagealulcer caused by dabigatran gastroenterological endoscopyvol pp “ m okada and k okada œexfoliative esophagitis and esophageal ulcer induced by dabigatran endoscopy vol supplement pp e23“e24 t vanassche j hirsh j ginsberg and j eikelboom œanspecific bleeding patterns of anticoagulant therapy lessonsfrom clinical trials thrombosis and haemostasis vol pp “ h endo k hosono m inamori œcharacteristics ofsmall bowel injury in symptomatic chronic lowdose aspirinusers the experience of two medical centers in capsule endoscopy journal of gastroenterology vol no pp “ i watari s oka s tanaka œcomparison of smallbowelmucosalinjury between lowdose aspirin and nonaspirinnonsteroidal anti‚ammatory drugs a capsule endoscopystudy digestion vol no pp “ h endo k hosono t higurashi œquantitative analysisof lowdose aspirinassociated small bowel injury using a capsule endoscopy scoring index digestive endoscopy vol no pp “ d kaufman g leslie n marya œsmallintestinalangioectasia characterization risk factors and rebleedingjournal of clinical gastroenterology vol no pp “ a igawa s oka s tanaka œmajor predictors and management of smallbowel angioectasia bmc gastroenterologyvol no 0c'

cancer7504

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: since leung reported a single case of lowdose aspirin lda induced multiple smallintestinal ulcers in many investigators have described ldainducedsmall intestinal mucosal injuries watanabe reportedthat all of their patients who used lda had small intestinallesions that were detected using capsule endoscopy 0cgastroenterology research and practicece iwamoto investigated patients whounderwent ce for occult bleeding and erosions wereobserved in cases of whom were taking lda ornonlda nonsteroidal anti‚ammatory drugs endo described small intestinal lesions in of subjects who took aspirin mg for weeks shiotani performed ce on young healthy individuals beforeand after medium doses of enteric aspirin were administered for days and rabeprazole mg was administered for week and found large erosions that included small intestinal ulcers in of the subjects recently direct oral anticoagulants doacs have beenadministered as alternatives to warfarin dabigatran is adirect thrombin inhibitor and rivaroxaban apixaban andedoxaban are factor xa inhibitors doacs have significantlyfewer side eï¬ects than warfarin including intracranial hemorrhage hence the number of patients taking doacs isgradually increasing [“] howeverthe findings frommetaanalyses have shown that compared with warfarinthe incidence of gastrointestinal gi bleeding is higher inassociation with doacs [ ] the causes of gi bleedingin association with doacs were bleeding from colon astric cancers and diverticular hemorrhages comparedwith warfarin dabigatran and rivaroxaban are associatedwith higher risks of gi bleeding depending on their dosesand the risk of gi bleeding associated with apixaban iscomparable edoxaban is associated with significantlyless gi bleeding at low doses mg once daily comparedwith warfarin but the risk is significantly greater at highdoses mg once daily but unlike western countries the rate of gi bleeding for both dabigatran and rivaroxaban is equivalent to warfarin in asian countries furthermore edoxaban tends to cause less digestive tractbleeding than warfarin esophageal ulcers caused bydabigatran have been described by toya kasai and okada and okada the tartaric acidcoating on dabigatran causes esophageal mucosal disorders because dabigatran persists in the midesophagus ifit is consumed without water however there havebeen no reports of smalllesions in patientswho receive doacs here we aimed to evaluate smallintestinal mucosal injuries in patients taking doacs usingvideo capsule endoscopy vceintestinal methodsthis study was a prospective openlabel nonblinded multicenter and observational study from september tomarch pat5ents taking doacs namely dabigatranrivaroxaban and apixaban for atrial fibrillation at saitamamedical university hospital keio university hospital saitama medical center and yokohama municipal citizen™shospital were enrolled patients with severe comorbiditiesincluding severe anemia and exacerbations of heart failurethat required blood transfusion crohn™s disease and ileuswere excludedthe hemoglobin hb and serum ferritin levels the esophagogastroduodenoscopy egd findings and colonoscopicfindings were examined vce pillcam sb2 given imagingfigure rednessfigure erosionltd yoqneam israel was performed to examine small intestinal lesions according to the doac used redness erosionulcer and angioectasia were checked redness was a red spotfigure and erosions were defined as small and superficialmucosal disruptions denuded of villi figure ulcers weredefined as large submucosal disruptions with a central areacovered with exudate and angioectasia was a patchy erythematous lesion figure the images were analyzed using theproprietary rapid software by an expert n h whohad performed more than vce examinations blindlythe type and location of smallbowel lesions were registeredalso the proportion of lesions detected between types ofdoac was evaluated and the hemoglobin hb and serumferritin levels were compared between patients with and without smallbowel lesionsthe study protocol accorded with the tenets of therevised declaration of helsinkiand it wasapproved by the institutional review boards at our institutions written informed consent was obtained from all ofthe patients this study was registered with the universityhospital medicalinformation network clinical trialsregistry umin000011527 october 0cgastroenterology research and practicetable patients™ characteristicsparametermean age years rangesex n malefemalecomorbid disease natrial fibrillationparoxysmal atrial fibrillationhypertensionhyperlipidemiacerebral infarctiondoac n years “dabigatranrivaroxabanapixabanbayaspirin ncelecoxib nppi nh2 blocker n “mean hb gdl rangedoac direct oral anticoagulant ppi proton pump inhibitor hbhemoglobinlower portion in patients table erosions wereobserved in patients and they were present in theupper portion in in the middle portion in and in the lower portion in patientstable erosions tended to occur less frequently in themiddle portion however the diï¬erence was not significantp compared with the upper portion p compared with the lower portion angioectasia was observedin patients and was present in the upper portionin patients and in the middle portion in patient and was absent from the lower portion table there were no ulcers in any patients erosions tended to bemore frequent in patients taking dabigatran or apixaban thanin patients taking rivaroxaban this diï¬erence was not significant p table no significant diï¬erences wereobserved regarding angioectasia among the patients takingthe diï¬erent doacs none of these patients had activebleeding from small intestinal lesionsthe mean hb concentrations in the patients with andlesions were gdl and gdlwithout smallbowelrespectively a diï¬erence which was not significant p the mean ferritin levels in the patients with and withoutsmallbowel lesions were mgdl and mgdl respectively a diï¬erence which was not significant p discussionthis study™s findings showed that of the patients who tookdoacs had redness had erosionsor small ulcers had angioectasia and had no abnormalities in their small bowel smallbowellesions were observed in of patients thereforethere was a high incidence of smallbowel lesions in patientsfigure angioectasiaibm®spss® statistical software version ibm corporation armonk ny usa was used for the statisticalanalyses the data were analyzed using ttests and fisher™sexact test resultsthirtythree patients were enrolled to participate in thisstudy but patients withdrew their consent and vce wasperformed on patients the patients™ mean age was years range “ years and males and females participated in this study twenty patients had atrial fibrillation had paroxysmal atrial fibrillation had hypertension hadhyperlipidemia and had cerebral infarction eight patientstook dabigatran took rivaroxaban and took apixabanthe mean duration of doac use was months months additionally patient took bayaspirin patienttook celecoxib patients took proton pump inhibitorsppis and patients took h2 receptor antagonists theaverage hb concentration was gdl range “ gdl table twentytwo patients underwent egdand atrophic gastritis was present in patients hiatal hernias in patients gastric polyps in patients erosive gastritisin patients gastric ulcer or ulcer scar in patients refluxesophagitis in patients endoscopic submucosal dissectionscar for early gastric cancer in patients and an esophagealulcer in patient nineteen patients underwent colonoscopyand colonic polyps were present in patients colonic diverticulum were present in patients and a rectal ulcer waspresent in patient none of these lesions detected by egdand colonoscopy had active bleedingthe patients evaluated with vce was redness in thelower esophagus was present in patient gastric erosionswere present in patients and gastric redness was presentin patient table the patient who had redness in thelower esophagus was taking apixaban smallbowel transitwas complete in of patients smallbowellesions were observed in of patients redness was observed throughout the small intestine in patients and it was present in the upper portionin in the middle portion in and in the 0cgastroenterology research and practicetable capsule endoscopy findingsentire small intestine observation rate n detection rate n esophagusstomachupper small intestinemiddle small intestinelower small intestineentire small intestineredness lower n erosions n redness n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions nangioectasia nno abnormalities n table findings at each small intestinal site according to the directoral anticoagulant useddabigatranrivaroxabanapixabansitefindingupper small intestineredness nerosions nangioectasia nmiddle small intestineredness nerosions nangioectasialower small intestineredness nerosions nangioectasia nentire small intestineredness nerosions nangioectasia ntaking doacs however none of these patients had activebleeding and most of the lesions were mild patients withsevere anemia or active bleeding were excluded from thisstudy hence only patients with mild symptoms wereincluded ldainduced lesions cause redness erosions andulcers [“] previous studies™ findings that describe the characteristics of smallbowel injuries associated with chroniclda use suggest that ulcers are observed mainly in the distalpart of the small bowel [“] in this study erosionstended to be observed less frequently in the middle portionof the small bowel in the patients taking doacs howeverthere were no significant diï¬erences regarding the distributions of the lesions there were no ulcers in any patientstherefore the intake of doac might not be related withsevere ulcers in the small intestinein this study angioectasia was observed in the upper andmiddle portions but not in the lower portion of the smallbowel kaufman used a transit timebased quartilemethod to evaluate patients with angioectasia whounderwent ce and found that most lesions were inthe first quartile igawa reported that while therewere no diï¬erences regarding the location of type 1a angioectasia among patients with occult gastrointestinal bleedingtype 1b angioectasia was relatively less frequent in the lowerportion compared with that in the upper and middle portionsof the small bowel the data reported before thereforeangioectasia might not be aï¬ected by the intake of doacscomparison of vce findings before and after the administration of d719acs is neededno significant relationships were determined in relationto the presence of the hb level or the serum ferritin levelbetween the patients with and without smallbowel lesionsin this study despite detecting abnormal findings in thesmall bowel no active bleeding was seen by vce and therewas no severe anemia in any patients in this study furthermore compared with warfarin the incidence of gi bleedingis higher in association with doacs the causes of gi bleeding in association with doacs are bleeding from colon astric cancers and diverticular hemorrhages however noneof these lesions detected by egd and colonoscopy had activebleeding doacs did not aï¬ect bleeding from the upper gitract and the colon in this studyno significant diï¬erences were observed among thedoacs in relation to smallbowel lesions the findings fromthe randomized evaluation of longterm anticoagulationtherapy trial of dabigatran showed that in the warfaringroup patients with gi tract bleeding had gastric canceror colonic cancer and that in the dabigatran group patients with gi tract bleeding had colonic cancer and patient had gastric cancer hence dabigatran mightinduce gi tract bleeding from colon cancer rivaroxaban apixaban and edoxaban compete directly with the s1pocket of factor xa and inhibit factor xa activity whereasdabigatran is a prodrug that is activated in the presence ofesterase in the gi tract plasma and liver the causes of themucosal damage by dabigatran were thought to be due todirect acting at the local area where it is absorbed in addition tartaric acid coats dabigatran tablets and the tabletscan cause mucosal damage if they are retained within theesophagus while we expected an increase in the frequencyof intestinal mucosal injury among the patients who tookdabigatran as a consequence of the tartaric acid coating this 0cgastroenterology research and practicestudy™s findings did not demonstrate a higher rate of smallbowel lesions associated with the use of this doac themechanisms underlying mucosal injuries caused by doacsother than dabigatran remain unclear smallbowel lesionsincluding redness erosions and angioectasia might be moreeasily detected by performing ce on patients who takedoacs because the doacs might cause bleeding thatcould facilitate the detection of the lesionsthis study has several limitations while this was a multicenter study the sample size was small hence more patientsshould be accrued and investigated in the near future moreover this study only included data that described the patients™ï¬ndings after the administration of the doacs and datadescribing the findings before their administration wereabsent therefore it remains unclear whether small intestinallesions are directly induced by doacs studies of patients™ï¬ndings before and after the administration of doacs areneeded in the near future furthermore patients with severeanemia and overt bleeding were excluded from this studyand most of the enrolled patients did not have bleeding orhad minor bleeding patients taking edoxaban were notincluded in this study because edoxaban was not available injapan when this study began conclusionlesionssmallbowelincluding redness erosions smallulcers and angioectasia were detected in of patientswho took doacs more patients using doacs should beinvestigated using ce in the near futureabbreviationsdoac direct oral anticoagulantvideo capsule endoscopyvceldalowdose aspirincapsule endoscopycegastrointestinalgihemoglobinhbegdesophagogastroduodenoscopyproton pump inhibitorppidata availabilitythis manuscript describes a study that was aimed at evaluating direct oral anticoagulant doac related to smallbowellesions using video capsule endoscopy we believe that ourstudy makes a significant contribution to the literaturebecause its findings showed that many patients takingdoacs had smallbowel lesions however most lesions wererelatively mild and they did not cause bleedingconflicts of interestthe authors have no conflicts of interest to disclose that arerelevant to this studyauthors™ contributionshiroyuki imaeda was responsible for the conception anddesign and final approval of the article minoru yamaokahiroyuki imaeda naoki hosoe kazuaki yoneno ryukanno hisashi mitsufuji takahiro sasaki and keijiyamamoto were responsible for enrollment of patientshiroyuki imaeda and naoki hosoe were responsible foranalysis and interpretation of the data minoru yamaokaand hiroyuki imaeda were responsible for drafting of thearticle naoki hosoe was responsible for the critical revision of the article for important intellectual content takanori kanai toshimasa yamamoto toshihide mimuraharuhiko ogata nobuo araki and hidetomo nakamotowere the supervisorsreferences w k leung i bjarnason v w s wong j j y sung andf k l chan œsmall bowel enteropathy associated withchronic lowdose aspirin therapy lancet vol no t watanabe s sugimori n kameda œsmall bowelinjury by lowdose entericcoated aspirin and treatment withmisoprostol a pilot study clinical gastroenterology andhepatology vol no pp “ j iwamoto y mizokami y saito œsmallbowel mucosalinjuries in lowdose aspirin users with obscure gastrointestinalbleeding world journal of gastroenterology vol no pp “ h endo k hosono m inamori œincidence of smallbowel injury induced by lowdose aspirin a crossover studyusing capsule endoscopy in healthy volunteers digestionvol no pp “ a shiotani k haruma r nishi œrandomized doubleblind pilot study of geranylgeranylacetone versus placebo inpatients taking lowdose entericcoated aspirin lowdoseaspirininduced small bowel damage scandinavian journalof gastroenterology vol no pp “ s j connolly m d ezekowitz s yusuf œdabigatranversus warfarin in patients with atrial fibrillation the newengland journal of medicine vol no pp “ m r patel k w mahaï¬ey j garg œrivaroxaban versuswarfarin in nonvalvular atrial fibrillation new england journal of medicine vol pp “ c b granger j h alexander j j v mcmurray œapixaban versus warfarin in patients with atrial fibrillation thenew england journal of medicine vol no pp “ c t ruï¬ r p giugliano e braunwald œcomparison ofthe efficacy and safety of new oral anticoagulants with warfarinin patients with atrial fibrillation a metaanalysis of randomised trials lancet vol no pp “ c s miller a dorreen m martel t huynh and a n barkun œrisk of gastrointestinal bleeding in patients taking nonvitamin k antagonist oral anticoagulants a systematic reviewand metaanalysis clinical gastroenterology and hepatologyvol no pp “1683e3 d caldeira m barra a ferreira œsystematic reviewwith metaanalysis the risk of major gastrointestinal bleeding 0cgastroenterology research and practicewith nonvitamin k antagonist oral anticoagulants alimentary pharmacology therapeutics vol no pp “ t yamashita y koretsune y yang œedoxaban vs warfarin in east asian patients with atrial fibrillationan engageaftimi subanalysis circulation journal vol no pp “ m hori m matsumoto n tanahashi œrivaroxaban vswarfarin in japanese patients with atrial fibrillation circulation journal vol no pp “ y toya s nakamura k tomita œdabigatraninducedesophagitis the prevalence and endoscopic characteristicsjournal of gastroenterology and hepatology vol no pp “ k kasai e ishida and y kobayashi œtwo cases of esophagealulcer caused by dabigatran gastroenterological endoscopyvol pp “ m okada and k okada œexfoliative esophagitis and esophageal ulcer induced by dabigatran endoscopy vol supplement pp e23“e24 t vanassche j hirsh j ginsberg and j eikelboom œanspecific bleeding patterns of anticoagulant therapy lessonsfrom clinical trials thrombosis and haemostasis vol pp “ h endo k hosono m inamori œcharacteristics ofsmall bowel injury in symptomatic chronic lowdose aspirinusers the experience of two medical centers in capsule endoscopy journal of gastroenterology vol no pp “ i watari s oka s tanaka œcomparison of smallbowelmucosalinjury between lowdose aspirin and nonaspirinnonsteroidal anti‚ammatory drugs a capsule endoscopystudy digestion vol no pp “ h endo k hosono t higurashi œquantitative analysisof lowdose aspirinassociated small bowel injury using a capsule endoscopy scoring index digestive endoscopy vol no pp “ d kaufman g leslie n marya œsmallintestinalangioectasia characterization risk factors and rebleedingjournal of clinical gastroenterology vol no pp “ a igawa s oka s tanaka œmajor predictors and management of smallbowel angioectasia bmc gastroenterologyvol no 0c' Answer:

Colon_Cancer

chrysoperla nipponensis okamoto which has the unique diapause phenotype distinguishable from nondiapause adult is an ideal model anism for studying the mechanism of reproductive diapause however there is no reliable and effective reference genes used for the reproductive diapause study of c a0nipponensis therefore in this study we evaluated the expression stability of candidate reference genes tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin αtub in adults under diapause and nondiapause induction conditions using four statistical algorithms including genorm normfinder bestkeeper and ˆ†ct method results showed that arp3 and tub1 were the most stable reference genes in all samples and in the adult tissues group arp3 and rps5 were the most stable reference genes in the development degree group αtub and ef1a were unstable reference genes under the conditions of this study meanwhile to verify the reliability of the reference genes we evaluated the relative expression levels of vg and vgr in different treatments significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was respectively observed when using arp3 as the reference gene but not when using an unstable reference gene the reference genes identified in this work provided not only the basis for future functional genomics research in diapause of c a0nipponensis and will also identify reliable normalization factors for realtime quantitative realtime polymerase chain reaction data for other related insectskey words chrysoperla nipponensis okamoto reference genes qrtpcr reproduction diapausedue to the advantages of high sensitivity rapidity specificity and accuracy bustin et a0al valasek et a0al vanguilder et a0al shakeel et a0al quantitative realtime polymerase chain reaction qrtpcr has been widely used in the study of animals plants and microanisms roy et a0al jia et a0al zhang et a0 al ding et a0 al sun et a0 al qrtpcr is the most commonly used method for the expression analysis of target genes however the reliability of qrtpcr results in different samples is determined by a variety of factors among which the use of stably expressed reference genes is an important link for accurate detection of gene expression changes by qrtpcr bustin et a0 al at present several commonly used reference genes for data normalization include tubulin actin ribosomal protein elongation factor 1α glyceraldehyde3phosphate dehydrogenase 18s ribosomal rna and other genes bustin vanguilder et a0al however more and more studies have found that these reference genes do not show consistent expression patterns under different experimental conditions and even affect the reliability of experimental results therefore in order to obtain stable and reliable normalization factors reference genes with stable expression are usually used for correction and standardization to reduce errors between samples selection and evaluation of reference genes have become a necessary step before quantifying the expression of target genes accuratelynowadays there have been many studies on the selection of reference genes for insects such as sesamia inferens helicoverpa armigera aphis gossypii myzus persicae etc lu et a0 al shakeel et a0al zhang et a0al ma et a0al kang et a0al in these studies four statistical algorithms including genorm normfinder bestkeeper and ˆ†ct method were mainly used to analyze the ct values obtained by qrtpcr of reference genes under various experimental conditions shakeel et a0 al finally the stability of the candidate reference genes was determined according to the geometric mean value of each gene ranked using different the authors published by oxford university press on behalf of entomological society of americathis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly cited for commercial reuse please contact spermissionsoupcom 0c of insect science vol no algorithms and the most suitable reference gene was selected for the target gene expression analysis xiao et a0al kang et a0al chrysoperla nipponensis okamoto as one of the important predatory natural enemies of agricultural and forestry pests prefers to eat aphids thrips and other pests okamoto nie et a0al because of its characteristically wide geographical distribution and broad range of host prey niijima syed et a0al it has good prospects for widespread application in biological control mcewen et a0 al memon et a0 al reproductive diapause is an important way for c a0nipponensis adults to escape from adverse environments xu et a0al at present there have been many reports on the diapause of c a0nipponensis xu et a0al found that the body color of c a0nipponensis was green during the reproductive period but turned brown and yellow during the diapause period chrysoperla nipponensis belongs to the photoperiodic sensitive insect the adult diapause was induced by short photoperiods xu et a0al chen et a0al found that different photoperiods affected the material content eg protein and glycogen of c a0nipponensis diapause induced by the short photoperiod was beneficial to the storage of c a0nipponensis chen et a0al we expect an exponential increase of diapause research on c a0nipponensis at the molecular level in the near future thus stable and reliable reference genes are important for accurately quantifying gene expression of c a0nipponensisribosomal proteins and ribosomal rna have been used as reference genes in previous diapause studies for example williams et a0al used ribosomal protein rp49 as a reference gene to study the natural variation of drosophila melanogaster diapause williams et a0 al and sim and denlinger used ribosomal protein large subunit rpl19 as a reference gene in a study of ovarian development of culex pipiens during overwintering diapause sim and denlinger this indicates that under the same experimental conditions the selected reference genes in different species research are also differentin this research candidate reference genes were selected including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub whose expression profiles were measured by the qrtpcr the stability was analyzed by four statistical algorithms genorm normfinder bestkeeper and ˆ†ct method in different developmental stages reproductive and diapause of adults and among different tissues the optimal reference genes under different conditions were determined which contributed to the accurate expression of target genes for future researchmaterials and a0methodsinsecta stable population of c a0 nipponensis was maintained in an artificial climate chamber rsz intelligent artificial climate chamber changzhou guohua jiangsu province under the following conditions a0± °c temperature a0± relative humidity rh and long photoperiod of l d h in our laboratory the eggs were collected by cutting the stalk and incubated in fingertip tubes a0cm in diameter and a0cm in height the primary hatching larvae were fed megoura japonica matsumura whose host plant was vicia faba l a0the adults were paired immediately after emergence in a bottle a0cm in diameter and a0cm in height fed a dry brewer™s yeast feed mixed with sucrose in a ratio of and then minced in a mortar and sifted through mesh and honey water the diapause adults used in this study were kept under the conditions of short photoperiod of ld h in all processes from eggs larvae pupae to adults whereas the nondiapause adults were kept under the conditions of long photoperiod of ld a0hsample collection development degree samples from individuals from varying developmental stages included female adults in the diapause induction period “ d under the short photoperiod the diapause maintenance period “ d under the short photoperiod the diapause termination period “ d under the short photoperiod and the reproduction period “ d under the long photoperiod each sample which included three to four females was independently replicated three times as three biological replicates adult tissues reproduction seven different tissues were collected from reproductive adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of reproductive adults under long photoperiod were collected on the 10th day after emergence each tissue required about “ a0mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues diapause seven different tissues were collected from diapause adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of diapause adults under short photoperiod were collected on the 20th day after emergence each tissue required about “ a0 mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues samples from reproductive and diapause adult tissues all samples samples from group and all treatments were immediately frozen with liquid nitrogen and stored in an ultralow temperature refrigerator at ˆ’°c prior to rna extractiontotal rna extraction and cdna synthesisin this study total rna was extracted using minibest universal rna extraction kit takara japan and dnase i a0 was used for digestion of the membrane rna integrity was estimated by agarose gel electrophoresis rna concentration and purity were measured with a nanodrop one spectrophotometer thermo scientific then 1μg rna was reversetranscribed into the firststrand complementary dna cdna according to the hiscript ii q rt supermix for qpcr gdna wipers vazyme nanjing china instructions and stored at ˆ’°c all cdna was diluted 10fold with dnasernasefree sterile water before usecandidate reference gene selection and primer a0designaccording to several commonly used reference genes candidate reference gene sequences were obtained by screening from the existing transcriptome of c a0 nipponensis in the laboratory namely tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub in order to ensure the predictive accuracy of the selected sequences we conducted blast alignment all primers were designed using primer premier based on the following criteria gc content “ annealing temperature “°c and primers length “ a0 bp and the specificity of each pair of amplicons was determined by qrtpcr followed by agarose gel electrophoresis and melting curve analysis the amplification efficiency of the pcr was calculated by using the formula e a0 a0 ˆ’slopeˆ’ the slope was obtained by the standard curve which was generated by qrtpcr of a series of continuously diluted cdna samples 0c of insect science vol no realtime qrtpcr analysisthe 20µl total reaction volume were configured according to the protocol of chamq sybr qpcr master mix vazyme contained 10µl a0× chamq sybr qpcr master mix a0µl a0μm of each gene specific primer a0µl of cdna and a0µl of ddh2o the amplification reaction program was set as follows predenaturation at °c for a0s followed by cycles of denaturation at °c for a0s annealing at °c for a0 s the melting curves were analyzed in the “°c temperature range after amplification step the reaction was performed on a roche lightcycler96 instrument to obtain ct values amplification curves melting curves and standard curves all samples were carried out in four technical and three biological replicates and the negative control no template was performed in paralleldata analysisct values for all samples were exported into an excel spreadsheet and were used to analyze the stability of candidate reference gene expression by genorm normfinder bestkeeper and ˆ†ct method the comprehensive ranking were performed following methods adapted from xiao et a0 al the optimal number of genes was determined by the pairwise variation vnn1 between the normalization factors calculated by genorm among the four algorithms genorm and normfinder need to convert the original ct value according to the corresponding requirements before analysis in genorm the stability ranking of genes was determined by the expression stability value m value in bestkeeper the stability ranking of genes was determined by the coefficient of variation cv and sd in normfinder the stability ranking of genes was determined by the gene expression stability value sv in ˆ†ct method the stability of genes was ranked according to the sd of genes ˆ†ct values in four statistical algorithms genes with the lowest value were the most stably expressedvalidation of reference a0genesin most insects vitellogenin vg and vitellogenin receptor vgr play important roles in the reproductive process of female insects vg is taken up by developing oocytes through receptormediated endocytosis rme thereby promoting the development of oocytes and the formation of eggs in this process vgr is the main receptor mediating endocytosis previous studies have shown that reproductive diapause arrests development of oogenesis and vitellogenesis tatar and yin and the expression levels of the vg and the vgr in nondiapause female were significantly higher than those in reproductive diapause female jiang et a0al in order to evaluate the effectiveness of the selected reference genes the expression levels of the target genes vg and vgr were respectively detected by qrtpcr in the different development degree and tissues of adults and the most unstable reference gene was used for comparison in parallel the reaction system and program were the same as for qrtpcr of reference genes and four technical and three biological replicates were performed for each treatment the relative expression levels of vg and vgr were respectively calculated in excel using the ˆ†ˆ†ct method the differences of target genes™ expression levels were analyzed by tukey™s test using spss software spss inc under different experimental conditionsresultsselection and primer performance of candidate reference a0genesthe candidate reference genes including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub were selected to identify the normalization factors for qrtpcr analysis and the sequence information has been submitted to genbank and the accession numbers are shown in supp table online only to determine the amplification specificity of the primers agarose gel electrophoresis and melting curve analysis were performed all primer pairs showed a single band and a single peak fig a0 to obtain correlation coefficient r2 and amplification efficiency e of pcr a standard curve was generated with the 10fold dilution series of cdna the results showed that the amplification efficiency ranged from to and the correlation coefficient varied from to supp table [online a0only]expression profiling of candidate reference a0genesto evaluate expression levels of the candidate reference genes the cycle threshold ct values under different groups were obtained by qrtpcr and represented by boxplot fig a0 the results indicated that ct values of all candidate reference genes were different under different conditions and also varied under the same condition overall ct values ranged from to among them the genes with higher expression abundance were tub1 “ rps26e “ and actin “ followed by 128up “ arp3 “ arpc5 “ rps5 “ and ef1a “ the genes with lower expression abundance were αtub “ and gapdh “ according to the ct value range of each candidate reference gene the genes with relatively stable expression were tub1 and arp3 whereas the most unstable genes were ef1a actin and αtubexpression stability of candidate reference genes under different conditionsin this study four statistical algorithms were used to analyze the expression stability of the candidate reference genes under different conditions including genorm normfinder bestkeeper and ˆ†ct method as different statistical algorithms would generate different ranking patterns the comprehensive ranking of genes was finally determined through the geometric mean of sequencing bestkeeper the original ct values were used for analysis evaluated the stabilities of the candidate reference genes according to the cv and sd of the ct values ˆ†ct method the difference values of original ct values were used for analysis performed stability ordering according to the mean sd of ˆ†ct value genorm and normfinder the original ct values were converted for analysis sequenced the candidate reference genes according to the stability values the lower the stability value the more stable the gene expressiondevelopment degree of a0adultsthe expression stability of the candidate reference genes at different periods of reproductive and diapause female showed that the top four ranked genes identified by the genorm bestkeeper normfinder and δct method were similar but the rank order was slightly different arp3 and rps5 were the first and second stably expressed genes in the four statistical algorithms supp table [online only] and comprehensive ranking analysis fig a0 as for the third and fourth ranked gene tub1 and actin identified by comprehensive ranking analysis were the same as those generated by genorm and normfinder while bestkeeper selected arpc5 and tub1 ˆ†ct method selected actin and rps26e supp table [online only] αtub was ranked by genorm bestkeeper normfinder and δct method as the least stable gene among the candidate reference 0c of insect science vol no fig specificity a and product length b of qrtpcr amplification for ten candidate reference genesgenes during different development degrees of adults supp table [online a0only]adult a0tissuesthe expression stability ranking of candidate reference genes in different tissues of reproductive and diapause females was varied according to the four statistical algorithms in different tissues of reproductive females the top four genes were rps5 arp3 arpc5 and tub1 respectively fig a0 but the rank order of the four genes was significantly different among different statistical algorithms rps5 was ranked first by genorm and ˆ†ct method and was ranked third and fourth by normfinder and bestkeeper respectively arp3 was ranked first by bestkeeper and was ranked second third and fifth by normfinder δct method and genorm respectively arpc5 was ranked first and second by genorm and δct method and was ranked fourth and fifth by normfinder and bestkeeper respectively tub1 was ranked first and second by normfinder and bestkeeper and was ranked fifth and sixth by δct method and genorm respectively supp table [online only] however the four statistical algorithms found that ef1a was ranked as the least stable gene in the tissues from reproductive females supp table [online only]in different tissues of diapause females the top four genes were different from those of different tissues of reproductive females tub1 was ranked first by the comprehensive ranking followed by rps26e arp3 and 128up fig a0 through analysis it was found that ˆ†ct method and normfinder displayed the same rankings for expression stability of candidate reference genes under this condition supp table [online only] tub1 was identified as the most stably expressed gene by genorm ˆ†ct method and normfinder although it was ranked fifth by bestkeeper rps26e ranked steadily among the four statistical algorithms was ranked second by ˆ†ct method and normfinder whereas it was ranked first and third by genorm and bestkeeper respectively arp3 was ranked first by bestkeeper with the smallest coefficient of variation whereas it was ranked third by ˆ†ct method and normfinder in addition to being ranked third by genorm 128up was ranked fourth by bestkeeper ˆ†ct method and normfinder supp table [online only] however in tissue from diapause females actin was consistently identified as the gene with the most unstable expression by the four statistical algorithms supp table [online only]in the reproductive and diapause female tissues the expression stability of the candidate reference genes was different in order to accurately determine the expression of the target genes the expression stability of candidate reference genes under the two conditions was analyzed according to the comprehensive ranking 0c of insect science vol no fig expression profiles of candidate reference genes in c a0nipponensis expression data are displayed as ct values for each reference gene using a box and whisker plot in different experimental conditions the line across the box is the median the box indicates the 25th and 75th percentiles the whiskers represent the 10th and 90th percentilesfig expression stability and comprehensive ranking of reference gene measured by the geomean method a a0lower geomean value indicates more stable expressiontub1 and ef1a were the most stable and unstable genes respectively whereas arp3 128up and arpc5 were ranked second third and fourth respectively fig a0 tub1 was the best candidate reference gene identified by normfinder and ˆ†ct method and was ranked third and sixth by bestkeeper and genorm respectively arp3 was the most suitable candidate reference gene selected by bestkeeper and was ranked second by normfinder and ˆ†ct method and fifth by genorm 128up and arpc5 were the most stable candidate reference genes identified by genorm whereas they were ranked separately fifth and sixth by normfinder and ˆ†ct method and seventh and fifth by bestkeeper respectively supp table [online only] ef1a was identified as the least stable gene by genorm normfinder and δct method although bestkeeper selected actin as the least stable gene supp table [online only]all a0samplesin order to determine the best reference gene suitable for the different conditions of adults the stability of the ten candidate reference genes was ranked for all samples arp3 was identified as the most stable gene by the comprehensive ranking followed by tub1 arpc5 and rps5 whereas ef1a was identified as the least stable gene fig a0 0c of insect science vol no but the most and least stable genes identified by different statistical algorithms were slightly different arp3 was selected as the most stable reference gene by bestkeeper and ˆ†ct method although tub1 and arpc5 were selected as the most stable reference gene by normfinder and genorm respectively ef1a was selected as the least stable reference gene by genorm and ˆ†ct method despite it being ranked ninth by bestkeeper and normfinder supp table [online only]the best combination of candidate reference genes under different conditionsaccording to the pairwise variation vnn1 between the normalization factors and cutoff value calculated by genorm the number of reference genes required for optimum normalization in each experimental condition was determined the cutoff value of vnn1 a0 suggested that n reference genes were enough to make gene expression normalization otherwise n reference genes were needed the analysis results showed that all v23 were indicated that the optimal number of reference genes under each condition was two fig a0 more specifically arp3 and rps5 were the most stable gene combinations under adult developmental stage and reproductive adult tissues conditions tub1 and rps26e were the most stable gene combinations under adult diapause tissues conditions and arp3 and tub1 were the most stable gene combinations under adult tissues and all samples groups table a0relative expression levels of target genes vg and vgr genbank mt308983 mt522179 in the whole adult and from tissues of reproductive and diapause adults respectively when the most stable reference genes arp3 andor rps5 were used as normalization factors at different periods of reproduction and diapause the expression patterns of the target genes vg and vgr were consistent with low expression in the diapause period and rich expression in the late diapause and reproduction period however when the most unstable reference gene αtub was used as a normalization factor neither the target gene vg nor vgr showed a consistent expression pattern fig a0 under different tissue conditions when the most stable reference genes tub1 andor arp3 were used as the normalization factors the expression level of the target gene vg in the fat body of the reproductive female was significantly higher than that in the ovary of the reproductive female the expression level of the target gene vgr in the fat body of the reproductive female was lower than the ovary of the diapause female while when the most unstable reference gene ef1a was used as the normalization factor the expression pattern differed with normalization by tub1 and tub1arp3 fig a0 in general when the most stable reference genes were used as the normalization factors the accurate expression pattern of the target gene could be obtainedvalidation of reference a0genesthe stability of reference gene is very important for the analysis of expression level of target gene vg and vgr which are important for insect reproduction were selected to verify the applicability of the selected reference gene we examined the discussionqrtpcr has become an important means to explore gene expression level due to its high sensitivity rapidity specificity and accuracy and was widely used in physiology studies that investigated insect diapause such as drosophila melanogaster williams fig pairwise variations vnn1 was calculated by genorm to determine the optimal number of reference genes for accurate normalization in different conditions the cut off values under indicate that no additional genes are required for the normalization 0c of insect science vol no et a0al culex pipiens sim and denlinger leptinotarsa decemlineata lehmann et a0 al chrysopa septempunctata liu et a0al and pieris melete wu et a0al however the selection of appropriate reference genes was the key to accurately analyze the gene expression level for example under conditions of injury heatstressed and experimentally varied diets table recommendation for the best combination of reference genes based on the genorm and comprehensive rankings under various experimental conditionsgroupreference genemostdevelopment degreeadult tissues reproductionadult tissues diapauseadult tissuesall samplesarp3rps5tub1tub1 arp3 rps5arp3rps26earp3tub1leastαtubef1aactinef1aef1athe best reference gene was different in drosophila melanogaster ponton et a0al under biotic factors and abiotic stress inappropriate selection of the reference genes in locusta migratoria resulted in significant differences in the expression level of the target gene chitin synthase chs1 yang et a0al diapause of most insects was mainly affected by photoperiod and temperature in past studies the screening of reference genes of drosophila melanogaster ponton et a0al leptinotarsa decemlineata shi et a0al helicoverpa armigera zhang et a0al bombyx mori guo et a0al and harmonia axyridis qu et a0al under main environmental factors was completed by genorm normfinder bestkeeper and ˆ†ct method in this study the expression profiles of candidate reference genes of c a0nipponensis were analyzed under different conditions by the same four statistical algorithms genorm vandesompele et a0 al normfinder andersen et a0al bestkeeper pfaffl and ˆ†ct method silver et a0al different algorithms produced different stability rankings in order to obtain statistically consistent and accurate results we finally ranked the gene based on their stabilities determined by fig validation of selected reference genes under different periods a and tissues b of reproductive and diapause female in c a0 nipponensis relative expression levels of the vg and vgr in different samples using different normalization factors the most and least stable genes asterisks indicate significant differences in the expression levels of the vg and vgr r reproduction period d1 the diapause induction period d2 the diapause maintenance period d3 the diapause termination period 0c of insect science vol no comprehensive analysis method xiao et a0 al and selected the most stable reference genes under each condition as far as we know actin which played an important role in cell contraction and cytoskeletal maintenance was found in virtually all eukaryotic cells and was considered as an ideal reference gene for many anisms sürencastillo et a0al shakeel et a0al for example actin was used as a reference gene for normalization in the determination of genes related to reproductive and nutritional signaling such as vitellogenin of chrysopa septempunctata liu et a0al however in this study three genes related to actin were selected for analysis among which actin was similar to actin of c a0septempunctata which was also a member of the neuroptera in our study actin was the most unstable gene in the diapause female tissues but the actinrelated protein arp3 which was structurally homologous with actin showed better stability arp3 was selected as the most stable reference gene in adults of different developmental levels and all samples while it was the second most stable gene in the reproductive adult tissues and all adult tissues although arp3 was ranked as the third most stable gene in the diapause adult tissues it showed relatively stable expression in the expression profile tubulin which played an essential role in maintaining cell shape movement and intracellular material transport was also often used as a reference gene but different types of tubulin have different stability for example in the study of helicoverpa armigera the expression of βtub was relatively stable compared with that of αtub under almost all conditions zhang et a0 al similarly in this study αtub showed unstable expression and was the least stably expressed gene in adults of different developmental stages whereas tub1 was considered to be the most stably expressed reference gene in diapausing adult tissues and all adult tissues and was the second most stable gene in all samples ribosomal protein rp widely distributed in various tissues played an important role in protein biosynthesis and was widely used as a reference gene in many insects lu et a0al koyama et a0 al sun et a0 al in this study rps5 was considered to be stable in the tissues of reproductive females and ranked second among different developmental stages of adults elongation factors ef was a protein factor which promoted polypeptide chain to extension during the translation of mrna and was recommended as the ideal reference gene under different conditions of a variety of insects chapuis et a0 al ponton et a0 al however some studies showed that ef1α was one of the most unstable genes under certain conditions fu et a0 al in our study ef1a was found to be the most unstable gene in the reproductive adult tissues all tissues and all samples and the second least stable gene in the adults of different developmental stages and diapause adult tissues therefore it was not suitable for the study of c a0nipponensisrecently an increasing number of studies have demonstrated the importance of using multiple stably expressed reference genes for the accuracy of qrtpcr analysis ling and salvaterra yuan et a0 al kang et a0 al however this does not mean that the more reference genes increase the reliability of the results the study has indicated that either too few o

cancer7505

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: chrysoperla nipponensis okamoto which has the unique diapause phenotype distinguishable from nondiapause adult is an ideal model anism for studying the mechanism of reproductive diapause however there is no reliable and effective reference genes used for the reproductive diapause study of c a0nipponensis therefore in this study we evaluated the expression stability of candidate reference genes tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin αtub in adults under diapause and nondiapause induction conditions using four statistical algorithms including genorm normfinder bestkeeper and ˆ†ct method results showed that arp3 and tub1 were the most stable reference genes in all samples and in the adult tissues group arp3 and rps5 were the most stable reference genes in the development degree group αtub and ef1a were unstable reference genes under the conditions of this study meanwhile to verify the reliability of the reference genes we evaluated the relative expression levels of vg and vgr in different treatments significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was respectively observed when using arp3 as the reference gene but not when using an unstable reference gene the reference genes identified in this work provided not only the basis for future functional genomics research in diapause of c a0nipponensis and will also identify reliable normalization factors for realtime quantitative realtime polymerase chain reaction data for other related insectskey words chrysoperla nipponensis okamoto reference genes qrtpcr reproduction diapausedue to the advantages of high sensitivity rapidity specificity and accuracy bustin et a0al valasek et a0al vanguilder et a0al shakeel et a0al quantitative realtime polymerase chain reaction qrtpcr has been widely used in the study of animals plants and microanisms roy et a0al jia et a0al zhang et a0 al ding et a0 al sun et a0 al qrtpcr is the most commonly used method for the expression analysis of target genes however the reliability of qrtpcr results in different samples is determined by a variety of factors among which the use of stably expressed reference genes is an important link for accurate detection of gene expression changes by qrtpcr bustin et a0 al at present several commonly used reference genes for data normalization include tubulin actin ribosomal protein elongation factor 1α glyceraldehyde3phosphate dehydrogenase 18s ribosomal rna and other genes bustin vanguilder et a0al however more and more studies have found that these reference genes do not show consistent expression patterns under different experimental conditions and even affect the reliability of experimental results therefore in order to obtain stable and reliable normalization factors reference genes with stable expression are usually used for correction and standardization to reduce errors between samples selection and evaluation of reference genes have become a necessary step before quantifying the expression of target genes accuratelynowadays there have been many studies on the selection of reference genes for insects such as sesamia inferens helicoverpa armigera aphis gossypii myzus persicae etc lu et a0 al shakeel et a0al zhang et a0al ma et a0al kang et a0al in these studies four statistical algorithms including genorm normfinder bestkeeper and ˆ†ct method were mainly used to analyze the ct values obtained by qrtpcr of reference genes under various experimental conditions shakeel et a0 al finally the stability of the candidate reference genes was determined according to the geometric mean value of each gene ranked using different the authors published by oxford university press on behalf of entomological society of americathis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly cited for commercial reuse please contact spermissionsoupcom 0c of insect science vol no algorithms and the most suitable reference gene was selected for the target gene expression analysis xiao et a0al kang et a0al chrysoperla nipponensis okamoto as one of the important predatory natural enemies of agricultural and forestry pests prefers to eat aphids thrips and other pests okamoto nie et a0al because of its characteristically wide geographical distribution and broad range of host prey niijima syed et a0al it has good prospects for widespread application in biological control mcewen et a0 al memon et a0 al reproductive diapause is an important way for c a0nipponensis adults to escape from adverse environments xu et a0al at present there have been many reports on the diapause of c a0nipponensis xu et a0al found that the body color of c a0nipponensis was green during the reproductive period but turned brown and yellow during the diapause period chrysoperla nipponensis belongs to the photoperiodic sensitive insect the adult diapause was induced by short photoperiods xu et a0al chen et a0al found that different photoperiods affected the material content eg protein and glycogen of c a0nipponensis diapause induced by the short photoperiod was beneficial to the storage of c a0nipponensis chen et a0al we expect an exponential increase of diapause research on c a0nipponensis at the molecular level in the near future thus stable and reliable reference genes are important for accurately quantifying gene expression of c a0nipponensisribosomal proteins and ribosomal rna have been used as reference genes in previous diapause studies for example williams et a0al used ribosomal protein rp49 as a reference gene to study the natural variation of drosophila melanogaster diapause williams et a0 al and sim and denlinger used ribosomal protein large subunit rpl19 as a reference gene in a study of ovarian development of culex pipiens during overwintering diapause sim and denlinger this indicates that under the same experimental conditions the selected reference genes in different species research are also differentin this research candidate reference genes were selected including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub whose expression profiles were measured by the qrtpcr the stability was analyzed by four statistical algorithms genorm normfinder bestkeeper and ˆ†ct method in different developmental stages reproductive and diapause of adults and among different tissues the optimal reference genes under different conditions were determined which contributed to the accurate expression of target genes for future researchmaterials and a0methodsinsecta stable population of c a0 nipponensis was maintained in an artificial climate chamber rsz intelligent artificial climate chamber changzhou guohua jiangsu province under the following conditions a0± °c temperature a0± relative humidity rh and long photoperiod of l d h in our laboratory the eggs were collected by cutting the stalk and incubated in fingertip tubes a0cm in diameter and a0cm in height the primary hatching larvae were fed megoura japonica matsumura whose host plant was vicia faba l a0the adults were paired immediately after emergence in a bottle a0cm in diameter and a0cm in height fed a dry brewer™s yeast feed mixed with sucrose in a ratio of and then minced in a mortar and sifted through mesh and honey water the diapause adults used in this study were kept under the conditions of short photoperiod of ld h in all processes from eggs larvae pupae to adults whereas the nondiapause adults were kept under the conditions of long photoperiod of ld a0hsample collection development degree samples from individuals from varying developmental stages included female adults in the diapause induction period “ d under the short photoperiod the diapause maintenance period “ d under the short photoperiod the diapause termination period “ d under the short photoperiod and the reproduction period “ d under the long photoperiod each sample which included three to four females was independently replicated three times as three biological replicates adult tissues reproduction seven different tissues were collected from reproductive adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of reproductive adults under long photoperiod were collected on the 10th day after emergence each tissue required about “ a0mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues diapause seven different tissues were collected from diapause adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of diapause adults under short photoperiod were collected on the 20th day after emergence each tissue required about “ a0 mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues samples from reproductive and diapause adult tissues all samples samples from group and all treatments were immediately frozen with liquid nitrogen and stored in an ultralow temperature refrigerator at ˆ’°c prior to rna extractiontotal rna extraction and cdna synthesisin this study total rna was extracted using minibest universal rna extraction kit takara japan and dnase i a0 was used for digestion of the membrane rna integrity was estimated by agarose gel electrophoresis rna concentration and purity were measured with a nanodrop one spectrophotometer thermo scientific then 1μg rna was reversetranscribed into the firststrand complementary dna cdna according to the hiscript ii q rt supermix for qpcr gdna wipers vazyme nanjing china instructions and stored at ˆ’°c all cdna was diluted 10fold with dnasernasefree sterile water before usecandidate reference gene selection and primer a0designaccording to several commonly used reference genes candidate reference gene sequences were obtained by screening from the existing transcriptome of c a0 nipponensis in the laboratory namely tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub in order to ensure the predictive accuracy of the selected sequences we conducted blast alignment all primers were designed using primer premier based on the following criteria gc content “ annealing temperature “°c and primers length “ a0 bp and the specificity of each pair of amplicons was determined by qrtpcr followed by agarose gel electrophoresis and melting curve analysis the amplification efficiency of the pcr was calculated by using the formula e a0 a0 ˆ’slopeˆ’ the slope was obtained by the standard curve which was generated by qrtpcr of a series of continuously diluted cdna samples 0c of insect science vol no realtime qrtpcr analysisthe 20µl total reaction volume were configured according to the protocol of chamq sybr qpcr master mix vazyme contained 10µl a0× chamq sybr qpcr master mix a0µl a0μm of each gene specific primer a0µl of cdna and a0µl of ddh2o the amplification reaction program was set as follows predenaturation at °c for a0s followed by cycles of denaturation at °c for a0s annealing at °c for a0 s the melting curves were analyzed in the “°c temperature range after amplification step the reaction was performed on a roche lightcycler96 instrument to obtain ct values amplification curves melting curves and standard curves all samples were carried out in four technical and three biological replicates and the negative control no template was performed in paralleldata analysisct values for all samples were exported into an excel spreadsheet and were used to analyze the stability of candidate reference gene expression by genorm normfinder bestkeeper and ˆ†ct method the comprehensive ranking were performed following methods adapted from xiao et a0 al the optimal number of genes was determined by the pairwise variation vnn1 between the normalization factors calculated by genorm among the four algorithms genorm and normfinder need to convert the original ct value according to the corresponding requirements before analysis in genorm the stability ranking of genes was determined by the expression stability value m value in bestkeeper the stability ranking of genes was determined by the coefficient of variation cv and sd in normfinder the stability ranking of genes was determined by the gene expression stability value sv in ˆ†ct method the stability of genes was ranked according to the sd of genes ˆ†ct values in four statistical algorithms genes with the lowest value were the most stably expressedvalidation of reference a0genesin most insects vitellogenin vg and vitellogenin receptor vgr play important roles in the reproductive process of female insects vg is taken up by developing oocytes through receptormediated endocytosis rme thereby promoting the development of oocytes and the formation of eggs in this process vgr is the main receptor mediating endocytosis previous studies have shown that reproductive diapause arrests development of oogenesis and vitellogenesis tatar and yin and the expression levels of the vg and the vgr in nondiapause female were significantly higher than those in reproductive diapause female jiang et a0al in order to evaluate the effectiveness of the selected reference genes the expression levels of the target genes vg and vgr were respectively detected by qrtpcr in the different development degree and tissues of adults and the most unstable reference gene was used for comparison in parallel the reaction system and program were the same as for qrtpcr of reference genes and four technical and three biological replicates were performed for each treatment the relative expression levels of vg and vgr were respectively calculated in excel using the ˆ†ˆ†ct method the differences of target genes™ expression levels were analyzed by tukey™s test using spss software spss inc under different experimental conditionsresultsselection and primer performance of candidate reference a0genesthe candidate reference genes including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub were selected to identify the normalization factors for qrtpcr analysis and the sequence information has been submitted to genbank and the accession numbers are shown in supp table online only to determine the amplification specificity of the primers agarose gel electrophoresis and melting curve analysis were performed all primer pairs showed a single band and a single peak fig a0 to obtain correlation coefficient r2 and amplification efficiency e of pcr a standard curve was generated with the 10fold dilution series of cdna the results showed that the amplification efficiency ranged from to and the correlation coefficient varied from to supp table [online a0only]expression profiling of candidate reference a0genesto evaluate expression levels of the candidate reference genes the cycle threshold ct values under different groups were obtained by qrtpcr and represented by boxplot fig a0 the results indicated that ct values of all candidate reference genes were different under different conditions and also varied under the same condition overall ct values ranged from to among them the genes with higher expression abundance were tub1 “ rps26e “ and actin “ followed by 128up “ arp3 “ arpc5 “ rps5 “ and ef1a “ the genes with lower expression abundance were αtub “ and gapdh “ according to the ct value range of each candidate reference gene the genes with relatively stable expression were tub1 and arp3 whereas the most unstable genes were ef1a actin and αtubexpression stability of candidate reference genes under different conditionsin this study four statistical algorithms were used to analyze the expression stability of the candidate reference genes under different conditions including genorm normfinder bestkeeper and ˆ†ct method as different statistical algorithms would generate different ranking patterns the comprehensive ranking of genes was finally determined through the geometric mean of sequencing bestkeeper the original ct values were used for analysis evaluated the stabilities of the candidate reference genes according to the cv and sd of the ct values ˆ†ct method the difference values of original ct values were used for analysis performed stability ordering according to the mean sd of ˆ†ct value genorm and normfinder the original ct values were converted for analysis sequenced the candidate reference genes according to the stability values the lower the stability value the more stable the gene expressiondevelopment degree of a0adultsthe expression stability of the candidate reference genes at different periods of reproductive and diapause female showed that the top four ranked genes identified by the genorm bestkeeper normfinder and δct method were similar but the rank order was slightly different arp3 and rps5 were the first and second stably expressed genes in the four statistical algorithms supp table [online only] and comprehensive ranking analysis fig a0 as for the third and fourth ranked gene tub1 and actin identified by comprehensive ranking analysis were the same as those generated by genorm and normfinder while bestkeeper selected arpc5 and tub1 ˆ†ct method selected actin and rps26e supp table [online only] αtub was ranked by genorm bestkeeper normfinder and δct method as the least stable gene among the candidate reference 0c of insect science vol no fig specificity a and product length b of qrtpcr amplification for ten candidate reference genesgenes during different development degrees of adults supp table [online a0only]adult a0tissuesthe expression stability ranking of candidate reference genes in different tissues of reproductive and diapause females was varied according to the four statistical algorithms in different tissues of reproductive females the top four genes were rps5 arp3 arpc5 and tub1 respectively fig a0 but the rank order of the four genes was significantly different among different statistical algorithms rps5 was ranked first by genorm and ˆ†ct method and was ranked third and fourth by normfinder and bestkeeper respectively arp3 was ranked first by bestkeeper and was ranked second third and fifth by normfinder δct method and genorm respectively arpc5 was ranked first and second by genorm and δct method and was ranked fourth and fifth by normfinder and bestkeeper respectively tub1 was ranked first and second by normfinder and bestkeeper and was ranked fifth and sixth by δct method and genorm respectively supp table [online only] however the four statistical algorithms found that ef1a was ranked as the least stable gene in the tissues from reproductive females supp table [online only]in different tissues of diapause females the top four genes were different from those of different tissues of reproductive females tub1 was ranked first by the comprehensive ranking followed by rps26e arp3 and 128up fig a0 through analysis it was found that ˆ†ct method and normfinder displayed the same rankings for expression stability of candidate reference genes under this condition supp table [online only] tub1 was identified as the most stably expressed gene by genorm ˆ†ct method and normfinder although it was ranked fifth by bestkeeper rps26e ranked steadily among the four statistical algorithms was ranked second by ˆ†ct method and normfinder whereas it was ranked first and third by genorm and bestkeeper respectively arp3 was ranked first by bestkeeper with the smallest coefficient of variation whereas it was ranked third by ˆ†ct method and normfinder in addition to being ranked third by genorm 128up was ranked fourth by bestkeeper ˆ†ct method and normfinder supp table [online only] however in tissue from diapause females actin was consistently identified as the gene with the most unstable expression by the four statistical algorithms supp table [online only]in the reproductive and diapause female tissues the expression stability of the candidate reference genes was different in order to accurately determine the expression of the target genes the expression stability of candidate reference genes under the two conditions was analyzed according to the comprehensive ranking 0c of insect science vol no fig expression profiles of candidate reference genes in c a0nipponensis expression data are displayed as ct values for each reference gene using a box and whisker plot in different experimental conditions the line across the box is the median the box indicates the 25th and 75th percentiles the whiskers represent the 10th and 90th percentilesfig expression stability and comprehensive ranking of reference gene measured by the geomean method a a0lower geomean value indicates more stable expressiontub1 and ef1a were the most stable and unstable genes respectively whereas arp3 128up and arpc5 were ranked second third and fourth respectively fig a0 tub1 was the best candidate reference gene identified by normfinder and ˆ†ct method and was ranked third and sixth by bestkeeper and genorm respectively arp3 was the most suitable candidate reference gene selected by bestkeeper and was ranked second by normfinder and ˆ†ct method and fifth by genorm 128up and arpc5 were the most stable candidate reference genes identified by genorm whereas they were ranked separately fifth and sixth by normfinder and ˆ†ct method and seventh and fifth by bestkeeper respectively supp table [online only] ef1a was identified as the least stable gene by genorm normfinder and δct method although bestkeeper selected actin as the least stable gene supp table [online only]all a0samplesin order to determine the best reference gene suitable for the different conditions of adults the stability of the ten candidate reference genes was ranked for all samples arp3 was identified as the most stable gene by the comprehensive ranking followed by tub1 arpc5 and rps5 whereas ef1a was identified as the least stable gene fig a0 0c of insect science vol no but the most and least stable genes identified by different statistical algorithms were slightly different arp3 was selected as the most stable reference gene by bestkeeper and ˆ†ct method although tub1 and arpc5 were selected as the most stable reference gene by normfinder and genorm respectively ef1a was selected as the least stable reference gene by genorm and ˆ†ct method despite it being ranked ninth by bestkeeper and normfinder supp table [online only]the best combination of candidate reference genes under different conditionsaccording to the pairwise variation vnn1 between the normalization factors and cutoff value calculated by genorm the number of reference genes required for optimum normalization in each experimental condition was determined the cutoff value of vnn1 a0 suggested that n reference genes were enough to make gene expression normalization otherwise n reference genes were needed the analysis results showed that all v23 were indicated that the optimal number of reference genes under each condition was two fig a0 more specifically arp3 and rps5 were the most stable gene combinations under adult developmental stage and reproductive adult tissues conditions tub1 and rps26e were the most stable gene combinations under adult diapause tissues conditions and arp3 and tub1 were the most stable gene combinations under adult tissues and all samples groups table a0relative expression levels of target genes vg and vgr genbank mt308983 mt522179 in the whole adult and from tissues of reproductive and diapause adults respectively when the most stable reference genes arp3 andor rps5 were used as normalization factors at different periods of reproduction and diapause the expression patterns of the target genes vg and vgr were consistent with low expression in the diapause period and rich expression in the late diapause and reproduction period however when the most unstable reference gene αtub was used as a normalization factor neither the target gene vg nor vgr showed a consistent expression pattern fig a0 under different tissue conditions when the most stable reference genes tub1 andor arp3 were used as the normalization factors the expression level of the target gene vg in the fat body of the reproductive female was significantly higher than that in the ovary of the reproductive female the expression level of the target gene vgr in the fat body of the reproductive female was lower than the ovary of the diapause female while when the most unstable reference gene ef1a was used as the normalization factor the expression pattern differed with normalization by tub1 and tub1arp3 fig a0 in general when the most stable reference genes were used as the normalization factors the accurate expression pattern of the target gene could be obtainedvalidation of reference a0genesthe stability of reference gene is very important for the analysis of expression level of target gene vg and vgr which are important for insect reproduction were selected to verify the applicability of the selected reference gene we examined the discussionqrtpcr has become an important means to explore gene expression level due to its high sensitivity rapidity specificity and accuracy and was widely used in physiology studies that investigated insect diapause such as drosophila melanogaster williams fig pairwise variations vnn1 was calculated by genorm to determine the optimal number of reference genes for accurate normalization in different conditions the cut off values under indicate that no additional genes are required for the normalization 0c of insect science vol no et a0al culex pipiens sim and denlinger leptinotarsa decemlineata lehmann et a0 al chrysopa septempunctata liu et a0al and pieris melete wu et a0al however the selection of appropriate reference genes was the key to accurately analyze the gene expression level for example under conditions of injury heatstressed and experimentally varied diets table recommendation for the best combination of reference genes based on the genorm and comprehensive rankings under various experimental conditionsgroupreference genemostdevelopment degreeadult tissues reproductionadult tissues diapauseadult tissuesall samplesarp3rps5tub1tub1 arp3 rps5arp3rps26earp3tub1leastαtubef1aactinef1aef1athe best reference gene was different in drosophila melanogaster ponton et a0al under biotic factors and abiotic stress inappropriate selection of the reference genes in locusta migratoria resulted in significant differences in the expression level of the target gene chitin synthase chs1 yang et a0al diapause of most insects was mainly affected by photoperiod and temperature in past studies the screening of reference genes of drosophila melanogaster ponton et a0al leptinotarsa decemlineata shi et a0al helicoverpa armigera zhang et a0al bombyx mori guo et a0al and harmonia axyridis qu et a0al under main environmental factors was completed by genorm normfinder bestkeeper and ˆ†ct method in this study the expression profiles of candidate reference genes of c a0nipponensis were analyzed under different conditions by the same four statistical algorithms genorm vandesompele et a0 al normfinder andersen et a0al bestkeeper pfaffl and ˆ†ct method silver et a0al different algorithms produced different stability rankings in order to obtain statistically consistent and accurate results we finally ranked the gene based on their stabilities determined by fig validation of selected reference genes under different periods a and tissues b of reproductive and diapause female in c a0 nipponensis relative expression levels of the vg and vgr in different samples using different normalization factors the most and least stable genes asterisks indicate significant differences in the expression levels of the vg and vgr r reproduction period d1 the diapause induction period d2 the diapause maintenance period d3 the diapause termination period 0c of insect science vol no comprehensive analysis method xiao et a0 al and selected the most stable reference genes under each condition as far as we know actin which played an important role in cell contraction and cytoskeletal maintenance was found in virtually all eukaryotic cells and was considered as an ideal reference gene for many anisms sürencastillo et a0al shakeel et a0al for example actin was used as a reference gene for normalization in the determination of genes related to reproductive and nutritional signaling such as vitellogenin of chrysopa septempunctata liu et a0al however in this study three genes related to actin were selected for analysis among which actin was similar to actin of c a0septempunctata which was also a member of the neuroptera in our study actin was the most unstable gene in the diapause female tissues but the actinrelated protein arp3 which was structurally homologous with actin showed better stability arp3 was selected as the most stable reference gene in adults of different developmental levels and all samples while it was the second most stable gene in the reproductive adult tissues and all adult tissues although arp3 was ranked as the third most stable gene in the diapause adult tissues it showed relatively stable expression in the expression profile tubulin which played an essential role in maintaining cell shape movement and intracellular material transport was also often used as a reference gene but different types of tubulin have different stability for example in the study of helicoverpa armigera the expression of βtub was relatively stable compared with that of αtub under almost all conditions zhang et a0 al similarly in this study αtub showed unstable expression and was the least stably expressed gene in adults of different developmental stages whereas tub1 was considered to be the most stably expressed reference gene in diapausing adult tissues and all adult tissues and was the second most stable gene in all samples ribosomal protein rp widely distributed in various tissues played an important role in protein biosynthesis and was widely used as a reference gene in many insects lu et a0al koyama et a0 al sun et a0 al in this study rps5 was considered to be stable in the tissues of reproductive females and ranked second among different developmental stages of adults elongation factors ef was a protein factor which promoted polypeptide chain to extension during the translation of mrna and was recommended as the ideal reference gene under different conditions of a variety of insects chapuis et a0 al ponton et a0 al however some studies showed that ef1α was one of the most unstable genes under certain conditions fu et a0 al in our study ef1a was found to be the most unstable gene in the reproductive adult tissues all tissues and all samples and the second least stable gene in the adults of different developmental stages and diapause adult tissues therefore it was not suitable for the study of c a0nipponensisrecently an increasing number of studies have demonstrated the importance of using multiple stably expressed reference genes for the accuracy of qrtpcr analysis ling and salvaterra yuan et a0 al kang et a0 al however this does not mean that the more reference genes increase the reliability of the results the study has indicated that either too few o Answer:

Colon_Cancer

" micrornas mirnas have been reported to have important regulatory roles in the progression of several types of cancer including cervical cancer cc however the biological roles and regulatory mechanisms of mirnas in cc remain to be fully elucidated the aim of the present study was to examine the functions of mirnas in cc and the possible mechanisms using a microarray it was identified that mirna15a5p mir15a5p was one of the most downregulated mirnas in cc tissues compared with adjacent noncancerous tissues the low expression of mir15a5p was observed in cc tumor tissues with distant metastasis and in cc cell lines in addition the effects of mir15a5p upregulation on cell viability apoptosis invasion and migration of cc cells were investigated using cck‘ flow cytometry transwell and wound healing assays respectively it was demonstrated that upregulation of mir‘15a‘5p significantly suppressed the viability migration and invasion and promoted the apoptosis of siha and c33a cells furthermore yesassociated protein yap1 a well‘known oncogene was confirmed to be directly targeted by mir15a5p and was found to be negatively regulated by mir15a5p further correlation analysis indicated that mir15a5p expression was negatively correlated with yap1 expression in cc tissues notably overexpression of yap1 abrogated the tumor suppressive effects of mir15a5p in cc cells taken together these present findings indicated that the mir15a5pyap1 axis may provide a novel strategy for the clinical treatment of cccorrespondence to professor xu chen department of obstetrics and gynaecology huashan hospital north fudan university jingpohu road baoshan shanghai pr chinaemail xuchenccx163comcontributed equallykey words cervical cancer microrna15a5p cell viability migration invasion yesassociated protein introductioncervical cancer cc is a type of malignant tumor commonly presenting in women in cc cases are diagnosed each year and it accounts for of all female cancerassociated mortalities each year worldwide despite advances in the therapeutic strategies for cc including targeted therapies and immunotherapy the prognosis of cc remains poor due to the abnormal growth of epithelial cells thus it is imperative to clarify the molecular interactions occurring during the initiation and progression of ccmicrornas mirnas are a family of short noncoding rnas with an average length of nucleotides which negatively regulate target gene expression through either translation repression or rna degradation accumulating evidence has indicated that mirnas may function as oncogenes or tumor suppressors depending on their target mrna in various types of cancer including cc for example yang reported that mir214 inhibits the growth of cc cells by the regulation of its target enhancer of zeste homolog dong demonstrated a suppressive role of mir217 in the development of cc cells via targeting rhoassociated protein kinase chen reported that mir499a promotes the proliferation cell cycle progression colony formation migration and invasion of cc cells by targeting srybox transcription factor in addition several mirnas serve as diagnostic biomarkers in patients with cc such as mir152 and mir365 despite the aforementioned findings the roles of mirnas in the development of cc require further investigationin the present study a mirna microarray was performed to investigate the expression profiles of mirnas in cc tissues and the most downregulated mirna identified mir‘15a‘5p was selected for further analysis the potential role and underlying mechanism of mir15a5p in cc cells were also investigated the present results suggest that mir15a5p may serve as a therapeutic target for ccmaterials and methodspatients and samples in total paired cervical samples tumor tissues and adjacent noncancerous tissues were 0cchen mir15a inhibits cervical cancer cell growthobtained from female patients with cc who underwent cervical surgical resection without preoperative systemic therapy at the department of obstetrics and gynecology huashan hospital north of fudan university shanghai china between may and december the median age of the patients was years range years among all patients there were patients with metastatic cc and with nonmetastatic cc the matched nontumor adjacent tissue was obtained cm beyond the boundary of cc tissue all tissue samples were immediately snapfrozen in liquid nitrogen and stored at ‘Ëšc until use the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university written informed consent for participation in the study was obtained from all patientsmirna expression profiling total rna from cc tissues three randomly selected paired tumor tissues and adjacent noncancerous tissues was extracted using mirneasy mini kit qiagen gmbh the samples were assessed using the mircury lna„¢ array v180 agilent technologies inc the procedure and imaging processes were performed as described previously cell culture human cc cell lines hela c33a caski and siha 293t cells and normal cervical epithelial cells ect1e6e7 were obtained from the american type culture collection all cells were cultured in dmem sigmaaldrich merck kgaa supplemented with vv fbs sigmaaldrich merck kgaa plus uml penicillinstreptomycin at ˚c with co2reverse transcription‘quantitative pcr rt‘qpcr total rna was extracted from tissues or cell lines using trizol reagent invitrogen thermo fisher scientific inc for mirna rt cdna was generated from ng total rna samples using taqman„¢ microrna reverse transcription kit applied biosystems thermo fisher scientific inc at ˚c for min for mrna rt cdna was synthesized using primescript rt reagent kit takara bio inc at ˚c for min qpcr for mirna and mrna was performed using the sybrgreen i realtime pcr kit applied biosystems thermo fisher scientific inc on an abi system applied biosystems thermo fisher scientific inc the reaction was performed under the following conditions ˚c for min followed by cycles at ˚c for sec and ˚c for sec and a final extension at ˚c for sec the primers for qpcr analysis were as follows mir15a5p forward 'aat gtt gcc cgt aat gcc3' and reverse 'ccc aag cgg aga aag gaa3' u6 forward 'gct tcg gca gca cat ata cta aaa t3' and reverse 'cgc ttc acg aat ttg cgt gtc at3' yesassociated protein yap1 forward 'cgg tcc act tca gtc tcc3' and reverse 'gag tgt ggt gga cag gta ctg3' and gapdh forward 'gtg gtg aag acg cca gtg ga3' and reverse 'cga gcc aca tcg ctc aga ca3' the expression levels of mir15a5p and yap1 were normalized to the expression of u6 and gapdh respectively the relative expression of each gene was calculated using the ‘ˆ†ˆ†cq method cell transfection the mir15a5p mimic mimic negative control nc mir15a5p inhibitor inhibitor nc yap1 overexpression plasmid pcdnayap1 and pcdnavector were all provided by guangzhou ribobio co ltd when c33a and siha cells 5x105 cellswell in 6well plates grew to confluence mir‘15a‘5p mimic nm mimic nc nm mir15a5p inhibitor nm inhibitor nc nm pcdnayap1 µg or pcdnavector µg were transfected into cells at ˚c for h using lipofectamine® invitrogen thermo fisher scientific inc the sequences were as follows mir15a5p mimic 'uag cag cac aua aug guu ugu g3' mimic nc 'uuc ucc gaa cgu guc acg utt3' mir15a5p inhibitor 'cac aaa cca uua ugu gcu gcu a3' and inhibitor nc 'cag uac uuu ugu gua gua caa3'in addition small interfering rna targeting yap1 si‘yap1 and the negative control targeting a non‘specific sequence siscramble were provided by thermo fisher scientific inc siha and c‘33a cells were transfected with the sirnas nmoll using lipofectamine invitrogen thermo fisher scientific inc the sequences of si‘yap1 and siscramble were as follows siyap1 'ctc agg atg gag aaa ttt a3' and siscramble 'ttc tcc gaa cgt gtc acg t3' at h posttransfection the cells were harvested for further analysis and the inhibition efficiency was determined by western blottingcell viability the c33a and siha cells were seeded in 96well plates at a density of 5x103well overnight following transfection the cell viability was measured using a cck8 assay briefly µl cck‘ solution was added to each well and cultured for h at ˚c the absorbance of the samples at nm was detected using a microplate reader biorad laboratories inccaspase‘ activity following transfection c33a and siha cells were harvested and the caspase3 activity was measured using a caspase3 activity assay kit beyotime institute of biotechnology according to the manufacturer's protocolcell apoptosis the apoptosis of c33a and siha cells was examined using flow cytometry following transfection c33a and siha cells were collected and the apoptotic cells were identified using an annexin v‘fitc apoptosis detection kit abcam according to the manufacturer's protocol after washing with cold pbs the cells were resuspended in binding buffer followed by staining with annexin v and propidium iodide for min in the dark at room temperature the fluorescence was measured using a facscan flow cytometer beckman coulter inc and then analyzed by flowjo v871 software flowjo llcimmunofluorescence assay following transfection c33a and siha cells were fixed in absolute ethyl alcohol for min at room temperature after washing twice with pbs the fixed cells were stained with primary antibody targeting cleavedcaspase3 cat no c ell signaling technology inc for h at room temperature subsequently an antirabbit conjugated antibody with fitc cat no f0382 sigmaaldrich merck kgaa was added for h in the 0cinternational journal of molecular medicine dark fluorescence images were obtained using an inverted fluorescence microscope magnification x200cell invasion assays transwell chambers 8µm pore bd biosciences coated with matrigel bd biosciences were used for the invasion assay briefly c‘33a and siha cells 8x104 were seeded in the top chamber with serumfree medium while the lower chamber contained culture medium with fbs following incubation for h the cells were fixed in paraformaldehyde solution beyotime institute of biotechnology for min and stained with crystal violet beyotime institute of biotechnology for min at room temperature images were captured with an inverted microscope olympus corporation magnification x100wound healing assay for the wound healing assay c33a and siha cells were seeded onto 12well plates 2x105 cellswell and h after transfection a scratch was made using a 10µl pipette tip in the confluent cell monolayer then cells were washed twice with pbs and incubated in dmem without fbs the wound healing images were captured at and h after scratching using an inverted light microscope olympus corporation magnification x100 the wound healing rate was calculated using imagej software v146 national institutes of healthdual‘luciferase reporter assay mirna target prediction tools including miranda httpmirandaorguk and targetscan httptargetscanorg were used to search for the putative targets of mir15a5p pgl3yap1 widetype or pgl3yap1 mutant type pgl3yap1mut promega corporation were cotransfected with mir15a5p mimics into 293t cells in 24well plates 2x105well using lipofectamine invitrogen thermo fisher scientific inc at h post‘transfection the luciferase activities were analyzed using the dualluciferase reporter assay system promega corporation with renilla luciferase activity as an internal control western blot analysis western blotting was performed as previously described briefly cells were lysed using radio immunoprecipitation assay buffer beyotime institute of biotechnology and the protein concentration was determined using the bicinchoninic acid assay total protein µglane was separated by sdspage and electrophoretically transferred onto a polyvinylidene difluoride membrane emd millipore subsequently membranes were blocked with skim milk for h at ˚c overnight each membrane was probed with primary antibodies against yap1 cat no and β‘actin cat no at ˚c overnight all primary antibodies were obtained from cell signaling technology inc subsequently the membrane was incubated with horseradish peroxidaseconjugated goat antirabbit igg cat no abcam at room temperature for h βactin served as the loading control and for normalization of protein expression the protein bands were developed using ecl kit ge healthcare and expression levels were quantified using imagej v146 national institutes of healthstatistical analysis all data are presented as mean ± standard deviation the correlation between mir15a5p and yap1 levels was evaluated using spearman's correlation analysis pairwise comparisons were performed by student's ttest and comparisons among groups were analyzed by oneway anova followed by tukey's posthoc test p005 was considered to indicate a statistically significant differenceresultsmir‘15a‘5p is downregulated in cc to examine the potential involvement of mirnas in the development of cc microarray analysis was performed to evaluate the mirna expression profiles between cc tissues and adjacent noncancerous tissues of differently expressed mirnas identified in the tumor group mirnas exhibited decreased expression and mirnas demonstrated increased expression compared with that in adjacent noncancerous tissues fig 1a among the aberrant mirnas the present study focused on mir15a5p for subsequent experiments due to its suppressive role in a variety of other cancer types such as endometrial cancer and chronic myeloid leukemia subsequently rtqpcr was performed to detect the expression of mir15a5p in pairs of tumor tissues and adjacent noncancerous tissues the results revealed that the level of mir15a5p was significantly lower in tumor tissues compared with that in adjacent noncancerous tissues fig 1b it was also observed that mir15a5p was expressed at a significantly lower level in tumor tissues with distant metastasis compared with in tumors tissues without distant metastasis fig 1c indicating that mir15a5p downregulation is associated with cc metastasis in addition rtqpcr was used to examine the mir15a5p level in four cc cell lines hela c33a caski and siha and the normal cervical epithelial cell line ect1e6e7 which was used as a control as expected mir‘15a‘5p was significantly lower in the four cc cell lines compared with ect1e6e7 cells fig 1d siha and c33a cells were selected for further experiments as they demonstrated the lowest expression of mir15a5p among all cell lines examinedupregulation of mir‘15a‘5p inhibits cell viability and promotes cell apoptosis in an attempt to understand the biological function of mir15a5p mir15a5p expression was upregulated or downregulated in the cultured siha and c33a cells by transfection with mir15a5p mimic or inhibitor respectively mir‘15a‘5p expression was significantly increased after mir15a5p mimic transfection whereas it was significantly decreased following mir‘15a‘5p inhibitor transfection in both siha and c33a cells fig 2a the present study then investigated the effect of mir15a5p expression on cell viability and the results demonstrated that the viability of siha and c‘33a cells was significantly inhibited by overexpression of mir15a5p whereas it was significantly enhanced by knockdown of mir‘15a‘5p compared with the negative control group fig 2b and c to assess the effects of mir15a5p upregulation on the apoptosis of siha and c33a cells caspase3 expression level and activity were analyzed by immunofluorescence and caspase‘ activity assays respectively as presented in fig 2d and e the expression of cleaved caspase3 and caspase3 activity was increased in siha and c33a cells transfected with 0cchen mir15a inhibits cervical cancer cell growthfigure mir‘15a‘5p is downregulated in cc tissues and cell lines a heat map presents significant differentially expressed mirnas in cc tissues and matched adjacent noncancerous tissues n3 green indicates downregulation and red indicates upregulation b mir15a5p expression was measured by rtqpcr in pairs of cc tissues and matched adjacent noncancerous tissues c mir15a5p expression was measured in tumor tissues with distant metastasis and tumors tissues without distant metastasis by rtqpcr d mir15a5p expression was detected in four cervical cancer cell lines hela c33a caski and siha and the normal cervical epithelial cells ect1e6e7 data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs ect1e6e7 cells mir microrna cc cervical cancer rtqpcr reverse transcriptionquantitative pcr mir15a5p mimic compared with the mimic nc groups furthermore the results of flow cytometry demonstrated that the extent of apoptosis was significantly increased after mir15a5p mimic transfection compared with the mimic nc groups fig 2f taken together these results indicate that overexpression of mir15a5p inhibits cell viability by inducing cell apoptosisupregulation of mir‘15a‘5p inhibits the invasion and migra‘tion of cc cells the present study further investigated whether overexpression of mir15a5p could reduce the invasiveness and migratory potential of cc cells using a transwell assay it was identified that the invasive capacities of siha and c‘33a cells were significantly inhibited by mir‘15a‘5p mimic whereas they were increased by mir15a5p inhibitor compared with the nc groups furthermore the wound healing assay results also demonstrated a significant reduction of cell migration in siha and c33a cells following mir15a5p overexpression however the migration of siha and c‘33a cells was significantly enhanced by mir15a5p inhibition fig 3c and d collectively the present data suggest that overexpression of mir15a5p suppresses the invasive and migratory abilities of cc cellsyap1 is a direct target of mir‘15a‘5p using the targetscan and miranda algorithms yap1 was found to have a putative target site of mir15a5p in its 'utr fig 4a to validate the possibility that yap1 is a direct target gene of mir15a5p a luciferase reporter assay was then performed the data revealed that mir‘15a‘5p mimic significantly inhibited the luciferase activity in the constructs containing the wildtype 0cinternational journal of molecular medicine figure overexpression of mir15a5p suppresses cell viability and promotes cell apoptosis siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis a transfection efficiency was assessed by reverse transcription‘quantitative pcr cell viability was measured by cck8 assay at indicated times for b siha and c c33a cells d the expression of cleaved caspase3 was determined by immunofluorescence assay magnification x200 e the caspase‘ activity was detected by a commercial caspase‘ activity kit f cell apoptosis was measured by flow cytometry data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs mimic nc p005 p001 vs inhibitor nc mir microrna nc negative control od optical density pi propidium iodidebinding site of yap13'utr while it had no evident effects on the activity of yap13'utrmut by contrast mir15a5p inhibitor significantly increased luciferase activity without any evident effects on yap13'utrmut activity fig 4b subsequently to further detect the potential regulation of yap1 by mir15a5p the expression of yap1 protein was measured in cc cells by western blotting as presented in fig 4c the expression of yap1 was significantly decreased upon ectopic expression of mir15a5p suggesting that high expression of yap1 was partly due to the downregulation of mir15a5p in cc cells in addition it was identified that the mrna level of yap1 was significantly increased in cervical cancer compared with the control and inversely correlated with mir15a5p expression levels in cancer tissues fig 4d and e these results indicated that yap1 is a downstream gene of mir15a5p in cc 0cchen mir15a inhibits cervical cancer cell growthfigure overexpression of mir15a5p suppresses cell invasion and migration siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis invasion of a siha and b c‘33a cells was measured by a transwell assay magnification x200 the migration of c siha and d c33a cells was assessed by a wound healing assay the images were taken at and h after gaps were generated wound healing was quantified by the distance of the wounded region with an absence of cells data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs mimics nc p001 vs inhibitor nc mir microrna nc negative controlyap1 inhibition suppresses cell viability promotes cell apop‘tosis and inhibits invasion and migration previous evidence has shown that yap1 exerts an oncogenic function in several types of human cancer such as breast and lung cancer as the findings of the present study revealed that yap1 is upregulated in cc it was hypothesized that yap1 may act as an oncogenic gene in cc to confirm this hypothesis siha and c33a cells were transfected with siyap1 or siscramble western blot assay revealed that yap1 was notably downregulated following transfection with siyap1 fig 5a functionally yap1‘knockdown significantly suppressed the cell viability and induced cell apoptosis compared with the siscramble group fig 5b and c furthermore knockdown of yap1 significantly suppressed the invasive and migratory abilities of siha and c33a cells fig 5d and e suggesting that yap1 may play an oncogene role in the development of ccoverexpression of yap1 moderates the negative functions of mir‘15a‘5p on cell viability migration and invasion to ascertain whether yap1 is involved in the inhibitory effects of mir15a5p on cc cells the present study cotransfected pcdnayap1 andor mir15a5p mimic as well as their controls into siha and c33a cells the overexpression efficiency was verified by western blotting as shown in fig 6a yap1 was notably increased in siha and c33a cells after pcdna‘yap1 transfection subsequently the cell viability apoptosis invasion and migration were evaluated overexpression of yap1 significantly abolished the inhibitory effects of mir15a5p upregulation on the viability of siha and c33a cells fig 6b the increased apoptosis induced by mir15a5p overexpression was also reversed by overexpression of yap1 fig 6c furthermore overexpression of yap1 significantly reversed the inhibitory effects of mir‘15a‘5p on cell invasion and migration fig 6d and e in addition it was identified that overexpression of yap1 alone significantly promoted cc cell viability inhibited cell apoptosis and enhanced the invasion and migration compared with blank control group suggesting the oncogenic role of yap1 in cc cells these results indicate that mir15a5p exerts its tumor suppressive role in cc at least partially through yap1 0cinternational journal of molecular medicine figure yap1 is a direct target of mir15a5p a schematic of the yap1 'utr containing the mir15a5p binding sites b luciferase assay of 293t cells co‘transfected with firefly luciferase constructs containing the yap1 wt or mut '‘utrs and mir‘15a‘5p mimics mimics nc mir‘15a‘5p inhibitor or inhibitor nc as indicated n3 p001 c siha and c33a cells were transfected with the mir15a5p mimic and mimic nc for h and the expression levels of yap1 protein were determined by western blotting p001 vs mimic nc d yap1 expression was measured by reverse transcriptionquantitative pcr in cc tissues and matched adjacent noncancerous tissues n40 p001 e spearman's analysis was used to analyze the correlation between the expression of yap1 and the expression of mir15a5p expression in cervical cancer tissues r p001 data are expressed at the mean ± standard deviation n3 of one representative experiment yap1 yesassociated protein mir microrna 'utr 'untranslated region wt wildtype mut mutant nc negative controldiscussionin the present study mir15a5p was shown to be decreased in cc tissues and cell lines and associated with cc metastasis furthermore overexpression of mir15a5p inhibited the cc cell viability invasion and migration and promoted cell apoptosis while inhibition of mir15a5p demonstrated the opposite effects additionally yap1 was confirmed as a functional target of mir15a5p ectopic expression of which significantly reversed suppression of mir‘15a‘5p the present data indicated that mir15a5p may function as a tumor suppressor in cc progression by inhibiting yap1 expressiona number of studies have shown that mirnas participate in the development of cc for example xia reported that mir374b overexpression suppresses cell proliferative and invasive abilities via affecting forkhead box m1 expression yao also demonstrated that mir641 upregulation restricts cc cell growth in vitro and in vivo xu reported that mir2185p suppresses the progression of cc via the lynnfκb signaling pathway yuan demonstrated that overexpression of mir138 suppresses cc cell growth in vivo these findings suggest that targeting mirnas may be an effective therapeutic strategy for cc in the present study based on microarray expression data it was identified that mir15a5p is one of the most markedly downregulated mirnas in cc tissues notably previous studies have reported that mir15a5p functions as a tumor suppressor in several human cancer types although mir15a5p has been found to be downregulated in cc to the best of our knowledge the tumorigenic role and mechanism remain unknown therefore the present study focused on mir15a5p in cc for molecular analyses in the 0cchen mir15a inhibits cervical cancer cell growthfigure yap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration siha and c33a cells were transfected with siyap1 or siscramble and then cells were harvested for further study a the expression of yap1 was measured by western blotting b cell viability was measured by cck‘ assay c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration assessed by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs siscramble yap1 yesassociated protein mir microrna si small interfering rnafigure mir15a5p inhibits cell viability and induces cell apoptosis by targeting yap1 a siha and c33a cells were transfected with the pcdnayap1 plasmid for h and then the protein expression of yap1 was measured by western blotting subsequently siha and c33a cells were cotransfected with the pcdnayap1 plasmid and mir15a5p mimic for h and then cells were used for analysis b viability of siha and c33a cells was measured by cck8 assay at indicated times c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration was measured by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs blank group p001 mir microrna yap1 yesassociated protein microarray expression data the expression levels of numerous mirnas exhibited significant changes such as mir137 which demonstrated the most significant upregulation in cc tissues miao reported that mir137 upregulation inhibits cc cell invasion migration and epithelialmesenchymal transition by suppressing the tgfβsmad pathway 0cinternational journal of molecular medicine notably mir15a3p has also reported to exhibit differential expression and induce apoptosis in human cc cells although the present study did not detect the expression change of mir15a3p in the microarray expression data the expression of mir15a3p in four cc cell lines was examined and the results demonstrated that mir15a3p was also downregulated in cc cells compared with ect1e6e7 cells data not shown however the role and regulatory mechanisms of mir15a3p on invasion and migration remain unclear the function of more mirnas in cc will be investigated in the futureprevious studies have reported that mir15a5p has the potential to suppress cell growth and inhibit the progression of human cancers by regulating its downstream target genes for example luo demonstrated that overexpression of mir15a5p causes cellular growth inhibition and suppression of migration by targeting cyclin e1 in breast cancer wu and guo found that mir15a overexpression suppressed the cell proliferation and invasion by suppression of bmi1 translation in gastric cancer gc as well as pancreatic cancer pc of note several studies have reported aberrant expression of mir15a5p in cc tissues or cells however the role and mechanism of mir15a5p in cc remain largely unknown the present results demonstrated that overexpression of mir15a5p inhibited cell viability cell migration and invasion and induced cell apoptosis in siha and c33a cells while inhibition of mir15a5p demonstrated the opposite effects indicating that mir15a5p may serve as tumor suppressive role in cc yap1 a transcriptional coactivator and oncogene has been found to play an important role in different types of carcinoma for example liu reported that yap1 overexpression promotes the invasion migration and growth of colon cancer cells yu demonstrated that knockdown of yap1 causes a significant inhibition of the growth and migration of renal cell carcinoma cells in vitro and in vivo notably yap1 has been verified to target mir‘15a‘5p to suppress cell growth and metastasis in gastric adenocarcinoma and colon cancer however whether yap1 is a target of mir15a5p in cc remains unclear in the present study yap1 was confirmed to be a target of mir‘15a‘5p and its protein expression levels were negatively regulated by mir15a5p further investigation indicated that yap1 was significantly increased in cc tissues and inversely correlated with mir15a5p in cc tissues furthermore yap1 was confirmed to act as an oncogene gene in cc cells and its overexpression partly abrogated the inhibitory effect induced by enhanced expression of mir15a5p in cc cells taken together the present study demonstrates that mir15a5p exerts its tumor suppressive role in cc cells by targeting yap1due to the limitation in experimental conditions and funds further research in the future is required to investigate whether mir15a5p serves its role via other downstream targets in addition the present study investigated the cellular function of mir15a5p and its underlying mechanism in cc however in vivo studies and clinical trial data are required to validate the preliminary in vitro results obtained therefore the function of mir15a5p in cc needs to be further investigated in vivoin conclusion the present results demonstrated that mir15a5p suppresses the viability migration and invasion of cc cells by directly targeting yap1 based on these findings it is proposed that the mir15a5pyap1 axis may serve as a novel biomarker for new targets in cc therapyacknowledgementsnot applicablefundingfunding was received from the scientific research project of shanghai science and technology commission grant nos and availability of data and materialsthe datasets used andor analysed during the current study are available from the corresponding author on reasonable request authors' contributionsrc hl tz xy and sx performed the experiments contributed to data analysis and wrote the paper rc hl tz xy and sx analysed the data xc conceptualized the study design and contributed to data analysis and experimental materials all authors read and approved the final manuscriptethics approval and consent to participateall individuals provided written informed consent for the use of human specimens for clinical research the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests references alldredge jk and tewari ks clinical trials of antiangiogenesis therapy in recurrentpersistent and metastatic cervical cancer oncologist tsikouras p zervoudis s manav b tomara e iatrakis g romanidis c bothou a and galazios g cervical cancer screening diagnosis and staging j buon fang j zhang h and jin s epigenetics and cervical cancer from pathogenesis to therapy tumour biol wang j liu y wang x li j we j wang y song w and zhang z mir1266 promotes cell proliferation migration and invasion in cervical cancer by targeting dab2ip biochim biophys acta mol basis dis zhu l zhu l s

cancer7506

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " micrornas mirnas have been reported to have important regulatory roles in the progression of several types of cancer including cervical cancer cc however the biological roles and regulatory mechanisms of mirnas in cc remain to be fully elucidated the aim of the present study was to examine the functions of mirnas in cc and the possible mechanisms using a microarray it was identified that mirna15a5p mir15a5p was one of the most downregulated mirnas in cc tissues compared with adjacent noncancerous tissues the low expression of mir15a5p was observed in cc tumor tissues with distant metastasis and in cc cell lines in addition the effects of mir15a5p upregulation on cell viability apoptosis invasion and migration of cc cells were investigated using cck‘ flow cytometry transwell and wound healing assays respectively it was demonstrated that upregulation of mir‘15a‘5p significantly suppressed the viability migration and invasion and promoted the apoptosis of siha and c33a cells furthermore yesassociated protein yap1 a well‘known oncogene was confirmed to be directly targeted by mir15a5p and was found to be negatively regulated by mir15a5p further correlation analysis indicated that mir15a5p expression was negatively correlated with yap1 expression in cc tissues notably overexpression of yap1 abrogated the tumor suppressive effects of mir15a5p in cc cells taken together these present findings indicated that the mir15a5pyap1 axis may provide a novel strategy for the clinical treatment of cccorrespondence to professor xu chen department of obstetrics and gynaecology huashan hospital north fudan university jingpohu road baoshan shanghai pr chinaemail xuchenccx163comcontributed equallykey words cervical cancer microrna15a5p cell viability migration invasion yesassociated protein introductioncervical cancer cc is a type of malignant tumor commonly presenting in women in cc cases are diagnosed each year and it accounts for of all female cancerassociated mortalities each year worldwide despite advances in the therapeutic strategies for cc including targeted therapies and immunotherapy the prognosis of cc remains poor due to the abnormal growth of epithelial cells thus it is imperative to clarify the molecular interactions occurring during the initiation and progression of ccmicrornas mirnas are a family of short noncoding rnas with an average length of nucleotides which negatively regulate target gene expression through either translation repression or rna degradation accumulating evidence has indicated that mirnas may function as oncogenes or tumor suppressors depending on their target mrna in various types of cancer including cc for example yang reported that mir214 inhibits the growth of cc cells by the regulation of its target enhancer of zeste homolog dong demonstrated a suppressive role of mir217 in the development of cc cells via targeting rhoassociated protein kinase chen reported that mir499a promotes the proliferation cell cycle progression colony formation migration and invasion of cc cells by targeting srybox transcription factor in addition several mirnas serve as diagnostic biomarkers in patients with cc such as mir152 and mir365 despite the aforementioned findings the roles of mirnas in the development of cc require further investigationin the present study a mirna microarray was performed to investigate the expression profiles of mirnas in cc tissues and the most downregulated mirna identified mir‘15a‘5p was selected for further analysis the potential role and underlying mechanism of mir15a5p in cc cells were also investigated the present results suggest that mir15a5p may serve as a therapeutic target for ccmaterials and methodspatients and samples in total paired cervical samples tumor tissues and adjacent noncancerous tissues were 0cchen mir15a inhibits cervical cancer cell growthobtained from female patients with cc who underwent cervical surgical resection without preoperative systemic therapy at the department of obstetrics and gynecology huashan hospital north of fudan university shanghai china between may and december the median age of the patients was years range years among all patients there were patients with metastatic cc and with nonmetastatic cc the matched nontumor adjacent tissue was obtained cm beyond the boundary of cc tissue all tissue samples were immediately snapfrozen in liquid nitrogen and stored at ‘Ëšc until use the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university written informed consent for participation in the study was obtained from all patientsmirna expression profiling total rna from cc tissues three randomly selected paired tumor tissues and adjacent noncancerous tissues was extracted using mirneasy mini kit qiagen gmbh the samples were assessed using the mircury lna„¢ array v180 agilent technologies inc the procedure and imaging processes were performed as described previously cell culture human cc cell lines hela c33a caski and siha 293t cells and normal cervical epithelial cells ect1e6e7 were obtained from the american type culture collection all cells were cultured in dmem sigmaaldrich merck kgaa supplemented with vv fbs sigmaaldrich merck kgaa plus uml penicillinstreptomycin at ˚c with co2reverse transcription‘quantitative pcr rt‘qpcr total rna was extracted from tissues or cell lines using trizol reagent invitrogen thermo fisher scientific inc for mirna rt cdna was generated from ng total rna samples using taqman„¢ microrna reverse transcription kit applied biosystems thermo fisher scientific inc at ˚c for min for mrna rt cdna was synthesized using primescript rt reagent kit takara bio inc at ˚c for min qpcr for mirna and mrna was performed using the sybrgreen i realtime pcr kit applied biosystems thermo fisher scientific inc on an abi system applied biosystems thermo fisher scientific inc the reaction was performed under the following conditions ˚c for min followed by cycles at ˚c for sec and ˚c for sec and a final extension at ˚c for sec the primers for qpcr analysis were as follows mir15a5p forward 'aat gtt gcc cgt aat gcc3' and reverse 'ccc aag cgg aga aag gaa3' u6 forward 'gct tcg gca gca cat ata cta aaa t3' and reverse 'cgc ttc acg aat ttg cgt gtc at3' yesassociated protein yap1 forward 'cgg tcc act tca gtc tcc3' and reverse 'gag tgt ggt gga cag gta ctg3' and gapdh forward 'gtg gtg aag acg cca gtg ga3' and reverse 'cga gcc aca tcg ctc aga ca3' the expression levels of mir15a5p and yap1 were normalized to the expression of u6 and gapdh respectively the relative expression of each gene was calculated using the ‘ˆ†ˆ†cq method cell transfection the mir15a5p mimic mimic negative control nc mir15a5p inhibitor inhibitor nc yap1 overexpression plasmid pcdnayap1 and pcdnavector were all provided by guangzhou ribobio co ltd when c33a and siha cells 5x105 cellswell in 6well plates grew to confluence mir‘15a‘5p mimic nm mimic nc nm mir15a5p inhibitor nm inhibitor nc nm pcdnayap1 µg or pcdnavector µg were transfected into cells at ˚c for h using lipofectamine® invitrogen thermo fisher scientific inc the sequences were as follows mir15a5p mimic 'uag cag cac aua aug guu ugu g3' mimic nc 'uuc ucc gaa cgu guc acg utt3' mir15a5p inhibitor 'cac aaa cca uua ugu gcu gcu a3' and inhibitor nc 'cag uac uuu ugu gua gua caa3'in addition small interfering rna targeting yap1 si‘yap1 and the negative control targeting a non‘specific sequence siscramble were provided by thermo fisher scientific inc siha and c‘33a cells were transfected with the sirnas nmoll using lipofectamine invitrogen thermo fisher scientific inc the sequences of si‘yap1 and siscramble were as follows siyap1 'ctc agg atg gag aaa ttt a3' and siscramble 'ttc tcc gaa cgt gtc acg t3' at h posttransfection the cells were harvested for further analysis and the inhibition efficiency was determined by western blottingcell viability the c33a and siha cells were seeded in 96well plates at a density of 5x103well overnight following transfection the cell viability was measured using a cck8 assay briefly µl cck‘ solution was added to each well and cultured for h at ˚c the absorbance of the samples at nm was detected using a microplate reader biorad laboratories inccaspase‘ activity following transfection c33a and siha cells were harvested and the caspase3 activity was measured using a caspase3 activity assay kit beyotime institute of biotechnology according to the manufacturer's protocolcell apoptosis the apoptosis of c33a and siha cells was examined using flow cytometry following transfection c33a and siha cells were collected and the apoptotic cells were identified using an annexin v‘fitc apoptosis detection kit abcam according to the manufacturer's protocol after washing with cold pbs the cells were resuspended in binding buffer followed by staining with annexin v and propidium iodide for min in the dark at room temperature the fluorescence was measured using a facscan flow cytometer beckman coulter inc and then analyzed by flowjo v871 software flowjo llcimmunofluorescence assay following transfection c33a and siha cells were fixed in absolute ethyl alcohol for min at room temperature after washing twice with pbs the fixed cells were stained with primary antibody targeting cleavedcaspase3 cat no c ell signaling technology inc for h at room temperature subsequently an antirabbit conjugated antibody with fitc cat no f0382 sigmaaldrich merck kgaa was added for h in the 0cinternational journal of molecular medicine dark fluorescence images were obtained using an inverted fluorescence microscope magnification x200cell invasion assays transwell chambers 8µm pore bd biosciences coated with matrigel bd biosciences were used for the invasion assay briefly c‘33a and siha cells 8x104 were seeded in the top chamber with serumfree medium while the lower chamber contained culture medium with fbs following incubation for h the cells were fixed in paraformaldehyde solution beyotime institute of biotechnology for min and stained with crystal violet beyotime institute of biotechnology for min at room temperature images were captured with an inverted microscope olympus corporation magnification x100wound healing assay for the wound healing assay c33a and siha cells were seeded onto 12well plates 2x105 cellswell and h after transfection a scratch was made using a 10µl pipette tip in the confluent cell monolayer then cells were washed twice with pbs and incubated in dmem without fbs the wound healing images were captured at and h after scratching using an inverted light microscope olympus corporation magnification x100 the wound healing rate was calculated using imagej software v146 national institutes of healthdual‘luciferase reporter assay mirna target prediction tools including miranda httpmirandaorguk and targetscan httptargetscanorg were used to search for the putative targets of mir15a5p pgl3yap1 widetype or pgl3yap1 mutant type pgl3yap1mut promega corporation were cotransfected with mir15a5p mimics into 293t cells in 24well plates 2x105well using lipofectamine invitrogen thermo fisher scientific inc at h post‘transfection the luciferase activities were analyzed using the dualluciferase reporter assay system promega corporation with renilla luciferase activity as an internal control western blot analysis western blotting was performed as previously described briefly cells were lysed using radio immunoprecipitation assay buffer beyotime institute of biotechnology and the protein concentration was determined using the bicinchoninic acid assay total protein µglane was separated by sdspage and electrophoretically transferred onto a polyvinylidene difluoride membrane emd millipore subsequently membranes were blocked with skim milk for h at ˚c overnight each membrane was probed with primary antibodies against yap1 cat no and β‘actin cat no at ˚c overnight all primary antibodies were obtained from cell signaling technology inc subsequently the membrane was incubated with horseradish peroxidaseconjugated goat antirabbit igg cat no abcam at room temperature for h βactin served as the loading control and for normalization of protein expression the protein bands were developed using ecl kit ge healthcare and expression levels were quantified using imagej v146 national institutes of healthstatistical analysis all data are presented as mean ± standard deviation the correlation between mir15a5p and yap1 levels was evaluated using spearman's correlation analysis pairwise comparisons were performed by student's ttest and comparisons among groups were analyzed by oneway anova followed by tukey's posthoc test p005 was considered to indicate a statistically significant differenceresultsmir‘15a‘5p is downregulated in cc to examine the potential involvement of mirnas in the development of cc microarray analysis was performed to evaluate the mirna expression profiles between cc tissues and adjacent noncancerous tissues of differently expressed mirnas identified in the tumor group mirnas exhibited decreased expression and mirnas demonstrated increased expression compared with that in adjacent noncancerous tissues fig 1a among the aberrant mirnas the present study focused on mir15a5p for subsequent experiments due to its suppressive role in a variety of other cancer types such as endometrial cancer and chronic myeloid leukemia subsequently rtqpcr was performed to detect the expression of mir15a5p in pairs of tumor tissues and adjacent noncancerous tissues the results revealed that the level of mir15a5p was significantly lower in tumor tissues compared with that in adjacent noncancerous tissues fig 1b it was also observed that mir15a5p was expressed at a significantly lower level in tumor tissues with distant metastasis compared with in tumors tissues without distant metastasis fig 1c indicating that mir15a5p downregulation is associated with cc metastasis in addition rtqpcr was used to examine the mir15a5p level in four cc cell lines hela c33a caski and siha and the normal cervical epithelial cell line ect1e6e7 which was used as a control as expected mir‘15a‘5p was significantly lower in the four cc cell lines compared with ect1e6e7 cells fig 1d siha and c33a cells were selected for further experiments as they demonstrated the lowest expression of mir15a5p among all cell lines examinedupregulation of mir‘15a‘5p inhibits cell viability and promotes cell apoptosis in an attempt to understand the biological function of mir15a5p mir15a5p expression was upregulated or downregulated in the cultured siha and c33a cells by transfection with mir15a5p mimic or inhibitor respectively mir‘15a‘5p expression was significantly increased after mir15a5p mimic transfection whereas it was significantly decreased following mir‘15a‘5p inhibitor transfection in both siha and c33a cells fig 2a the present study then investigated the effect of mir15a5p expression on cell viability and the results demonstrated that the viability of siha and c‘33a cells was significantly inhibited by overexpression of mir15a5p whereas it was significantly enhanced by knockdown of mir‘15a‘5p compared with the negative control group fig 2b and c to assess the effects of mir15a5p upregulation on the apoptosis of siha and c33a cells caspase3 expression level and activity were analyzed by immunofluorescence and caspase‘ activity assays respectively as presented in fig 2d and e the expression of cleaved caspase3 and caspase3 activity was increased in siha and c33a cells transfected with 0cchen mir15a inhibits cervical cancer cell growthfigure mir‘15a‘5p is downregulated in cc tissues and cell lines a heat map presents significant differentially expressed mirnas in cc tissues and matched adjacent noncancerous tissues n3 green indicates downregulation and red indicates upregulation b mir15a5p expression was measured by rtqpcr in pairs of cc tissues and matched adjacent noncancerous tissues c mir15a5p expression was measured in tumor tissues with distant metastasis and tumors tissues without distant metastasis by rtqpcr d mir15a5p expression was detected in four cervical cancer cell lines hela c33a caski and siha and the normal cervical epithelial cells ect1e6e7 data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs ect1e6e7 cells mir microrna cc cervical cancer rtqpcr reverse transcriptionquantitative pcr mir15a5p mimic compared with the mimic nc groups furthermore the results of flow cytometry demonstrated that the extent of apoptosis was significantly increased after mir15a5p mimic transfection compared with the mimic nc groups fig 2f taken together these results indicate that overexpression of mir15a5p inhibits cell viability by inducing cell apoptosisupregulation of mir‘15a‘5p inhibits the invasion and migra‘tion of cc cells the present study further investigated whether overexpression of mir15a5p could reduce the invasiveness and migratory potential of cc cells using a transwell assay it was identified that the invasive capacities of siha and c‘33a cells were significantly inhibited by mir‘15a‘5p mimic whereas they were increased by mir15a5p inhibitor compared with the nc groups furthermore the wound healing assay results also demonstrated a significant reduction of cell migration in siha and c33a cells following mir15a5p overexpression however the migration of siha and c‘33a cells was significantly enhanced by mir15a5p inhibition fig 3c and d collectively the present data suggest that overexpression of mir15a5p suppresses the invasive and migratory abilities of cc cellsyap1 is a direct target of mir‘15a‘5p using the targetscan and miranda algorithms yap1 was found to have a putative target site of mir15a5p in its 'utr fig 4a to validate the possibility that yap1 is a direct target gene of mir15a5p a luciferase reporter assay was then performed the data revealed that mir‘15a‘5p mimic significantly inhibited the luciferase activity in the constructs containing the wildtype 0cinternational journal of molecular medicine figure overexpression of mir15a5p suppresses cell viability and promotes cell apoptosis siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis a transfection efficiency was assessed by reverse transcription‘quantitative pcr cell viability was measured by cck8 assay at indicated times for b siha and c c33a cells d the expression of cleaved caspase3 was determined by immunofluorescence assay magnification x200 e the caspase‘ activity was detected by a commercial caspase‘ activity kit f cell apoptosis was measured by flow cytometry data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs mimic nc p005 p001 vs inhibitor nc mir microrna nc negative control od optical density pi propidium iodidebinding site of yap13'utr while it had no evident effects on the activity of yap13'utrmut by contrast mir15a5p inhibitor significantly increased luciferase activity without any evident effects on yap13'utrmut activity fig 4b subsequently to further detect the potential regulation of yap1 by mir15a5p the expression of yap1 protein was measured in cc cells by western blotting as presented in fig 4c the expression of yap1 was significantly decreased upon ectopic expression of mir15a5p suggesting that high expression of yap1 was partly due to the downregulation of mir15a5p in cc cells in addition it was identified that the mrna level of yap1 was significantly increased in cervical cancer compared with the control and inversely correlated with mir15a5p expression levels in cancer tissues fig 4d and e these results indicated that yap1 is a downstream gene of mir15a5p in cc 0cchen mir15a inhibits cervical cancer cell growthfigure overexpression of mir15a5p suppresses cell invasion and migration siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis invasion of a siha and b c‘33a cells was measured by a transwell assay magnification x200 the migration of c siha and d c33a cells was assessed by a wound healing assay the images were taken at and h after gaps were generated wound healing was quantified by the distance of the wounded region with an absence of cells data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs mimics nc p001 vs inhibitor nc mir microrna nc negative controlyap1 inhibition suppresses cell viability promotes cell apop‘tosis and inhibits invasion and migration previous evidence has shown that yap1 exerts an oncogenic function in several types of human cancer such as breast and lung cancer as the findings of the present study revealed that yap1 is upregulated in cc it was hypothesized that yap1 may act as an oncogenic gene in cc to confirm this hypothesis siha and c33a cells were transfected with siyap1 or siscramble western blot assay revealed that yap1 was notably downregulated following transfection with siyap1 fig 5a functionally yap1‘knockdown significantly suppressed the cell viability and induced cell apoptosis compared with the siscramble group fig 5b and c furthermore knockdown of yap1 significantly suppressed the invasive and migratory abilities of siha and c33a cells fig 5d and e suggesting that yap1 may play an oncogene role in the development of ccoverexpression of yap1 moderates the negative functions of mir‘15a‘5p on cell viability migration and invasion to ascertain whether yap1 is involved in the inhibitory effects of mir15a5p on cc cells the present study cotransfected pcdnayap1 andor mir15a5p mimic as well as their controls into siha and c33a cells the overexpression efficiency was verified by western blotting as shown in fig 6a yap1 was notably increased in siha and c33a cells after pcdna‘yap1 transfection subsequently the cell viability apoptosis invasion and migration were evaluated overexpression of yap1 significantly abolished the inhibitory effects of mir15a5p upregulation on the viability of siha and c33a cells fig 6b the increased apoptosis induced by mir15a5p overexpression was also reversed by overexpression of yap1 fig 6c furthermore overexpression of yap1 significantly reversed the inhibitory effects of mir‘15a‘5p on cell invasion and migration fig 6d and e in addition it was identified that overexpression of yap1 alone significantly promoted cc cell viability inhibited cell apoptosis and enhanced the invasion and migration compared with blank control group suggesting the oncogenic role of yap1 in cc cells these results indicate that mir15a5p exerts its tumor suppressive role in cc at least partially through yap1 0cinternational journal of molecular medicine figure yap1 is a direct target of mir15a5p a schematic of the yap1 'utr containing the mir15a5p binding sites b luciferase assay of 293t cells co‘transfected with firefly luciferase constructs containing the yap1 wt or mut '‘utrs and mir‘15a‘5p mimics mimics nc mir‘15a‘5p inhibitor or inhibitor nc as indicated n3 p001 c siha and c33a cells were transfected with the mir15a5p mimic and mimic nc for h and the expression levels of yap1 protein were determined by western blotting p001 vs mimic nc d yap1 expression was measured by reverse transcriptionquantitative pcr in cc tissues and matched adjacent noncancerous tissues n40 p001 e spearman's analysis was used to analyze the correlation between the expression of yap1 and the expression of mir15a5p expression in cervical cancer tissues r p001 data are expressed at the mean ± standard deviation n3 of one representative experiment yap1 yesassociated protein mir microrna 'utr 'untranslated region wt wildtype mut mutant nc negative controldiscussionin the present study mir15a5p was shown to be decreased in cc tissues and cell lines and associated with cc metastasis furthermore overexpression of mir15a5p inhibited the cc cell viability invasion and migration and promoted cell apoptosis while inhibition of mir15a5p demonstrated the opposite effects additionally yap1 was confirmed as a functional target of mir15a5p ectopic expression of which significantly reversed suppression of mir‘15a‘5p the present data indicated that mir15a5p may function as a tumor suppressor in cc progression by inhibiting yap1 expressiona number of studies have shown that mirnas participate in the development of cc for example xia reported that mir374b overexpression suppresses cell proliferative and invasive abilities via affecting forkhead box m1 expression yao also demonstrated that mir641 upregulation restricts cc cell growth in vitro and in vivo xu reported that mir2185p suppresses the progression of cc via the lynnfκb signaling pathway yuan demonstrated that overexpression of mir138 suppresses cc cell growth in vivo these findings suggest that targeting mirnas may be an effective therapeutic strategy for cc in the present study based on microarray expression data it was identified that mir15a5p is one of the most markedly downregulated mirnas in cc tissues notably previous studies have reported that mir15a5p functions as a tumor suppressor in several human cancer types although mir15a5p has been found to be downregulated in cc to the best of our knowledge the tumorigenic role and mechanism remain unknown therefore the present study focused on mir15a5p in cc for molecular analyses in the 0cchen mir15a inhibits cervical cancer cell growthfigure yap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration siha and c33a cells were transfected with siyap1 or siscramble and then cells were harvested for further study a the expression of yap1 was measured by western blotting b cell viability was measured by cck‘ assay c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration assessed by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs siscramble yap1 yesassociated protein mir microrna si small interfering rnafigure mir15a5p inhibits cell viability and induces cell apoptosis by targeting yap1 a siha and c33a cells were transfected with the pcdnayap1 plasmid for h and then the protein expression of yap1 was measured by western blotting subsequently siha and c33a cells were cotransfected with the pcdnayap1 plasmid and mir15a5p mimic for h and then cells were used for analysis b viability of siha and c33a cells was measured by cck8 assay at indicated times c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration was measured by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs blank group p001 mir microrna yap1 yesassociated protein microarray expression data the expression levels of numerous mirnas exhibited significant changes such as mir137 which demonstrated the most significant upregulation in cc tissues miao reported that mir137 upregulation inhibits cc cell invasion migration and epithelialmesenchymal transition by suppressing the tgfβsmad pathway 0cinternational journal of molecular medicine notably mir15a3p has also reported to exhibit differential expression and induce apoptosis in human cc cells although the present study did not detect the expression change of mir15a3p in the microarray expression data the expression of mir15a3p in four cc cell lines was examined and the results demonstrated that mir15a3p was also downregulated in cc cells compared with ect1e6e7 cells data not shown however the role and regulatory mechanisms of mir15a3p on invasion and migration remain unclear the function of more mirnas in cc will be investigated in the futureprevious studies have reported that mir15a5p has the potential to suppress cell growth and inhibit the progression of human cancers by regulating its downstream target genes for example luo demonstrated that overexpression of mir15a5p causes cellular growth inhibition and suppression of migration by targeting cyclin e1 in breast cancer wu and guo found that mir15a overexpression suppressed the cell proliferation and invasion by suppression of bmi1 translation in gastric cancer gc as well as pancreatic cancer pc of note several studies have reported aberrant expression of mir15a5p in cc tissues or cells however the role and mechanism of mir15a5p in cc remain largely unknown the present results demonstrated that overexpression of mir15a5p inhibited cell viability cell migration and invasion and induced cell apoptosis in siha and c33a cells while inhibition of mir15a5p demonstrated the opposite effects indicating that mir15a5p may serve as tumor suppressive role in cc yap1 a transcriptional coactivator and oncogene has been found to play an important role in different types of carcinoma for example liu reported that yap1 overexpression promotes the invasion migration and growth of colon cancer cells yu demonstrated that knockdown of yap1 causes a significant inhibition of the growth and migration of renal cell carcinoma cells in vitro and in vivo notably yap1 has been verified to target mir‘15a‘5p to suppress cell growth and metastasis in gastric adenocarcinoma and colon cancer however whether yap1 is a target of mir15a5p in cc remains unclear in the present study yap1 was confirmed to be a target of mir‘15a‘5p and its protein expression levels were negatively regulated by mir15a5p further investigation indicated that yap1 was significantly increased in cc tissues and inversely correlated with mir15a5p in cc tissues furthermore yap1 was confirmed to act as an oncogene gene in cc cells and its overexpression partly abrogated the inhibitory effect induced by enhanced expression of mir15a5p in cc cells taken together the present study demonstrates that mir15a5p exerts its tumor suppressive role in cc cells by targeting yap1due to the limitation in experimental conditions and funds further research in the future is required to investigate whether mir15a5p serves its role via other downstream targets in addition the present study investigated the cellular function of mir15a5p and its underlying mechanism in cc however in vivo studies and clinical trial data are required to validate the preliminary in vitro results obtained therefore the function of mir15a5p in cc needs to be further investigated in vivoin conclusion the present results demonstrated that mir15a5p suppresses the viability migration and invasion of cc cells by directly targeting yap1 based on these findings it is proposed that the mir15a5pyap1 axis may serve as a novel biomarker for new targets in cc therapyacknowledgementsnot applicablefundingfunding was received from the scientific research project of shanghai science and technology commission grant nos and availability of data and materialsthe datasets used andor analysed during the current study are available from the corresponding author on reasonable request authors' contributionsrc hl tz xy and sx performed the experiments contributed to data analysis and wrote the paper rc hl tz xy and sx analysed the data xc conceptualized the study design and contributed to data analysis and experimental materials all authors read and approved the final manuscriptethics approval and consent to participateall individuals provided written informed consent for the use of human specimens for clinical research the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests references alldredge jk and tewari ks clinical trials of antiangiogenesis therapy in recurrentpersistent and metastatic cervical cancer oncologist tsikouras p zervoudis s manav b tomara e iatrakis g romanidis c bothou a and galazios g cervical cancer screening diagnosis and staging j buon fang j zhang h and jin s epigenetics and cervical cancer from pathogenesis to therapy tumour biol wang j liu y wang x li j we j wang y song w and zhang z mir1266 promotes cell proliferation migration and invasion in cervical cancer by targeting dab2ip biochim biophys acta mol basis dis zhu l zhu l s Answer:

Colon_Cancer

during the last decade green synthesized cerium oxide nanops ceo2 nps attracted remarkable interest in various fields of science and technology this review explores the vast array of biological resources such as plants microbes and other biological products being used in synthesis of ceo2 nps it also discusses their biosynthetic mechanism current understandings and trends in the green synthesis of ceo2 nps novel therapies based on green synthesized ceo2 nps are illustrated in particular their antimicrobial potential along with attempts of their mechanistic elucidation overall the main objective of this review is to provide a rational insight of the major accomplishments of ceo2 nps as novel therapeutics agents for a wide range of microbial pathogens and combating other diseases keywords nanotechnology green synthesis cerium oxide nanops antimicrobial infections biomedicalintroductionnanotechnology has got a remarkable interest in every field of science and technology and is presently considered among one of the leading research avenues it has a multitude of applications in the field of electronics imaging industry and healthcare14 mostly in healthcare it has been exploited in diseases diagnostics treatment delivery and formulations of novel drugs14 it exploits nano size structures with size ranges from “ nm known as nanop nps these nano scale entities have unique physiochemical properties and have been utilized in various fields of physics biology and chemistry5among other nps cerium oxide ceo2 nps have been mostly exploited due to their unique surface chemistry high stability and biocompatibility67 it is mostly used in the fabrication of sensors cells catalysis therapeutics agents drug delivery careers and antiparasitic ointments figure presently ceo2 nps is mostly synthesized via two methods such as physical and chemical79 however these methods utilize toxic reducing solvents posing several threats to the biodiversity and ecosystem moreover the nps obtained with such approaches are toxic and unstable making them less efficient910 thus recently a safe less toxic method has been used by researchers known as green synthesis this method utilizes various biological resources such as plants microbes or any other biological derivative1115 these biological extracts have a rich source of phytochemicals international of nanomedicine “ nadeem this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomterms php and incorporate the creative commons attribution “ non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cnadeem dovepresslatter is extensively utilized for its biomedical pharmacological and food applications due to their safe and biocompatible nature9 moreover features like high yield everlasting stability and better morphologies can be obtained using a greener approach79green synthesis from plantsgreen syntheses of ceo2 nps have been reported using plant extracts microbial and other biological derivatives plants in this regard have been the most efficient source due to their abundance safe nature and rich source of reducing and stabilizing agents2629 various parts of plants such as leaves flower and stem have been used for the synthesis of ceo2 nps163031 till date the majority of green synthesis studies have been conducted on leaves extracts as it is a rich source of metabolites11163233 a broad variety of metabolitesphytochemicals in plant extracts such as ketones carboxylic acids phenols and ascorbic acid are used as reduction and stabilizing agents figure plants based ceo2 nps are produced through a simple approach in which bulk metal salt is mixed with the extract and the reaction completes in minutes to a few hours in ordinary lab conditions282934 the metallic salt solution is reduced into respective nanops via the phytochemicals whose synthesis is confirmed firstly through color change from colorless to yellowish brownish or whitish and then characterized through various spectroscopic and imaging techniques162935leaf extract of moringa oleifera l was used to synthesize ceo2 nps with spherical morphologies and size of nm the synthesized nps showed potential antimicrobial and wound healing properties36 gloriosa superba leaf extract was used as a reducing and stabilizing agent in synthesis of ceo2 nps and has shown potential antibacterial properties37 hibiscus sabdariffa natural extract yielded crystalline ceo2 with a diameter of nm30 spherical shaped nanops of size nm synthesized from gel extract of medicinally important plant aloe barbadensis38 the resultant ceo2 nps showed high antioxidant potential green synthesis of ceo2 nanops was demonstrated using jatropha curcus leaf extract and a monodispersed shape of “ nm10 spherical shaped cerium oxide nanops are synthesized using leaf extract of oleo europaea with a size of nm having high antimicrobial activity against both gramnegative and positive strains of bacteria16 origanum majorana extracts were used to synthesize ceo2 nps having pseudo photocatalytic activity having high figure general applications of ceo2 nanopssuch asketones amines enzymes and phenols which are believed to be responsible for the reduction and stabilization of bulk salts into respective nanops nps1619to date various applications of green synthesized ceo2 nps have been reported such as antimicrobial anti cancer antilarvicidal photocatalysis and antioxidant therapies162022 among other biomedical applications the antimicrobial potential is certainly the most exploited previously it has been reported that ceo2 nps to display their antimicrobial actions through various mechanisms9 but mostly ceo2 nps kill microbes via triggering the production of an excess of reactive oxygen species in cells916 however further studies need to be conducted to fully elucidate the complete mechanism of action here in this review we aim to focus on the following topics arrays of biological resources have been exploited to date for the synthesis of ceo2 nps moreover the synthesis mechanisms along their biomedical applications are discussed with special emphasis on the antimicrobial activitysynthesis of ceo2 npsnanops are synthesized through various physicochemical methods5 however both methods require toxic solvents high temperature and pressure which pose threats to the environment2325 moreover higher cost laborious downstream processing lesser biocompatibility instability and low yield make them further inefficient710 there is a growing need to fabricate nanostructures which have the potential to solve these problems59 presently researchers have exploited the green method to overcome all these challenges5 for instance plants microbes and other biological products have been used as reducing and or stabilizing agents in the fabrication of ecofriendly nps25 ceo2 nps have also been synthesized using various physical chemical and biological methods9 the submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure biosynthesis reduction stabilization and characterization of ceo2 nanopsspherical shape nm ftir confirmed that the reduction is attributed to the presence of different phenolic and flavonoids compounds in the extract18 ceo2 was synthesized using rubia cordifolia leaf fusions spectroscopic and microscopic analysis revealed hexagonal shaped nps having a size of nm the biogenic ceo2 nps also showed excellent anticancer potential33 nano rod size ranges from “ nm ceo2 nps resulted when pedalium murex l was added to the aqueous solution of salt at room temperature having high antibacterial activity32 china rose petal was used as a robust bio template for the facile fabrication of novel ceria nano sheet with a size of about nm39 the deviation in size and morphology noticed among the reported studies might be due to the different influence of reaction temperature ph time concentration of salt precursor or plants extracts and part of the plant being used13272840 moreover plants based ceo2 np™s showed excellent stability at diverse conditions for instance green mediated ceria np™s remain stable at liquid solution changes were observed1336 similarly biogenic ceo2 np™s also showed high thermal stability at high temperature and remained stable for a longer period of time which indicates their long durability and everlasting stability27283341 until now various plants have been used in the biogenic synthesis of ceo2 nps and are shown in table physiochemical and no green synthesis from microbesmicrobes also have an intrinsic potential to synthesize nanops as they are a rich source of secondary metabolites23 among other nanops ceo2 nps with various shapes and sizes have been synthesized in recent years from microbes table green synthesis of ceo2 from microbial species is a simple reliable costeffective and ecofriendly approach42 microbial metabolites such as enzymes proteins and heterocyclic derivatives play a crucial role in reducing and stabilizing of ceo2 bulk salt into respective nps4243 moreover microbiogenic ceo2 nps exhibited improved stability water dispensability and showed high fluorescent properties and were less agglomerated43aspergillus niger extract yielded cubic fluorite nps with spherical morphology and an average size of nm ftir analysis revealed the presence of an hydroxyl group carboxylic group and phenol group which are supposed to be involved in the reduction of nps21 curvularia lunata extract has also been used to synthesize spherical shaped ceo2 nps with size ranges from “ nm color change from white to yellow brown indicated initial reaction the nps were tested against microbial pathogens and showed excellent antibacterial potential spherical shaped ceo2 nps of size ranges from “ nm were made using fusarium solani extract which showed effective growth inhibition and biofilm formation of pathogenic bacterial strains42 shadab ali khan observed the biosynthesis of spherical shaped “ nm ceo2 nps by using a thermophilic fungus humicola capping agent43 the resultant nps were characterized by uv xps pl spectroscopy tem ftir and xrd moreover these nps showed excellent potential in treatment of neurodegenerative disorders such as alzheimer™s and parkinson™s diseases bacterial extract has also been exploited in the fabrication of ceo2 nps for instance bacillus shaped nps with an average size of nm the bacterial mediated nps also showed excellent antioxidant potential in vitro44 despite all these applications the microbial route of synthesis has certain shortcomings such as the high probability of pathogenicity laborious culturing and contamination issues however it offers a lot of promise in the field of nanotechnology and could become a leading avenue in subtilis extract yielded spherical international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable ceo2 nanops made from various plants speciesplant namepartcharacterizationshapesize nmftir groupoh ceoh hoh ocoleafxrd xps tem ftir uv vissphericalflower hrtem sem xrd xps eds ftirceohleafleafleafsem xrd ftir tgasphericalxrd sem tem uv vis ftir tgasphericaluvvis psa ftir xrd xps hrtemrtesphericalco oh hhoh ch nooh ceoseedxrd uvvis ftir fesem tgasphericalceo oceo ch co []acalypha indicaleafxrd sem tem eds ftiraloe barbadensisleafxrd tem ftir psasphericalspherical“ oh ceoh ceoce“ch co ch cf ch cclaloe veraleafftir xps hrtemspherical““ch “ccrubia cordifolialeafuv vis xrd xps sem ftir edaxhexagonalprosopis farctaaerialuv“vis pxrd tem fesem ftirsphericalchina rosepetalfesem fetem afm xrd xpsnanosheetcentella asiatica“uvvis dls sem hrtem edssphericaloh ceooh ceo and no__ walnutshellleafxrd sem tem eds ftiruvvis xrd xps fesem tem hrtem eds uvdrs ftirsphericalspherical“ceo and oh“ceoc oh chstempxrd sem tem ftir plflaky“oh coo and chs nogloriosa superbahibiscus sabdariffaoliveoleo europaeaprosopis jujliflorasalvia macrosiphon boissazadirachta indicaeuphorbia tirucallipetroselinum crispummoringa oleiferaref[][][][][][][][][][][][][][][][][][][][]lemon grassgrassxrd pl tem saed““leucas asperaleafpxrd sem uvvis tem saedmicrosphere“uvvis ir xrd sem temspherical“peeluv vis ftir xrd hrtemspherical watermelonfruit juicecarrageenanpxrd ftir uvvis semirregularpxrd ftir fesem uv“vis and tga dtaspherical___ceo ceoceceo co co ch oh“so3 ceo hoh c“o []continuedsubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem table continued plant namepartcharacterizationshapeleafxrd fesem tem ftirsphericalxrd fesem eds vsmspherical“size nmftir group__ceratonia siliquastevia rebaudianasalvadora persicamorus nigraannona muricatajusticia adhatodas nopxrd ftir uvvis tem fesem edsspherical“0h ch cc ceo co cfruitfruittem xrd uvvisxrd ftir uvdrs fesemirregular_ ___leafxrd sem tem ftir uvdrssticklike“oh ch co no cc co cncjatropha curcusleafxrd tem uvvismonodispersed ˆ’_origanum majoranapedalium murexelaeagnus angustifoliaeuphorbia amygdaloidesleaftem fesem xrd ftirsphericaloh ch coleafxrd ftir uvvis drs semnanorod“oh ch co co cn cn ceoleafxrd sem tem ftirspherical“ch cotem sem xrd uvvisspherical“orangepeelxrd tem ftir uvviscubic structure“coh or corpiper betleleafxrd ftir sem eds xps tem“nh no cnref[][][][][][][][][][][][][]nanomedicine but is yet to be explored particularly these microbial based nps can be used in designing novel fertilizers fabricating sterile surfaces polymers and medical accessories moreover these biogenic nps can also be exploited in disease management drug synthesis and deliverytable ceo2 nanops synthesized from various fungus speciess nomicrobe namecurvularia lunatacharacterizationshapesize nmftir grouptgdta xrd raman pl ftir uvvis and temspherical“_humicola spuvvis xps pls tem ftir xrdspherical“ceo ceocefusarium solaniftir pl tgdta fesem xrd edax tem xps saed clsmspherical“oh cn cocaspergillus nigeruvvis ftir xps xrd tgdta pl temspherical“hoh oco ce oref[][][][]international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepressgreen synthesis from biological productsapart from the synthesis of nanomaterial from eukaryotes and prokaryotes anisms nps have also synthesized from biological derivatives23 they also play a crucial role in the reduction and stabilization of nps2325 in contrast to plants and the microbial approach bioproduct based ceo2 are much safer scalable and have shown excellent biocompatibility4547 for instance egg white protein was used in order to synthesize ceo2 nps having size ranges from “ nm with spherical morphologies48 these nps were characterized and confirmed by uv™s ftir tgadta and pxrd ftir analysis revealed that the phenol ether hydroxyl and amide groups were responsible for the reduction of these nps it also showed a good in vitro cytotoxicity effect towards human periodontal fibroblasts cells agarose is a natural matrix and has been used as a stabilizing and capping agent for ceo2 nps nps obtained have spherical morphologies with a diameter of nm nps were characterized using various methods including uv fesem ftir tgadta pxrd and tgadta techniques as revealed by ftir analysis it was found that the hydroxyl ether phenol and amide groups were involved in biosynthesis45 starch has also been exploited as a novel source for the synthesis of nanoceria with the results revealing spherical shape nps with a diameter of nm12spherical shaped ceo2 nps with a size of “ nm were synthesized from dextran the resultant nps exhibited strong anticancer potential46 gum tragacanth reported by darroudi 49 was used in the biosynthesis of ceo2 nps these nps were monodispersed shape with an average size range from “ nm the ceo2nps exhibited very low cytotoxic effects on neuro2a cell lines making them suitable candidates for various biomedical and pharmacological applications49 some other biological products which have been used for synthesis of ceo2 nps are listed in table despite their biological applications these biogenic nps could be used as a promising candidate in diseases treatment drug delivery and packing food some other products have also been explored for the synthesis of ceo2 nps which are shown in table biological activity of greenmediated cerium oxide nanopsantimicrobial activity of greenmediated cerium oxide nanopsin the last few years nanotechnologybased therapies have been exploited in disease diagnostics treatment and table biomediated ceo2 nanops from different sources of biological productss nonamecharacterizationshapeegg proteinuvvis fesem ftir tgadta pxrdsphericalsize nm“honeyuvvis fesem ftir tgadta eds pxrdsphericalagaroseuvvis fesem ftir tgadta pxrdsphericalstarchdextranpolyethylene glycoluvvis pxrd temtem dls xps uvvishrtem tem uvvis slsdlschitosanxrd hrtem ftir tgadta uvvispectindls fesem edss xrd ftir nmr pl fesem edss uvvissphericalsphericalsphericalsphericalspherical“ ‰¤ ftir groupoh ceo ceoce ch nooh ceo ceoce ceoc nooh ceo ceoce no____oh ch co och3 coc no ceotannic acidftir xps xrd hrtem uvvisiblepolycrystalline _ref[][][][][][][][][]submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure schematic representation of antibacterial activity of ceo2 nanops cell wall disruption cell membrane disintegration free radicals productions loss of protein peptides dna fragmentation vital enzymes inhibition loss of cellular fluids and disruption in electron transportformulations of novel drugs1 for instance the antimicrobial potential of nps has been mostly exploited and has showed substantial outcomes2324 presently ceo2 nps have attracted great interest as an antimicrobial agent in particular against bacterial pathogens151650 the exact mechanism of killing microbes is yet not clearly elucidated however it is proposed that ceo2 nps mostly kill microbes via a massive production of reactive oxygen species ros in cells as shown in figure the bactericidal potential of ceo2 nps is attributed to strong electrostatic properties distinctive morphologies small size and low band energy1652 due to strong electrostatic potential ceo2 nps interact with membrane proteins thiols groups which results in protein denaturation membrane impermeability eventually leads to microbial death4253 figure exposure to ceo2 nps kills microbes via membrane collapse by attachment with mesosomes malfunctioning of cellular compartments and bio anic molecules which ultimately lead to abnormal metabolism and physiology1642 similarly green mediated nps killpathogens in a similar fashion and various biological species have been exploited and tested against a wide variety of microbes1316 table however biogenic ceo2 nps unique morphologies small size and biocompatible nature were found to be more effcient and have the potential to range of pathogenic bacterial species1113151642 moreover it also has the potential to kill both grampositive and gramnegative bacteria but due to structural complexity of gramnegative bacteria™s membranes it is more sensitive against grampositive species1321374754 in antimicrobial in electrostatics between nps and the bacterial wall plant species and wall compositiondifference to differences treat a wide activity the is due international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable various microbes tested against biogenic ceo2 nanopss nosourcemicrobes testedolea europaeafungus aspergillus flavus fusarium solani and aspergillus niger bacterial sps staphylococcus aureus escherichia coli pseudomonas aeruginosa and klebsiella pneumoniamoringa oleiferas aureus and e colicurvularia lunatastaphylococcus aureus streptococcus pneumoniae and bacillus subtilis pseudomonas aeruginosa proteus vulgaris and klebsiella pneumoniaeleucas asperaklebsiella aerogenes escherichia coli pseudomonas desmolyticum and staphylococcus aureusacalypha indicaescherichia coli and staphylococcus aureusannona muricataenterococcusfaecalis staphylococcus aureus klebsiella pneumonia and escherichia coligloriosa superbastaphylococcus aureus and streptococcus pneumonia ecoli proteus vulgaris klebsiella pneumonia shigella dysenteriae and pseudomonas aeruginosaaspergillus nigerstreptococcus pneumoniae bacillus subtilis proteus vulgaris and escherichia colifusarium solanistaphylococcus aureus pseudomonas aeruginosa escherichia coli and klebsiella pneumoniajusticia adhatodastaphylococcus aureus and escherichia colieuphorbia amygdaloidesp acidilacticipectine coli and b subtilisref[][][][][][][][][][][][]resistance to confer due to rapid evolution of the bacterial genome bacteria have evolved to antimicrobial agents5556 thus in quest of a new treatment biogenic ceo2 nps has shown promising results in treating multi drug resistance bacteria and could be a promising candidate against such refractory pathogenesis26 ceo2 nps along other conjugates have been amalgamated with various anic and inanic hybrids to enhance the antimicrobial response1746 similarly biomediated ceo2 nps kills fungi by producing a mass number of free radicals and ros which causes distorted structure and physiology and leads to fungus death16 however a few studies have only been conducted on fungi despite the increasing knowledge on the antimicrobial activity of ceo2 nps much remains unknown about their exact mechanism of encountering bacteria toxicity in vivo studies and environmental concerns which needs to be addressed moreover the low band energy potential of ceo2 nps could be used in fabricating sterile surfaces at hospital or lab settings and will diminish nosocomial and other acquired infectionsother potential biomedical applicationsbeside antimicrobial therapies ceo2 nps have also been used in management of other ailments3557 for instance siliqua showed high biogenic ceo2 nps have been mostly used in treatment of various cancers such as osteosarcoma colon cervical and breast cancers1820274652 results indicated that these nps exhibited strong anticancer potential and could be used as a chemotherapeutic agent thanks to their minimal toxicity capacity to induce apoptosis andor necrosis in cancer cells46 ceo2 nps synthesized from origanum majorana and ceratonia antioxidant activity183157 results showed higher expression of antioxidant enzymes which in turn eradicated free radicals and improved cellular functions31 furthermore antioxidant potential was higher when compared to commercial synthetic antioxidants18 ceo2 nps synthesized from fruit extract of morus nigra exhibited excellent antidiabetic activity on l6 cell lines the treatment was dosage dependent and nps with lesser size resulted in higher uptake of glucose in vitro35 though biogenic ceo2 nps have shown excellent pharmacological potential however the mechanism of action minimum inhibitory concentration and best possible delivery system should need to be determined moreover cytotoxicity and genotoxicity should be tested in vivo models to evaluate the compatibility in both in vivo and in vitro modelssubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem to their unique conclusions and future prospectsin this paper we have reviewed the recent trends and understandings of biogenic ceo2 nps and their pharmacological applications various sources such as plants microbes and other biological products have been discussed with the mechanism of synthesis and their biomedical applications due surface morphologies crystal small nature and biocompatible nature biogenic ceo2 nps have got phenomenal interest in biomedical and other fields for instance it has been used in treating various cancers antimicrobial and antioxidant therapies in particular the green synthesized nanops have shown significant antimicrobial potential against a wide range of bacterial species the mechanism of combating such pathogens have also been elucidated and supposed to be due to the mass production of reactive oxygen species and deactivation of scavenging enzymes the ros impedes the membranes disrupts the cellular compartments and disintegrates the bio anic molecules and hampers the associated functions and ultimately causes death it has also shown promising results against multidrug bacteria and could be a potential antimicrobial agent in future against such refractory pathogens however further studies should conduct in vivo models to reveal the full mechanism alongside any sideeffects moreover it has also shown excellent anticancer and antioxidant potential in vitro setups but their toxicity and dosage are yet unknown which needs to be addressed despite their role in various therapies their mechanism of synthesis needs to be optimized whereas in vivo evaluation as well as toxicity should be further screenedabbreviationsceo2 nps cerium oxide nanops dga differential thermal analysis dsl dynamic light scattering eds energydispersive xray spectroscopy fesem field emission scanning electron microscopy ftir fourier transform infrared spectroscopy hrtem highresolution transmission electron microscopy pl photoluminescence pxrd powder xray diffraction ros reactive oxygen species sls static light scattering tem transmission electron microscopy tga thermal gravimetric analysis uvvis uvvisible spectroscopy xps xray photoelectron spectrometry xrd xray diffractiondisclosurethe authors report no conflicts of interest for this workreferences kubik t boguniakubik k sugisaka m nanotechnology on duty in medical applications curr pharm biotechnol “ doi1021741389201053167248 bhushan b springer handbook of nanotechnology springer jianrong c yuqing m nongyue h et al nanotechnology and biosensors biotechnol adv “ doi101016j biotechadv200403004 smith dm simon jk baker jr jr jr applications of nanotechnology for immunology nat rev immunol “ doi101038nri3488 mohanraj v chen y nanopsa review trop j pharm res “ das s dowding jm klump ke cerium oxide nanops applications and prospects in nanomedicine nanomedicine “ doi102217nnm13133 he l su y lanhong j et al recent advances of cerium oxide nanops in synthesis luminescence and biomedical studies a review j rare earths “ doi101016s1002 walkey c das s seal s catalytic properties and biomedical applications of cerium oxide nanops environ sci “ doi101039c4en00138a rajeshkumar s naik p synthesis and biomedical applications of cerium oxide nanops“a review biotechnol rep “ doi101016jbtre201711008 magudieshwaran r ishii j raja kcn et al green and chemical synthesized ceo2 nanops for photocatalytic indoor air pollutant degradation mater lett “ doi101016jmatlet arunachalam t karpagasundaram m rajarathinam n ultrasound assisted green synthesis of cerium oxide nanops using prosopis juliflora leaf e

cancer7507

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: during the last decade green synthesized cerium oxide nanops ceo2 nps attracted remarkable interest in various fields of science and technology this review explores the vast array of biological resources such as plants microbes and other biological products being used in synthesis of ceo2 nps it also discusses their biosynthetic mechanism current understandings and trends in the green synthesis of ceo2 nps novel therapies based on green synthesized ceo2 nps are illustrated in particular their antimicrobial potential along with attempts of their mechanistic elucidation overall the main objective of this review is to provide a rational insight of the major accomplishments of ceo2 nps as novel therapeutics agents for a wide range of microbial pathogens and combating other diseases keywords nanotechnology green synthesis cerium oxide nanops antimicrobial infections biomedicalintroductionnanotechnology has got a remarkable interest in every field of science and technology and is presently considered among one of the leading research avenues it has a multitude of applications in the field of electronics imaging industry and healthcare14 mostly in healthcare it has been exploited in diseases diagnostics treatment delivery and formulations of novel drugs14 it exploits nano size structures with size ranges from “ nm known as nanop nps these nano scale entities have unique physiochemical properties and have been utilized in various fields of physics biology and chemistry5among other nps cerium oxide ceo2 nps have been mostly exploited due to their unique surface chemistry high stability and biocompatibility67 it is mostly used in the fabrication of sensors cells catalysis therapeutics agents drug delivery careers and antiparasitic ointments figure presently ceo2 nps is mostly synthesized via two methods such as physical and chemical79 however these methods utilize toxic reducing solvents posing several threats to the biodiversity and ecosystem moreover the nps obtained with such approaches are toxic and unstable making them less efficient910 thus recently a safe less toxic method has been used by researchers known as green synthesis this method utilizes various biological resources such as plants microbes or any other biological derivative1115 these biological extracts have a rich source of phytochemicals international of nanomedicine “ nadeem this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomterms php and incorporate the creative commons attribution “ non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cnadeem dovepresslatter is extensively utilized for its biomedical pharmacological and food applications due to their safe and biocompatible nature9 moreover features like high yield everlasting stability and better morphologies can be obtained using a greener approach79green synthesis from plantsgreen syntheses of ceo2 nps have been reported using plant extracts microbial and other biological derivatives plants in this regard have been the most efficient source due to their abundance safe nature and rich source of reducing and stabilizing agents2629 various parts of plants such as leaves flower and stem have been used for the synthesis of ceo2 nps163031 till date the majority of green synthesis studies have been conducted on leaves extracts as it is a rich source of metabolites11163233 a broad variety of metabolitesphytochemicals in plant extracts such as ketones carboxylic acids phenols and ascorbic acid are used as reduction and stabilizing agents figure plants based ceo2 nps are produced through a simple approach in which bulk metal salt is mixed with the extract and the reaction completes in minutes to a few hours in ordinary lab conditions282934 the metallic salt solution is reduced into respective nanops via the phytochemicals whose synthesis is confirmed firstly through color change from colorless to yellowish brownish or whitish and then characterized through various spectroscopic and imaging techniques162935leaf extract of moringa oleifera l was used to synthesize ceo2 nps with spherical morphologies and size of nm the synthesized nps showed potential antimicrobial and wound healing properties36 gloriosa superba leaf extract was used as a reducing and stabilizing agent in synthesis of ceo2 nps and has shown potential antibacterial properties37 hibiscus sabdariffa natural extract yielded crystalline ceo2 with a diameter of nm30 spherical shaped nanops of size nm synthesized from gel extract of medicinally important plant aloe barbadensis38 the resultant ceo2 nps showed high antioxidant potential green synthesis of ceo2 nanops was demonstrated using jatropha curcus leaf extract and a monodispersed shape of “ nm10 spherical shaped cerium oxide nanops are synthesized using leaf extract of oleo europaea with a size of nm having high antimicrobial activity against both gramnegative and positive strains of bacteria16 origanum majorana extracts were used to synthesize ceo2 nps having pseudo photocatalytic activity having high figure general applications of ceo2 nanopssuch asketones amines enzymes and phenols which are believed to be responsible for the reduction and stabilization of bulk salts into respective nanops nps1619to date various applications of green synthesized ceo2 nps have been reported such as antimicrobial anti cancer antilarvicidal photocatalysis and antioxidant therapies162022 among other biomedical applications the antimicrobial potential is certainly the most exploited previously it has been reported that ceo2 nps to display their antimicrobial actions through various mechanisms9 but mostly ceo2 nps kill microbes via triggering the production of an excess of reactive oxygen species in cells916 however further studies need to be conducted to fully elucidate the complete mechanism of action here in this review we aim to focus on the following topics arrays of biological resources have been exploited to date for the synthesis of ceo2 nps moreover the synthesis mechanisms along their biomedical applications are discussed with special emphasis on the antimicrobial activitysynthesis of ceo2 npsnanops are synthesized through various physicochemical methods5 however both methods require toxic solvents high temperature and pressure which pose threats to the environment2325 moreover higher cost laborious downstream processing lesser biocompatibility instability and low yield make them further inefficient710 there is a growing need to fabricate nanostructures which have the potential to solve these problems59 presently researchers have exploited the green method to overcome all these challenges5 for instance plants microbes and other biological products have been used as reducing and or stabilizing agents in the fabrication of ecofriendly nps25 ceo2 nps have also been synthesized using various physical chemical and biological methods9 the submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure biosynthesis reduction stabilization and characterization of ceo2 nanopsspherical shape nm ftir confirmed that the reduction is attributed to the presence of different phenolic and flavonoids compounds in the extract18 ceo2 was synthesized using rubia cordifolia leaf fusions spectroscopic and microscopic analysis revealed hexagonal shaped nps having a size of nm the biogenic ceo2 nps also showed excellent anticancer potential33 nano rod size ranges from “ nm ceo2 nps resulted when pedalium murex l was added to the aqueous solution of salt at room temperature having high antibacterial activity32 china rose petal was used as a robust bio template for the facile fabrication of novel ceria nano sheet with a size of about nm39 the deviation in size and morphology noticed among the reported studies might be due to the different influence of reaction temperature ph time concentration of salt precursor or plants extracts and part of the plant being used13272840 moreover plants based ceo2 np™s showed excellent stability at diverse conditions for instance green mediated ceria np™s remain stable at liquid solution changes were observed1336 similarly biogenic ceo2 np™s also showed high thermal stability at high temperature and remained stable for a longer period of time which indicates their long durability and everlasting stability27283341 until now various plants have been used in the biogenic synthesis of ceo2 nps and are shown in table physiochemical and no green synthesis from microbesmicrobes also have an intrinsic potential to synthesize nanops as they are a rich source of secondary metabolites23 among other nanops ceo2 nps with various shapes and sizes have been synthesized in recent years from microbes table green synthesis of ceo2 from microbial species is a simple reliable costeffective and ecofriendly approach42 microbial metabolites such as enzymes proteins and heterocyclic derivatives play a crucial role in reducing and stabilizing of ceo2 bulk salt into respective nps4243 moreover microbiogenic ceo2 nps exhibited improved stability water dispensability and showed high fluorescent properties and were less agglomerated43aspergillus niger extract yielded cubic fluorite nps with spherical morphology and an average size of nm ftir analysis revealed the presence of an hydroxyl group carboxylic group and phenol group which are supposed to be involved in the reduction of nps21 curvularia lunata extract has also been used to synthesize spherical shaped ceo2 nps with size ranges from “ nm color change from white to yellow brown indicated initial reaction the nps were tested against microbial pathogens and showed excellent antibacterial potential spherical shaped ceo2 nps of size ranges from “ nm were made using fusarium solani extract which showed effective growth inhibition and biofilm formation of pathogenic bacterial strains42 shadab ali khan observed the biosynthesis of spherical shaped “ nm ceo2 nps by using a thermophilic fungus humicola capping agent43 the resultant nps were characterized by uv xps pl spectroscopy tem ftir and xrd moreover these nps showed excellent potential in treatment of neurodegenerative disorders such as alzheimer™s and parkinson™s diseases bacterial extract has also been exploited in the fabrication of ceo2 nps for instance bacillus shaped nps with an average size of nm the bacterial mediated nps also showed excellent antioxidant potential in vitro44 despite all these applications the microbial route of synthesis has certain shortcomings such as the high probability of pathogenicity laborious culturing and contamination issues however it offers a lot of promise in the field of nanotechnology and could become a leading avenue in subtilis extract yielded spherical international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable ceo2 nanops made from various plants speciesplant namepartcharacterizationshapesize nmftir groupoh ceoh hoh ocoleafxrd xps tem ftir uv vissphericalflower hrtem sem xrd xps eds ftirceohleafleafleafsem xrd ftir tgasphericalxrd sem tem uv vis ftir tgasphericaluvvis psa ftir xrd xps hrtemrtesphericalco oh hhoh ch nooh ceoseedxrd uvvis ftir fesem tgasphericalceo oceo ch co []acalypha indicaleafxrd sem tem eds ftiraloe barbadensisleafxrd tem ftir psasphericalspherical“ oh ceoh ceoce“ch co ch cf ch cclaloe veraleafftir xps hrtemspherical““ch “ccrubia cordifolialeafuv vis xrd xps sem ftir edaxhexagonalprosopis farctaaerialuv“vis pxrd tem fesem ftirsphericalchina rosepetalfesem fetem afm xrd xpsnanosheetcentella asiatica“uvvis dls sem hrtem edssphericaloh ceooh ceo and no__ walnutshellleafxrd sem tem eds ftiruvvis xrd xps fesem tem hrtem eds uvdrs ftirsphericalspherical“ceo and oh“ceoc oh chstempxrd sem tem ftir plflaky“oh coo and chs nogloriosa superbahibiscus sabdariffaoliveoleo europaeaprosopis jujliflorasalvia macrosiphon boissazadirachta indicaeuphorbia tirucallipetroselinum crispummoringa oleiferaref[][][][][][][][][][][][][][][][][][][][]lemon grassgrassxrd pl tem saed““leucas asperaleafpxrd sem uvvis tem saedmicrosphere“uvvis ir xrd sem temspherical“peeluv vis ftir xrd hrtemspherical watermelonfruit juicecarrageenanpxrd ftir uvvis semirregularpxrd ftir fesem uv“vis and tga dtaspherical___ceo ceoceceo co co ch oh“so3 ceo hoh c“o []continuedsubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem table continued plant namepartcharacterizationshapeleafxrd fesem tem ftirsphericalxrd fesem eds vsmspherical“size nmftir group__ceratonia siliquastevia rebaudianasalvadora persicamorus nigraannona muricatajusticia adhatodas nopxrd ftir uvvis tem fesem edsspherical“0h ch cc ceo co cfruitfruittem xrd uvvisxrd ftir uvdrs fesemirregular_ ___leafxrd sem tem ftir uvdrssticklike“oh ch co no cc co cncjatropha curcusleafxrd tem uvvismonodispersed ˆ’_origanum majoranapedalium murexelaeagnus angustifoliaeuphorbia amygdaloidesleaftem fesem xrd ftirsphericaloh ch coleafxrd ftir uvvis drs semnanorod“oh ch co co cn cn ceoleafxrd sem tem ftirspherical“ch cotem sem xrd uvvisspherical“orangepeelxrd tem ftir uvviscubic structure“coh or corpiper betleleafxrd ftir sem eds xps tem“nh no cnref[][][][][][][][][][][][][]nanomedicine but is yet to be explored particularly these microbial based nps can be used in designing novel fertilizers fabricating sterile surfaces polymers and medical accessories moreover these biogenic nps can also be exploited in disease management drug synthesis and deliverytable ceo2 nanops synthesized from various fungus speciess nomicrobe namecurvularia lunatacharacterizationshapesize nmftir grouptgdta xrd raman pl ftir uvvis and temspherical“_humicola spuvvis xps pls tem ftir xrdspherical“ceo ceocefusarium solaniftir pl tgdta fesem xrd edax tem xps saed clsmspherical“oh cn cocaspergillus nigeruvvis ftir xps xrd tgdta pl temspherical“hoh oco ce oref[][][][]international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepressgreen synthesis from biological productsapart from the synthesis of nanomaterial from eukaryotes and prokaryotes anisms nps have also synthesized from biological derivatives23 they also play a crucial role in the reduction and stabilization of nps2325 in contrast to plants and the microbial approach bioproduct based ceo2 are much safer scalable and have shown excellent biocompatibility4547 for instance egg white protein was used in order to synthesize ceo2 nps having size ranges from “ nm with spherical morphologies48 these nps were characterized and confirmed by uv™s ftir tgadta and pxrd ftir analysis revealed that the phenol ether hydroxyl and amide groups were responsible for the reduction of these nps it also showed a good in vitro cytotoxicity effect towards human periodontal fibroblasts cells agarose is a natural matrix and has been used as a stabilizing and capping agent for ceo2 nps nps obtained have spherical morphologies with a diameter of nm nps were characterized using various methods including uv fesem ftir tgadta pxrd and tgadta techniques as revealed by ftir analysis it was found that the hydroxyl ether phenol and amide groups were involved in biosynthesis45 starch has also been exploited as a novel source for the synthesis of nanoceria with the results revealing spherical shape nps with a diameter of nm12spherical shaped ceo2 nps with a size of “ nm were synthesized from dextran the resultant nps exhibited strong anticancer potential46 gum tragacanth reported by darroudi 49 was used in the biosynthesis of ceo2 nps these nps were monodispersed shape with an average size range from “ nm the ceo2nps exhibited very low cytotoxic effects on neuro2a cell lines making them suitable candidates for various biomedical and pharmacological applications49 some other biological products which have been used for synthesis of ceo2 nps are listed in table despite their biological applications these biogenic nps could be used as a promising candidate in diseases treatment drug delivery and packing food some other products have also been explored for the synthesis of ceo2 nps which are shown in table biological activity of greenmediated cerium oxide nanopsantimicrobial activity of greenmediated cerium oxide nanopsin the last few years nanotechnologybased therapies have been exploited in disease diagnostics treatment and table biomediated ceo2 nanops from different sources of biological productss nonamecharacterizationshapeegg proteinuvvis fesem ftir tgadta pxrdsphericalsize nm“honeyuvvis fesem ftir tgadta eds pxrdsphericalagaroseuvvis fesem ftir tgadta pxrdsphericalstarchdextranpolyethylene glycoluvvis pxrd temtem dls xps uvvishrtem tem uvvis slsdlschitosanxrd hrtem ftir tgadta uvvispectindls fesem edss xrd ftir nmr pl fesem edss uvvissphericalsphericalsphericalsphericalspherical“ ‰¤ ftir groupoh ceo ceoce ch nooh ceo ceoce ceoc nooh ceo ceoce no____oh ch co och3 coc no ceotannic acidftir xps xrd hrtem uvvisiblepolycrystalline _ref[][][][][][][][][]submit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem figure schematic representation of antibacterial activity of ceo2 nanops cell wall disruption cell membrane disintegration free radicals productions loss of protein peptides dna fragmentation vital enzymes inhibition loss of cellular fluids and disruption in electron transportformulations of novel drugs1 for instance the antimicrobial potential of nps has been mostly exploited and has showed substantial outcomes2324 presently ceo2 nps have attracted great interest as an antimicrobial agent in particular against bacterial pathogens151650 the exact mechanism of killing microbes is yet not clearly elucidated however it is proposed that ceo2 nps mostly kill microbes via a massive production of reactive oxygen species ros in cells as shown in figure the bactericidal potential of ceo2 nps is attributed to strong electrostatic properties distinctive morphologies small size and low band energy1652 due to strong electrostatic potential ceo2 nps interact with membrane proteins thiols groups which results in protein denaturation membrane impermeability eventually leads to microbial death4253 figure exposure to ceo2 nps kills microbes via membrane collapse by attachment with mesosomes malfunctioning of cellular compartments and bio anic molecules which ultimately lead to abnormal metabolism and physiology1642 similarly green mediated nps killpathogens in a similar fashion and various biological species have been exploited and tested against a wide variety of microbes1316 table however biogenic ceo2 nps unique morphologies small size and biocompatible nature were found to be more effcient and have the potential to range of pathogenic bacterial species1113151642 moreover it also has the potential to kill both grampositive and gramnegative bacteria but due to structural complexity of gramnegative bacteria™s membranes it is more sensitive against grampositive species1321374754 in antimicrobial in electrostatics between nps and the bacterial wall plant species and wall compositiondifference to differences treat a wide activity the is due international of nanomedicine submit your manuscript wwwdovepresscom dovepress 0cnadeem dovepresstable various microbes tested against biogenic ceo2 nanopss nosourcemicrobes testedolea europaeafungus aspergillus flavus fusarium solani and aspergillus niger bacterial sps staphylococcus aureus escherichia coli pseudomonas aeruginosa and klebsiella pneumoniamoringa oleiferas aureus and e colicurvularia lunatastaphylococcus aureus streptococcus pneumoniae and bacillus subtilis pseudomonas aeruginosa proteus vulgaris and klebsiella pneumoniaeleucas asperaklebsiella aerogenes escherichia coli pseudomonas desmolyticum and staphylococcus aureusacalypha indicaescherichia coli and staphylococcus aureusannona muricataenterococcusfaecalis staphylococcus aureus klebsiella pneumonia and escherichia coligloriosa superbastaphylococcus aureus and streptococcus pneumonia ecoli proteus vulgaris klebsiella pneumonia shigella dysenteriae and pseudomonas aeruginosaaspergillus nigerstreptococcus pneumoniae bacillus subtilis proteus vulgaris and escherichia colifusarium solanistaphylococcus aureus pseudomonas aeruginosa escherichia coli and klebsiella pneumoniajusticia adhatodastaphylococcus aureus and escherichia colieuphorbia amygdaloidesp acidilacticipectine coli and b subtilisref[][][][][][][][][][][][]resistance to confer due to rapid evolution of the bacterial genome bacteria have evolved to antimicrobial agents5556 thus in quest of a new treatment biogenic ceo2 nps has shown promising results in treating multi drug resistance bacteria and could be a promising candidate against such refractory pathogenesis26 ceo2 nps along other conjugates have been amalgamated with various anic and inanic hybrids to enhance the antimicrobial response1746 similarly biomediated ceo2 nps kills fungi by producing a mass number of free radicals and ros which causes distorted structure and physiology and leads to fungus death16 however a few studies have only been conducted on fungi despite the increasing knowledge on the antimicrobial activity of ceo2 nps much remains unknown about their exact mechanism of encountering bacteria toxicity in vivo studies and environmental concerns which needs to be addressed moreover the low band energy potential of ceo2 nps could be used in fabricating sterile surfaces at hospital or lab settings and will diminish nosocomial and other acquired infectionsother potential biomedical applicationsbeside antimicrobial therapies ceo2 nps have also been used in management of other ailments3557 for instance siliqua showed high biogenic ceo2 nps have been mostly used in treatment of various cancers such as osteosarcoma colon cervical and breast cancers1820274652 results indicated that these nps exhibited strong anticancer potential and could be used as a chemotherapeutic agent thanks to their minimal toxicity capacity to induce apoptosis andor necrosis in cancer cells46 ceo2 nps synthesized from origanum majorana and ceratonia antioxidant activity183157 results showed higher expression of antioxidant enzymes which in turn eradicated free radicals and improved cellular functions31 furthermore antioxidant potential was higher when compared to commercial synthetic antioxidants18 ceo2 nps synthesized from fruit extract of morus nigra exhibited excellent antidiabetic activity on l6 cell lines the treatment was dosage dependent and nps with lesser size resulted in higher uptake of glucose in vitro35 though biogenic ceo2 nps have shown excellent pharmacological potential however the mechanism of action minimum inhibitory concentration and best possible delivery system should need to be determined moreover cytotoxicity and genotoxicity should be tested in vivo models to evaluate the compatibility in both in vivo and in vitro modelssubmit your manuscript wwwdovepresscom dovepress international of nanomedicine 0cdovepress nadeem to their unique conclusions and future prospectsin this paper we have reviewed the recent trends and understandings of biogenic ceo2 nps and their pharmacological applications various sources such as plants microbes and other biological products have been discussed with the mechanism of synthesis and their biomedical applications due surface morphologies crystal small nature and biocompatible nature biogenic ceo2 nps have got phenomenal interest in biomedical and other fields for instance it has been used in treating various cancers antimicrobial and antioxidant therapies in particular the green synthesized nanops have shown significant antimicrobial potential against a wide range of bacterial species the mechanism of combating such pathogens have also been elucidated and supposed to be due to the mass production of reactive oxygen species and deactivation of scavenging enzymes the ros impedes the membranes disrupts the cellular compartments and disintegrates the bio anic molecules and hampers the associated functions and ultimately causes death it has also shown promising results against multidrug bacteria and could be a potential antimicrobial agent in future against such refractory pathogens however further studies should conduct in vivo models to reveal the full mechanism alongside any sideeffects moreover it has also shown excellent anticancer and antioxidant potential in vitro setups but their toxicity and dosage are yet unknown which needs to be addressed despite their role in various therapies their mechanism of synthesis needs to be optimized whereas in vivo evaluation as well as toxicity should be further screenedabbreviationsceo2 nps cerium oxide nanops dga differential thermal analysis dsl dynamic light scattering eds energydispersive xray spectroscopy fesem field emission scanning electron microscopy ftir fourier transform infrared spectroscopy hrtem highresolution transmission electron microscopy pl photoluminescence pxrd powder xray diffraction ros reactive oxygen species sls static light scattering tem transmission electron microscopy tga thermal gravimetric analysis uvvis uvvisible spectroscopy xps xray photoelectron spectrometry xrd xray diffractiondisclosurethe authors report no conflicts of interest for this workreferences kubik t boguniakubik k sugisaka m nanotechnology on duty in medical applications curr pharm biotechnol “ doi1021741389201053167248 bhushan b springer handbook of nanotechnology springer jianrong c yuqing m nongyue h et al nanotechnology and biosensors biotechnol adv “ doi101016j biotechadv200403004 smith dm simon jk baker jr jr jr applications of nanotechnology for immunology nat rev immunol “ doi101038nri3488 mohanraj v chen y nanopsa review trop j pharm res “ das s dowding jm klump ke cerium oxide nanops applications and prospects in nanomedicine nanomedicine “ doi102217nnm13133 he l su y lanhong j et al recent advances of cerium oxide nanops in synthesis luminescence and biomedical studies a review j rare earths “ doi101016s1002 walkey c das s seal s catalytic properties and biomedical applications of cerium oxide nanops environ sci “ doi101039c4en00138a rajeshkumar s naik p synthesis and biomedical applications of cerium oxide nanops“a review biotechnol rep “ doi101016jbtre201711008 magudieshwaran r ishii j raja kcn et al green and chemical synthesized ceo2 nanops for photocatalytic indoor air pollutant degradation mater lett “ doi101016jmatlet arunachalam t karpagasundaram m rajarathinam n ultrasound assisted green synthesis of cerium oxide nanops using prosopis juliflora leaf e Answer:

Colon_Cancer

colorectal carcinoma crc as one of the most commongastrointestinal malignancies has developed the world™sfourth most deadly cancer with a high rate of incidence andmortality [ ] liver metastasis which is the most commonform for crc metastasis is the leading cause of high mortality for this severe malignancy however few clinical prevention and treatment measures could be available for tumormetastasis therefore it is really urgent to develop new biomarkers and explore the underlying mechanism for crcmetastasis and eventually develop new therapeutic strategiesfor crc patientsduring cancer progression metabolic reprograming withincreasing glucose utilization is termed as warburg aï¬ectwhich is accompanied by altered pyruvate and mitochondrialmetabolism the fate of pyruvate is the core manifestationto distinguish normal cells and tumor cells through metabolism in normal cells pyruvate was used for efficient atpproduction directly into mitochondria however pyruvatewas converted into lactate in cytosol despite of normoxicand hypoxic conditions in cancer cells this may be dueto the impaired process of pyruvate from the cytosol intothe mitochondrial matrix which is a critical metabolic steplinking glycolysis and mitochondrial oxidative phosphorylation the mitochondrial pyruvate carriermpc a 0c of immunology researchmultimeric complex containing two distinct proteins mpc1and mpc2 which is located in the inner mitochondrialmembrane is responsible for efficient mitochondrial pyruvate uptake loss of mpc expression or activity blockspyruvate entry into the tca cycle which results in a metabolism switch to increase glycolysis and the compensatoryusage of glutamine [ ]existed studies have reported that mpc1 was related withimmunoregulation stemness metabolism cellular morphology etc [ “] currently the important role of mpc1was uncovered in several tumors in crc and esophagealsquamous cell carcinoma decreased mpc1 results in accelerated aerobic glycolysis and malignant progression [ ] inlung adenocarcinoma mpc1 deficiency accelerates lung adenocarcinoma progression through the stat3 pathway in prostate cancer mpc1 was reported to be involved instemness and metabolism which regulated by couptfii[ ] in renal cell carcinoma hypoxiainduced loss ofmpc1 enhanced the expression of mmp7 and mmp9 to promote cell invasion collectively these data suggestedthat mpc1 maybe serves as a suppressor to disrupt tumormalignancy however whether mpc1 is involved in crcmetastasis and the underlying mechanisms remain to beillustratedin the present study we figured out the relationshipbetween the mpc1 expression and crc liver metastasiswe identified that decreased mpc1 was closely correlatedwith patient™s metastasis as well as led to poor prognosisfunctionally mpc1 overexpression could attenuate themigration and invasion capacities of crc cells both in vitroand in vivo mechanically mpc1 suppressed crc metastasisthrough mediating the wntcatenin signaling thus ourfinding firstly revealed a critical role of mpc1 in crc livermetastasis materials and methods data mining seven geo datasets gse21510 gse5206gse20916 gse9348 gse89393 gse67675 and gse4183and tcga were used to analyze the mpc1 expression pattern in crc the primary data for tcga datasets weredownloaded wwwcancergov the primary data feo datasets were downloaded at wwwncbinlmnihgovgeo the oncolnc database httpwwwoncolnc was used to detect the prognostic value of mpc1 inthe tcga cohort patients and clinical specimens in our study a total of patients containing paired crc tissues and adjacentnontumor tissues were enrolled from the department of gastrointestinal surgery renji hospital school of medicineshanghai jiao tong university among them cases ofliver metastases were collected and only cases wereavailable with complete followup data for survival analysisinformed consents were signed by all patients the researchwas approved by the research ethics committee of renjihospital and carried out in accordance with ethical standardsas formulated in the helsinki declarationtable sequences of primers used for realtime pcrprimermmp7 forwardmmp7 reverseecadherin forwardecadherin reversesnail1 forwardsnail1 reversemyc forwardmyc reversegapdh forwardgapdh reversesequence ²²gagtgagctacagtgggaacactatgacgcgggagtttaacatatgagtgtcccccggtatcttcacgagcagagaatcataaggcggtatccagagctgtttggaaacattttcctcccaggccatcacagccctcactcacacagattccacaaggtgcctgggctacactgagcaccaagtggtcgttgagggcaatg cell culture and cell transduction human crc celllines hct116 ht29 sw620 rko sw480 and lovoand mouse crc cell line mc38 were gained from the cellbank of the chinese academy of sciences shanghai chinaall cells were cultured in dmem medium supplemented°with fetal bovine serum and antibiotics at c in ahumidified incubator with co2mc38 cells were transfected with lentivirus containing aluciferase reporter plasmid stable transfection cells werescreened with μgml blastisidin for days which termedmc38luc in addition one short hairpin rna shrnasequence against mpc1 was packaged as lentivirus andtransfected into mc38luc cells lovo and sw480 were transfected with lentivirus containing fulllength human mpc1cdna or empty vehicle control stable transfection cells werescreened under μgml puromycin for days and verifiedby western blot in those assays all lentiviral transfectionswere performed in the presence of μgml polybrene immunohistochemical ihc the protocol of this assayand quantify the mpc1 protein expression level were performed according to previously reported primary antibodies used as follows mmp7 yt2663 immunoway ecadherin cst snail1 ab53519 abcam and mycyp0861 immunoway cellular immunofluorescence assays were performedaccording to the previous description briefly cells wereincubated with antibodies against catenin ab32572abcam and incubated with alexa 488conjugated secondaryantibody the nuclei were stained with ²6diamidino2phenylindole dapi western blotting wholecell lysates or nuclear proteinwas extracted using a protein extraction buï¬er beyotimeshanghai china or nucleoprotein extraction kit sangonbiotech c500009 respectively proteins were resolved bysdspage and transferred onto nitrocellulose nc membranes using standard methods primary antibodies used asfollows mpc1 ab74871 abcam catenin ab32572abcamlamin ac ab8984 abcam and gapdhab9485 abcam speciesspecific secondary antibodies used 0c of immunology researchtable related enzyme and carrier of pyruvate analyzed in this studygenepdha1pdhbpdk4pdhxmpc2pdk2pdk1pdk3mpc1pcpdp1pdprpdha2pdp2pklrdescriptionpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase lipoamide betapyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase complex component xmitochondrial pyruvate carrier pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme mitochondrial pyruvate carrier pyruvate carboxylasepyruvate dehydrogenase phosphatase catalytic subunit pyruvate dehydrogenase phosphatase regulatory subunitpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase phosphatase catalytic subunit pyruvate kinase liver and rbclocationmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrionas follows irdye goat antimouse igg licor andirdye goat antirabbit igg licorttest was used for comparison between groups p wasconsidered as statistically significantsynthesized using the primescript realtime pcr total rna was extracted using trizoltakara according to the manufacturer™sinstructionscdna wasreversetranscriptionpolymerasechain reaction rtpcr kittakara the qpcr was performed using sybr green masˆ’–ct method was used to analyze theter mix roche the data and gapdh was used as a loading control the primersequences are listed in table liver metastasis model this study was performed inaccordance with the recommendations in the guide for thecare and use of laboratory animals and relevant chineselaws and regulations the protocol was approved by theinstitutional animal care and use committee iacucof shanghai jiao tong university the mc38 cells transplanting luciferaseexpressing were injected into the spleensof c57bl6n mice n with a concentration of cellsmouse two weeks later the mice were killed andthe liver metastasis tissues were harvested luciferase reporter assay the protocols of this assaywere operated in accordance with previous reported ng top reporter plasmid wntcatenin signalingor ng fop reporter plasmid negative control ofwntcatenin signaling and ng renilla were mixedand transfected into crc cells using lipofectamine dualluciferase reporter assay system promega was usedto detect the firefly and renilla luciferase activities statistical analysesdata are shown as means ± sd spss chicago il usaand graphpad prism software were used to manipulate statistical analyses kaplanmeier method was used to calculatecumulative survival time the chisquare test or student™s results mpc1 expression was aberrantly decreased in crcpyruvate is a pivotal intermediate in the process of cellmetabolism which connects glycolysis and the tca cycleto determine the potential maladjustment genes involvedin pyruvate metabolism which is located in mitochondriaas shown in table we analyzed the tcga dataset containing crc and their normal counterparts the resultsshowed that multiple genes are significantly upregulated ordownregulated in crc t tissues compared to normal colonn notably mpc1 had a log2 fold change less than figures 1a and 1b as known to us pyruvate translocation from the cytoplasm to the mitochondria is the first stepinto the tca cycle which needs mpc1mpc2 heterodimerin the analysis no significant change was found in mpc2therefore mpc1 was selected for further study the expression pattern of mpc1 was further analyzed in five independent geo datasetsgse21510 gse5206 gse20916gse9348 and gse4183 consistently we found thatmpc1 was downregulated with statistical diï¬erence in crctissues figure 1c and inflammatory tissue supplementary figure in comparison to their normal counterpartsmeanwhile we found decreased mpc1 expression inhuman crc tissues compared to their normal counterpartsfigure 1d in addition a similar phenomenon wasrevealed in aomdss induced mouse crc modelsfigure 1e then we evaluated the protein level of mpc1used a tissue microarray containing matching cancerand corresponding adjacent nontumortissues whichsubjected ihc staining the expression of mpc1 wasscored as œ  based on the staining area andintensity figure 1f we found that more lower mpc1expression score as œ and œ was presented in crc 0c of immunology researcheulavp gol“tcga t nmpc1“mpc2“log2 fold changenoisserpxe cpm evitalertcgaŽŽŽn normaltumornoisserpxe cpm evitalergeo datasetsŽŽŽŽŽŽŽŽŽŽŽŽgse21510 gse5206 gse20916 gse9348normaltumorabhuman samplecrcnormalaom dssnormalccrcesacesac“esacesacdempc1 lower expressionmpc1 higher expressionfŽŽ egatnecrepromutlamron“gfigure expression pattern of mpc1 in crc a volcano plot showed fold changes xaxis and corresponding p values log10 yaxis ofpyruvate metabolismrelated genes located in mitochondria analyzed in the tcga dataset between paired normal and crc samplesstudent™s ttest b the comparison of mpc1 expression in tumor and matched normal tissues using the tcga dataset student™s ttest ˆ—ˆ—ˆ—p c expression analysis of mpc1 in tumors and corresponding normal tissue using four independent geo datasetsgse21510 gse5206 gse20916 and gse9348 student™s ttest ˆ—ˆ—ˆ—p d ihc staining of mpc1 expression in matched crctumor and nontumor tissues scale bar μm e ihc staining of mpc1 expression in the aomdss induced crc mouse models andcontrol animal scale bar μm f representative ihc staining of mpc1 expression in tissue microarray from the ren ji cohortwhich contained crc patients and paired adjacent normal tissue n the tumor tissues were divided into two groups based on theexpression level which scored as œ  scale bar μm g the percentage of tissue displaying diï¬erent expression level ofmpc1 in crc tumor and adjacent nontumor tissues fisher™s exact test ˆ—ˆ—p tissues figure 1g overall these results revealed thatmpc1 was disrupted during crc decreased mpc1 enhanced tumor metastasis capabilityand predicated poor prognosis in crc it is well known thatcrc is one of the most malignant tumors given its strongmetastasis ability so we tried to figure out whether thempc1 expression was correlated with metastasis we foundthat lower mpc1 expression was closely correlated withmetastasis p lymph node invasion p and tnm stage p which revealed by the analysisbetween the clinical significance and mpc1 expression incrc table next to illuminate the expression patternof mpc1 in diï¬erent process of crc data mining was carried out by two independent geo datasets gse21510 andgse89393 which contained normal tissue primary crcand metastatic lesion in the liver mcrc as shown infigures 2a and 2b mpc1 expression was gradually downregulated in patients with an increase in metastasis ability asimilar result was found in mice cells ct26 with high livermetastasis hmct26 or poor liver metastasis pmct26figure 2c which isolated by in vivo selection in an orthotopic mouse model of colon cancer metastasis to the liverfurther analysis showed that mpc1 protein expression was 0c of immunology researchtable clinicopathological correlation of mpc1 expression in theren ji crc cohortclinicopathological featureexpression ofmpc1lowhighp value χ2testage years‰¥gendermalefemalemetastasisyesnolymph node invasionyesnotumor size cm‰¥ cmtnm stageiiiiiiivkras mutationmutationno mutationgradually downregulated in normal tissue primary crc andliver metastasis crc crlm tissues figure 2d furthermore survival analysis showed that patients with lowermpc1 expression had a worse outcome compared to thepatients with higher mpc1 expression using the tcgacohort figure 2e and ren ji cohort figure 2f additionally among patients with metastasis worse prognosiswas emerged in the mpc1 low cases figure 2g while nosignificant association was observed in patients withoutmetastasis figure 2h mpc1 overexpression impaired crc cells motility bothin vitro and in vivo to evaluate the role of mpc1 on themotility of crc cells the transwell assay was performedfirstly we examined low mpc1 protein expression in humanand high mpc1 protein expression in mouse mc38 crccells by western blot figure 3a mpc1overexpressingstable cell lines were established using a lentivirus carrying the mpc1 gene in lovo and sw480 cells and theoverexpression efficiency was confirmed by immunoblotsfigure 3b mpc1 knockdown in lucmc38 cells wereestablished and verified by wb figure 3c mpc1 overexpression exhibited significantly weaker migration and invasion ability than the control cells in both lovo figure 3dand sw480 figure 3e cells following the liver metastasismodel of crc was established by spleen orthotopicallyinjecting mc38 cells transplanting luciferaseexpressingwhich would simulate mc38 metastasis to the liver throughsplenic veinportal vein the results revealed that the mpc1knockdown promoted mc38 cells metastasis to the liverthrough detecting the luminescence intensity monitoredby bioluminescence imaging figure 3f notablythenumber of metastatic liver nodules in the mpc1 silencinggroup wasin the control groupfigure 3g histological examination also proved thatmpc1 knockdown decreased the metastatic potential ofcrc in vivo figure 3gthan thatsmaller decreased mpc1 activated the wntcatenin pathwayby promoting nuclear translocation of catenin to furtherexplore the underlying mechanism of mpc1mediated inhibition of crc metastasis the tcga database was used toperform gsea analysis the results indicated that mpc1was involved in the wntcatenin signaling when set themrna expression median as a cutoï¬ figure 4a anddualluciferase reporter gene assay revealed that mpc1 overexpression obviously inhibited the activity of wntcateninpathway figure 4b which confirmed the result abovewe then tested the distribution changes of catenin innuclear and cytoplasmic which was a crucial step inwntcatenin pathway as shown in figure 4c no significant diï¬erence was emerged in the total amount ofcatenin between mpc1 overexpression cells and the control cells figure 4c however obviously decreased distribution of nuclear catenin was presented in mpc1overexpression cells compared to that in the control cellsas revealed by the stronger gray corresponding to cateninfigures 4c and 4d consistently immunofluorescenceif staining showed that mpc1 overexpression weakenednuclear catenin localization in both lovo and sw480 cellswhen compared to control cells figures 4e and 4f asimilar phenomenon was observed in mouse liver metastasistissues by ihc figure 4g following qpcr analyses displayed some downstream target genes of catenin such asmmp7 ecadherin snail1 and myc obviously increasedin ecadherin and decreased in mmp7 snail1 and mycwere observed after the mpc1 overexpression in both lovoand sw480 cells compared to that in the control cellsfigures 4h and 4i meanwhilethe opposite trendwas observed in mc38 cell after mpc1 knockdownfigure 4j and similar results were observed in mouseliver tissue detected by ihc figures 4k“4n takentogether the data above indicated that mpc1 mediatedcrc cell metastasis through the wntcatenin pathway discussionaccumulating evidences have shown that reprogrammedenergy metabolism conduces to the tumor malignant propertiesincluding enhanced crc liver metastatic capacity[“] mitochondria as the primary site of energy production regulate the pyruvate metabolism under both physiologic and pathologic conditions the first step of the tcacycle is mediated by mpc which transports pyruvate into 0cnoisserpxe cpm evitalergse21510ŽŽŽnormal crc mcrcnoisserpxe cpm evitalernormalacrccrlmesacesac of immunology researchgse67675ŽŽŽpmct26 hmct26ctcgan n p hr time daysgse89393ŽŽnormal crcmcrcbnoisserpxe cpm evitaler““Ž egatnecreplavivrus llarevolamroncrcmlrc“lavivrus llarevoren ji cohortn p hr n time monthsfdlavivrus llarevolavivrus llarevocrc with metastasis n n p hr n time monthsmpc1 higher expressionmpc1 lower expressiongecrc without metastasis n n n p hr time monthshfigure decreased mpc1 enhances tumor metastasis and predicts poor prognosis in crc a b data mining showed mpc1 expressionwas gradually decreased in normal tissue primary crc and liver lesion of metastatic crc mcrc from two independent geo datasetsoneway anova a gse21510 ˆ—ˆ—ˆ—p b gse89393 ˆ—ˆ—p c data from gse67675 revealed that mpc1 expressionwas lower in high liver metastasis ct26 cells hmct26 than that in poor liver metastasis ct26 cells pmct26 student™s ttestˆ—ˆ—ˆ—p d gradually decreased mpc1 expression was presented in normal tissue primary crc and liver metastasis crccrlm tissue scale bar μm n fisher™s exact test ˆ—p e kaplanmeier overall survival os curves in the tcgadataset of crc patients according to the mrna expression of mpc1 the lower quartile value of expression was utilized as a cutoï¬logrank test p f kaplanmeier os curve for the mpc1 expression in the ren ji cohort logrank test p gkaplanmeier os curve for the mpc1 expression in patients with metastasis logrank test p h kaplanmeier os curve forthe mpc1 expression in patients without metastasis logrank test p mitochondrial from cytoplasm in the beginning tcgawas used to analyze the relative genes involved in pyruvatemetabolism which is located in mitochondria the resultsrevealed that mpc1 expression was significantly downregulated in crc tissues meanwhile the geo datasets analysisas well as ihc staining on crc patients™ tissue and mousemodels confirmed this trend these phenomena may indicate that loss of mpc activity enhanced tumorigenic glucoseutilization by blocking mitochondrial pyruvate uptake andoxidation interestingly in the course of data analysis wefound that the expression of mpc1 was decreased at thestage of intestinal inflammation which was not diï¬erentfrom that in tumor tissue mpc1 has been reported to beinvolved in immune regulation of peripheral t cell homeostasis through metabolic regulation and a decrease inmpc1 was found at the earliest stages of crc hence 0c of immunology researchhumanmouselovosw480lovosw480mpc1gapdhmpc1““mpc1gapdhmpc1““mpc1gapdhwsthwsovolokrcmtchablovoŽŽrotcevitnelcpmitnelrotcevitnelcpmitneldlefi rep sllec edavnidlefi rep sllec edavnirotcevitnelcpmitneldsw480rotcevitnelcpmitnelcncpmŽcncpmdlefi rep sllec detargimdlefi rep sllec detargimcŽŽŽcncpmŽŽcncpmmc38shnc shmpc1¨¯ŽŽ sp xufl latotcnhscpmhscnhscpmhsradiancepseccm2srfemc38ŽŽsuledon sisatsatemcnhscpmhsgfigure mpc1 overexpression inhibits the motility of crc cells in vitro and in vivo a mpc1 expression in human and mouse crc celllines examined by western blot gapdh serves as loading control b mpc1 overexpression in lovo and sw480 cells c mpc1 silencing by shrnampc1 in mouse mc38 cells d e transwell assays showed that upregulated mpc1 suppressed the invasion and migration ability of lovod and sw480 e cells quantification of invaded and migrated cells was performed for five randomly selected fields values are means ± sd frepresentative bioluminescence photograph of mice spleen implanted with luciferaseexpressing mc38 cells treated with shmpc1 or controlvector total flux was quantified by the ivis system to verify the ability of liver metastasis g representative image of liver metastases andquantified by the nodules in mice inoculated with mc38 cells treated with shmpc1 or control vector as well as representative images ofhe staining of the liver metastatic lesions scale bar μm student™s ttest ˆ—p ˆ—ˆ—p ˆ—ˆ—ˆ—p 0c of immunology researchse erocs tnemhcirnewnt signaling pathwaynes “p mpc1 highmpc1 lowoitar pof pot evitaler𝛽cateninlamin ac𝛽cateningapdhmpc1alovosw480““csuelcunlatot icanmalnnetac𝛽iŽŽŽŽlovoncmpc1bŽsw480Žlovosw480ncmpc1d𝛽catenindapimerge𝛽catenindapimerge𝛽catenincpmcnovolnoisserpxe evitaleregnahc doflelovoŽŽŽŽlianscymŽpmmncmpc1nirehdacehcpmcnwsfsw480ŽŽŽlianscymŽŽpmmncmpc1nirehdaceifigure continuedcmcnhscpmhsmc38gŽŽlianscymŽŽpmmŽnirehdaceshncshmpc1jnoisserpxe evitaleregnahc doflnoisserpxe evitaleregnahc dofl 0c of immunology researchmmp7ecadherinsnail1myccmcnhscpmhscmcnhscpmhscmcnhscpmhscmcnhscpmhsklmnfigure decreased mpc1 activates the wntcatenin pathway by promoting nuclear translocation of catenin a gsea analysis ofmpc1 expression in crc using the tcga dataset nes normalized enrichment score b luciferase reporter gene assay of crc cellstreated with mpc1 overexpression or not c the expression of total catenin and nuclear catenin was detected in control and mpc1overexpression crc cells respectively gapdh and lamin ac were used as the loading control of total and nuclear protein respectivelyd the gray value analysis of nuclear catenin in mpc1overexpression cells and control cells e f mpc1 overexpression could inhibitthe nuclear translocation of catenin in crc cells scale bar μm g ihc staining of catenin in mouse liver metastatic lesionsinoculated with mc38 cells treatment with shmpc1 or control vector scale bar μm h i relative mrna expression level of catenin target genes in crc cells with mpc1 overexpression or control vector j relative mrna expression level of catenin targetgenes in mc38 cells with shmpc1 or control vector k“n relative protein expression level of catenin target genes in mouse livertissue detected by ihc scale bar μm student™s ttest ˆ—p ˆ—ˆ—p we suspect that mpc1 is involved in bowel inflammation totumorigenesis and more studies need to be devised to illustrate this processfollowing in the analysis between the clinical significance and mpc1 expression in crc we found that mpc1expression was especially correlated with metastasis inspiredby this we detected the mpc1 expression pattern in normaltissue primary crc and metastasis crc by geo datasetsand patients™ tissue all results revealed that gradually downregulated mpc1 was in patients with an increase in metastasis ability survival analysis indicated that worse outcome waspresented in patients with lower mpc1 expression especiallyin patients with metastasis additionally function assays verified that mpc1 overexpression could attenuate the migration and invasion capacities of crc cells in vitro andmpc1 knockdown could enhance the metastasis capacityin vivo and existing studies have revealed that the mpc1was participated in metastatic dissemination of pgc1αtransduced cholangiocarcinoma through elevating reactiveoxygen species ros production besides mpc inhibitor uk5099 treatment could trigger strong invasive capacitythrough blocking pyruvate translocation into the mitochondria so as to attenuate mitochondrial oxidative phosphorylation and trigger aerobic glycolysis these phenomenatogether with our results indicate that mpc1 could act as atumor suppressor through inhibiting tumor metastasis andexisting studies have shown that mpc1 could alter the maintenance and fate of stem cells through regulating cancermetabolism in crc however the relationship betweenthe metabolism and tumor metastasis regulated by mpc1was not being mentioned thus further evidence should bereceived to confirm thissubsequently the underlying mechanism of mpc1 inregulating metastasis was explored for this purpose gseaanalysis was performed the results indicated that mpc1was involved in the wntcatenin signaling the next seriesof experiments also confirmed that mpc1 could mediate thewntcatenin pathway by redistribution of catenin previous reports showed that cytoplasmic catenin phosphorylated in nterminally localized to sites of cellcell contact isassociated with ecadherin and was required for intact cellcell adhesions without any change detected in the levels oftotal catenin [“] simultaneously cell“cell adhesionbased on cadherin binding with catenin limited wnt signals in addition catenin was reported to interact withusp9x to inhibit the degradation of catenin through thedeubiquitination of catenin in breast cancer a constitutive irs1 and catenin protein interaction activated mycexpression in acute lymphoblastic leukemia cells inhcc catenin was reported to interact with yap1 to leadto rapid tumorigenesis hence it is reasonable to guessthat accumulated cytoplasmic catenin maybe crosstalkwith other genes or involved in other biological processeswhat is more some downstream target genes of cateninsuch as mmp7 ecadherin snail1 and myc were changedin expression as known to us mmp7 is a member of theproteolytic enzyme family which promotes the invasionand metastasis of tumor cells by degrading the basementmembrane and extracellular matrix and previous studies had evidenced for involvement of mmp7 activation incolorectal cancer liver metastases [ ] ecadherin andsnail1 were considered as the epithelialmesenchymal transition emt marker which was involved in metastasis ofmalignant tumor moreover ecadherin was reportedto be involved in cellcell junction to regulate cancer invasionand metastasis [ ] and the gsea analysis also revealedthat mpc1 could aï¬ect the cellcell contacts data notshown as described previously mmp7 could facilitatemorphological transition by cleaving ecadherin thecommunication between the cells is disrupted when ecadherin was shredded leading to destructed cell adhesionand induction of emt followed by increased cell migration inspired by this we assumed that mpc1 could mediatetumor cell motility through aï¬ecting mmp7 activity cellcell 0c of immunology researchcontacts and emt however further studies need to be performed to clarify the detailed underlying mechanisms conclusionsin conclusion we firstly demonstrated that decreased mpc1was closely correlated with patient™s metastasis as well as ledto poor outcome moreover mpc1driven nuclear translocation of catenin contributed to crc cell motility thismeans that mpc1 has the potential to be a diagnostic biomarker and therapeutic target for metastasis patientsabbreviationscrc colorectal carcinomampc mitochondrial pyruvate carrieremt epithelialmesenchymal transitiongsea gene set enrichment analysisdata availabilitythe data used to support the findings of this study are available from the corresponding author upon requestconflicts of interestall authors declare no conflicts of interestauthors™ contributionsyahui wang and guangang tian conceived and designedthe study chunjie xu and kaixia zhou obtained and anized the data guangang tian analyzed the data zhigangzhang and jianren gu contributed reagentsmaterialsanalysis tools xueli zhang and guangang tian wrote themanuscript guangang tian chunjie xu and kaixiazhou contributed equally to this workacknowledgmentsthis work was supported by the national natural sciencefoundation of china no to zhigang zhangand the natural science foundation of shanghai no18zr1436900 to xuelili zhangsupplementary materialsœsupplementary figure expression analysis of mpc1 innormal ibd and tumor tissues using the gse4183 datasetoneway anova was used to analyze the statistical diï¬erences between ibd adenoma and crc tissues ns no significance student™s ttest ˆ—ˆ—ˆ—p ˆ—ˆ—p ˆ—p supplementary materialsreferences h brody œcolorectal cancer nature vol no r l siegel k d miller s a fedewa et al œcolorectal cancerstatistics  ca a cancer for clinicians vol no pp “ a j rauckhorst and e b taylor œmitochondrial pyruvatecarrier function and cancer metabolism current opinion ingenetics development vol pp “ m g vander heiden l c cantley and c b thompsonœunderstanding the warburg eï¬ect the metabolic requirements of cell proliferation science vol no pp “ s herzig e raemy s montessuit et al œidentification andfunctional expression of the mitochondrial pyruvate carrierscience vol no pp “ c yang b ko c t hensley et al œglutamine oxidationmaintains the tca cycle and cell survival during impairedmitochondrial pyruvate transport molecular cell vol no pp “ t bender g pena and j c martinou œregulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes the embo vol no pp “ a g ramstead j a wallace sh lee et al œmitochondrialpyruvate carrier promotes peripheral t cell homeostasisthrough metabolic regulation of thymic development cellreports vol no pp “2899e6 x zhou z j xiong s m xiao et al œoverexpression ofmpc1 inhibits the proliferation migration invasion and stemcelllike properties of gastric cancer cells oncotargets andtherapy vol pp “ d li c wang p ma et al œpgc1α promotes cholangiocarcinoma metastasis by upregulating pdha1 and mpc1 expression to reverse the warburg eï¬ect cell death diseasevol no j c schell k a olson l jiang et al œa role for the mitochondrial pyruvate carrier as a repressor of the warburg eï¬ectand colon cancer cell growth molecular cell vol no pp “ y li x li q kan et al œmitochondrial pyruvate carrierfunction is negatively linked to warburg phenotype in vitroand malignant features in esophageal squamous cell carcinomas oncotarget vol no pp “ h zou q chen a zhang et al œmpc1 deficiency accelerateslung adenocarcinoma progression through the stat3 pathway cell death disease vol no l wang m xu j qin et al œmpc1 a key gene in cancermetabolism is regulated by couptfii in human prostatecancer oncotarget vol no pp “ y zhong x li d yu et al œapplication of mitochondrialpyruvate carrier blocker uk5099 creates metabolic reprogramand greater stemlike properties in lncap prostate cancer cellsin vitro oncotarget vol no pp “ x p tang q chen y li et al œmitochondrial pyruvatecarrier functions as a tumor suppressor and predicts theprognosis of human renal cell carcinoma laboratory investigation vol no pp “ g a tian c c zhu x x zhang et al œccbe1 promotesgist developmentthrough enhancing angiogenesis andmediating resistance to imatinib scientific reports vol no article c xu g tian c jiang et al œnptx2 promotes colorectalcancer growth and live

cancer7508

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: colorectal carcinoma crc as one of the most commongastrointestinal malignancies has developed the world™sfourth most deadly cancer with a high rate of incidence andmortality [ ] liver metastasis which is the most commonform for crc metastasis is the leading cause of high mortality for this severe malignancy however few clinical prevention and treatment measures could be available for tumormetastasis therefore it is really urgent to develop new biomarkers and explore the underlying mechanism for crcmetastasis and eventually develop new therapeutic strategiesfor crc patientsduring cancer progression metabolic reprograming withincreasing glucose utilization is termed as warburg aï¬ectwhich is accompanied by altered pyruvate and mitochondrialmetabolism the fate of pyruvate is the core manifestationto distinguish normal cells and tumor cells through metabolism in normal cells pyruvate was used for efficient atpproduction directly into mitochondria however pyruvatewas converted into lactate in cytosol despite of normoxicand hypoxic conditions in cancer cells this may be dueto the impaired process of pyruvate from the cytosol intothe mitochondrial matrix which is a critical metabolic steplinking glycolysis and mitochondrial oxidative phosphorylation the mitochondrial pyruvate carriermpc a 0c of immunology researchmultimeric complex containing two distinct proteins mpc1and mpc2 which is located in the inner mitochondrialmembrane is responsible for efficient mitochondrial pyruvate uptake loss of mpc expression or activity blockspyruvate entry into the tca cycle which results in a metabolism switch to increase glycolysis and the compensatoryusage of glutamine [ ]existed studies have reported that mpc1 was related withimmunoregulation stemness metabolism cellular morphology etc [ “] currently the important role of mpc1was uncovered in several tumors in crc and esophagealsquamous cell carcinoma decreased mpc1 results in accelerated aerobic glycolysis and malignant progression [ ] inlung adenocarcinoma mpc1 deficiency accelerates lung adenocarcinoma progression through the stat3 pathway in prostate cancer mpc1 was reported to be involved instemness and metabolism which regulated by couptfii[ ] in renal cell carcinoma hypoxiainduced loss ofmpc1 enhanced the expression of mmp7 and mmp9 to promote cell invasion collectively these data suggestedthat mpc1 maybe serves as a suppressor to disrupt tumormalignancy however whether mpc1 is involved in crcmetastasis and the underlying mechanisms remain to beillustratedin the present study we figured out the relationshipbetween the mpc1 expression and crc liver metastasiswe identified that decreased mpc1 was closely correlatedwith patient™s metastasis as well as led to poor prognosisfunctionally mpc1 overexpression could attenuate themigration and invasion capacities of crc cells both in vitroand in vivo mechanically mpc1 suppressed crc metastasisthrough mediating the wntcatenin signaling thus ourfinding firstly revealed a critical role of mpc1 in crc livermetastasis materials and methods data mining seven geo datasets gse21510 gse5206gse20916 gse9348 gse89393 gse67675 and gse4183and tcga were used to analyze the mpc1 expression pattern in crc the primary data for tcga datasets weredownloaded wwwcancergov the primary data feo datasets were downloaded at wwwncbinlmnihgovgeo the oncolnc database httpwwwoncolnc was used to detect the prognostic value of mpc1 inthe tcga cohort patients and clinical specimens in our study a total of patients containing paired crc tissues and adjacentnontumor tissues were enrolled from the department of gastrointestinal surgery renji hospital school of medicineshanghai jiao tong university among them cases ofliver metastases were collected and only cases wereavailable with complete followup data for survival analysisinformed consents were signed by all patients the researchwas approved by the research ethics committee of renjihospital and carried out in accordance with ethical standardsas formulated in the helsinki declarationtable sequences of primers used for realtime pcrprimermmp7 forwardmmp7 reverseecadherin forwardecadherin reversesnail1 forwardsnail1 reversemyc forwardmyc reversegapdh forwardgapdh reversesequence ²²gagtgagctacagtgggaacactatgacgcgggagtttaacatatgagtgtcccccggtatcttcacgagcagagaatcataaggcggtatccagagctgtttggaaacattttcctcccaggccatcacagccctcactcacacagattccacaaggtgcctgggctacactgagcaccaagtggtcgttgagggcaatg cell culture and cell transduction human crc celllines hct116 ht29 sw620 rko sw480 and lovoand mouse crc cell line mc38 were gained from the cellbank of the chinese academy of sciences shanghai chinaall cells were cultured in dmem medium supplemented°with fetal bovine serum and antibiotics at c in ahumidified incubator with co2mc38 cells were transfected with lentivirus containing aluciferase reporter plasmid stable transfection cells werescreened with μgml blastisidin for days which termedmc38luc in addition one short hairpin rna shrnasequence against mpc1 was packaged as lentivirus andtransfected into mc38luc cells lovo and sw480 were transfected with lentivirus containing fulllength human mpc1cdna or empty vehicle control stable transfection cells werescreened under μgml puromycin for days and verifiedby western blot in those assays all lentiviral transfectionswere performed in the presence of μgml polybrene immunohistochemical ihc the protocol of this assayand quantify the mpc1 protein expression level were performed according to previously reported primary antibodies used as follows mmp7 yt2663 immunoway ecadherin cst snail1 ab53519 abcam and mycyp0861 immunoway cellular immunofluorescence assays were performedaccording to the previous description briefly cells wereincubated with antibodies against catenin ab32572abcam and incubated with alexa 488conjugated secondaryantibody the nuclei were stained with ²6diamidino2phenylindole dapi western blotting wholecell lysates or nuclear proteinwas extracted using a protein extraction buï¬er beyotimeshanghai china or nucleoprotein extraction kit sangonbiotech c500009 respectively proteins were resolved bysdspage and transferred onto nitrocellulose nc membranes using standard methods primary antibodies used asfollows mpc1 ab74871 abcam catenin ab32572abcamlamin ac ab8984 abcam and gapdhab9485 abcam speciesspecific secondary antibodies used 0c of immunology researchtable related enzyme and carrier of pyruvate analyzed in this studygenepdha1pdhbpdk4pdhxmpc2pdk2pdk1pdk3mpc1pcpdp1pdprpdha2pdp2pklrdescriptionpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase lipoamide betapyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase complex component xmitochondrial pyruvate carrier pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme mitochondrial pyruvate carrier pyruvate carboxylasepyruvate dehydrogenase phosphatase catalytic subunit pyruvate dehydrogenase phosphatase regulatory subunitpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase phosphatase catalytic subunit pyruvate kinase liver and rbclocationmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrionas follows irdye goat antimouse igg licor andirdye goat antirabbit igg licorttest was used for comparison between groups p wasconsidered as statistically significantsynthesized using the primescript realtime pcr total rna was extracted using trizoltakara according to the manufacturer™sinstructionscdna wasreversetranscriptionpolymerasechain reaction rtpcr kittakara the qpcr was performed using sybr green masˆ’–ct method was used to analyze theter mix roche the data and gapdh was used as a loading control the primersequences are listed in table liver metastasis model this study was performed inaccordance with the recommendations in the guide for thecare and use of laboratory animals and relevant chineselaws and regulations the protocol was approved by theinstitutional animal care and use committee iacucof shanghai jiao tong university the mc38 cells transplanting luciferaseexpressing were injected into the spleensof c57bl6n mice n with a concentration of cellsmouse two weeks later the mice were killed andthe liver metastasis tissues were harvested luciferase reporter assay the protocols of this assaywere operated in accordance with previous reported ng top reporter plasmid wntcatenin signalingor ng fop reporter plasmid negative control ofwntcatenin signaling and ng renilla were mixedand transfected into crc cells using lipofectamine dualluciferase reporter assay system promega was usedto detect the firefly and renilla luciferase activities statistical analysesdata are shown as means ± sd spss chicago il usaand graphpad prism software were used to manipulate statistical analyses kaplanmeier method was used to calculatecumulative survival time the chisquare test or student™s results mpc1 expression was aberrantly decreased in crcpyruvate is a pivotal intermediate in the process of cellmetabolism which connects glycolysis and the tca cycleto determine the potential maladjustment genes involvedin pyruvate metabolism which is located in mitochondriaas shown in table we analyzed the tcga dataset containing crc and their normal counterparts the resultsshowed that multiple genes are significantly upregulated ordownregulated in crc t tissues compared to normal colonn notably mpc1 had a log2 fold change less than figures 1a and 1b as known to us pyruvate translocation from the cytoplasm to the mitochondria is the first stepinto the tca cycle which needs mpc1mpc2 heterodimerin the analysis no significant change was found in mpc2therefore mpc1 was selected for further study the expression pattern of mpc1 was further analyzed in five independent geo datasetsgse21510 gse5206 gse20916gse9348 and gse4183 consistently we found thatmpc1 was downregulated with statistical diï¬erence in crctissues figure 1c and inflammatory tissue supplementary figure in comparison to their normal counterpartsmeanwhile we found decreased mpc1 expression inhuman crc tissues compared to their normal counterpartsfigure 1d in addition a similar phenomenon wasrevealed in aomdss induced mouse crc modelsfigure 1e then we evaluated the protein level of mpc1used a tissue microarray containing matching cancerand corresponding adjacent nontumortissues whichsubjected ihc staining the expression of mpc1 wasscored as œ  based on the staining area andintensity figure 1f we found that more lower mpc1expression score as œ and œ was presented in crc 0c of immunology researcheulavp gol“tcga t nmpc1“mpc2“log2 fold changenoisserpxe cpm evitalertcgaŽŽŽn normaltumornoisserpxe cpm evitalergeo datasetsŽŽŽŽŽŽŽŽŽŽŽŽgse21510 gse5206 gse20916 gse9348normaltumorabhuman samplecrcnormalaom dssnormalccrcesacesac“esacesacdempc1 lower expressionmpc1 higher expressionfŽŽ egatnecrepromutlamron“gfigure expression pattern of mpc1 in crc a volcano plot showed fold changes xaxis and corresponding p values log10 yaxis ofpyruvate metabolismrelated genes located in mitochondria analyzed in the tcga dataset between paired normal and crc samplesstudent™s ttest b the comparison of mpc1 expression in tumor and matched normal tissues using the tcga dataset student™s ttest ˆ—ˆ—ˆ—p c expression analysis of mpc1 in tumors and corresponding normal tissue using four independent geo datasetsgse21510 gse5206 gse20916 and gse9348 student™s ttest ˆ—ˆ—ˆ—p d ihc staining of mpc1 expression in matched crctumor and nontumor tissues scale bar μm e ihc staining of mpc1 expression in the aomdss induced crc mouse models andcontrol animal scale bar μm f representative ihc staining of mpc1 expression in tissue microarray from the ren ji cohortwhich contained crc patients and paired adjacent normal tissue n the tumor tissues were divided into two groups based on theexpression level which scored as œ  scale bar μm g the percentage of tissue displaying diï¬erent expression level ofmpc1 in crc tumor and adjacent nontumor tissues fisher™s exact test ˆ—ˆ—p tissues figure 1g overall these results revealed thatmpc1 was disrupted during crc decreased mpc1 enhanced tumor metastasis capabilityand predicated poor prognosis in crc it is well known thatcrc is one of the most malignant tumors given its strongmetastasis ability so we tried to figure out whether thempc1 expression was correlated with metastasis we foundthat lower mpc1 expression was closely correlated withmetastasis p lymph node invasion p and tnm stage p which revealed by the analysisbetween the clinical significance and mpc1 expression incrc table next to illuminate the expression patternof mpc1 in diï¬erent process of crc data mining was carried out by two independent geo datasets gse21510 andgse89393 which contained normal tissue primary crcand metastatic lesion in the liver mcrc as shown infigures 2a and 2b mpc1 expression was gradually downregulated in patients with an increase in metastasis ability asimilar result was found in mice cells ct26 with high livermetastasis hmct26 or poor liver metastasis pmct26figure 2c which isolated by in vivo selection in an orthotopic mouse model of colon cancer metastasis to the liverfurther analysis showed that mpc1 protein expression was 0c of immunology researchtable clinicopathological correlation of mpc1 expression in theren ji crc cohortclinicopathological featureexpression ofmpc1lowhighp value χ2testage years‰¥gendermalefemalemetastasisyesnolymph node invasionyesnotumor size cm‰¥ cmtnm stageiiiiiiivkras mutationmutationno mutationgradually downregulated in normal tissue primary crc andliver metastasis crc crlm tissues figure 2d furthermore survival analysis showed that patients with lowermpc1 expression had a worse outcome compared to thepatients with higher mpc1 expression using the tcgacohort figure 2e and ren ji cohort figure 2f additionally among patients with metastasis worse prognosiswas emerged in the mpc1 low cases figure 2g while nosignificant association was observed in patients withoutmetastasis figure 2h mpc1 overexpression impaired crc cells motility bothin vitro and in vivo to evaluate the role of mpc1 on themotility of crc cells the transwell assay was performedfirstly we examined low mpc1 protein expression in humanand high mpc1 protein expression in mouse mc38 crccells by western blot figure 3a mpc1overexpressingstable cell lines were established using a lentivirus carrying the mpc1 gene in lovo and sw480 cells and theoverexpression efficiency was confirmed by immunoblotsfigure 3b mpc1 knockdown in lucmc38 cells wereestablished and verified by wb figure 3c mpc1 overexpression exhibited significantly weaker migration and invasion ability than the control cells in both lovo figure 3dand sw480 figure 3e cells following the liver metastasismodel of crc was established by spleen orthotopicallyinjecting mc38 cells transplanting luciferaseexpressingwhich would simulate mc38 metastasis to the liver throughsplenic veinportal vein the results revealed that the mpc1knockdown promoted mc38 cells metastasis to the liverthrough detecting the luminescence intensity monitoredby bioluminescence imaging figure 3f notablythenumber of metastatic liver nodules in the mpc1 silencinggroup wasin the control groupfigure 3g histological examination also proved thatmpc1 knockdown decreased the metastatic potential ofcrc in vivo figure 3gthan thatsmaller decreased mpc1 activated the wntcatenin pathwayby promoting nuclear translocation of catenin to furtherexplore the underlying mechanism of mpc1mediated inhibition of crc metastasis the tcga database was used toperform gsea analysis the results indicated that mpc1was involved in the wntcatenin signaling when set themrna expression median as a cutoï¬ figure 4a anddualluciferase reporter gene assay revealed that mpc1 overexpression obviously inhibited the activity of wntcateninpathway figure 4b which confirmed the result abovewe then tested the distribution changes of catenin innuclear and cytoplasmic which was a crucial step inwntcatenin pathway as shown in figure 4c no significant diï¬erence was emerged in the total amount ofcatenin between mpc1 overexpression cells and the control cells figure 4c however obviously decreased distribution of nuclear catenin was presented in mpc1overexpression cells compared to that in the control cellsas revealed by the stronger gray corresponding to cateninfigures 4c and 4d consistently immunofluorescenceif staining showed that mpc1 overexpression weakenednuclear catenin localization in both lovo and sw480 cellswhen compared to control cells figures 4e and 4f asimilar phenomenon was observed in mouse liver metastasistissues by ihc figure 4g following qpcr analyses displayed some downstream target genes of catenin such asmmp7 ecadherin snail1 and myc obviously increasedin ecadherin and decreased in mmp7 snail1 and mycwere observed after the mpc1 overexpression in both lovoand sw480 cells compared to that in the control cellsfigures 4h and 4i meanwhilethe opposite trendwas observed in mc38 cell after mpc1 knockdownfigure 4j and similar results were observed in mouseliver tissue detected by ihc figures 4k“4n takentogether the data above indicated that mpc1 mediatedcrc cell metastasis through the wntcatenin pathway discussionaccumulating evidences have shown that reprogrammedenergy metabolism conduces to the tumor malignant propertiesincluding enhanced crc liver metastatic capacity[“] mitochondria as the primary site of energy production regulate the pyruvate metabolism under both physiologic and pathologic conditions the first step of the tcacycle is mediated by mpc which transports pyruvate into 0cnoisserpxe cpm evitalergse21510ŽŽŽnormal crc mcrcnoisserpxe cpm evitalernormalacrccrlmesacesac of immunology researchgse67675ŽŽŽpmct26 hmct26ctcgan n p hr time daysgse89393ŽŽnormal crcmcrcbnoisserpxe cpm evitaler““Ž egatnecreplavivrus llarevolamroncrcmlrc“lavivrus llarevoren ji cohortn p hr n time monthsfdlavivrus llarevolavivrus llarevocrc with metastasis n n p hr n time monthsmpc1 higher expressionmpc1 lower expressiongecrc without metastasis n n n p hr time monthshfigure decreased mpc1 enhances tumor metastasis and predicts poor prognosis in crc a b data mining showed mpc1 expressionwas gradually decreased in normal tissue primary crc and liver lesion of metastatic crc mcrc from two independent geo datasetsoneway anova a gse21510 ˆ—ˆ—ˆ—p b gse89393 ˆ—ˆ—p c data from gse67675 revealed that mpc1 expressionwas lower in high liver metastasis ct26 cells hmct26 than that in poor liver metastasis ct26 cells pmct26 student™s ttestˆ—ˆ—ˆ—p d gradually decreased mpc1 expression was presented in normal tissue primary crc and liver metastasis crccrlm tissue scale bar μm n fisher™s exact test ˆ—p e kaplanmeier overall survival os curves in the tcgadataset of crc patients according to the mrna expression of mpc1 the lower quartile value of expression was utilized as a cutoï¬logrank test p f kaplanmeier os curve for the mpc1 expression in the ren ji cohort logrank test p gkaplanmeier os curve for the mpc1 expression in patients with metastasis logrank test p h kaplanmeier os curve forthe mpc1 expression in patients without metastasis logrank test p mitochondrial from cytoplasm in the beginning tcgawas used to analyze the relative genes involved in pyruvatemetabolism which is located in mitochondria the resultsrevealed that mpc1 expression was significantly downregulated in crc tissues meanwhile the geo datasets analysisas well as ihc staining on crc patients™ tissue and mousemodels confirmed this trend these phenomena may indicate that loss of mpc activity enhanced tumorigenic glucoseutilization by blocking mitochondrial pyruvate uptake andoxidation interestingly in the course of data analysis wefound that the expression of mpc1 was decreased at thestage of intestinal inflammation which was not diï¬erentfrom that in tumor tissue mpc1 has been reported to beinvolved in immune regulation of peripheral t cell homeostasis through metabolic regulation and a decrease inmpc1 was found at the earliest stages of crc hence 0c of immunology researchhumanmouselovosw480lovosw480mpc1gapdhmpc1““mpc1gapdhmpc1““mpc1gapdhwsthwsovolokrcmtchablovoŽŽrotcevitnelcpmitnelrotcevitnelcpmitneldlefi rep sllec edavnidlefi rep sllec edavnirotcevitnelcpmitneldsw480rotcevitnelcpmitnelcncpmŽcncpmdlefi rep sllec detargimdlefi rep sllec detargimcŽŽŽcncpmŽŽcncpmmc38shnc shmpc1¨¯ŽŽ sp xufl latotcnhscpmhscnhscpmhsradiancepseccm2srfemc38ŽŽsuledon sisatsatemcnhscpmhsgfigure mpc1 overexpression inhibits the motility of crc cells in vitro and in vivo a mpc1 expression in human and mouse crc celllines examined by western blot gapdh serves as loading control b mpc1 overexpression in lovo and sw480 cells c mpc1 silencing by shrnampc1 in mouse mc38 cells d e transwell assays showed that upregulated mpc1 suppressed the invasion and migration ability of lovod and sw480 e cells quantification of invaded and migrated cells was performed for five randomly selected fields values are means ± sd frepresentative bioluminescence photograph of mice spleen implanted with luciferaseexpressing mc38 cells treated with shmpc1 or controlvector total flux was quantified by the ivis system to verify the ability of liver metastasis g representative image of liver metastases andquantified by the nodules in mice inoculated with mc38 cells treated with shmpc1 or control vector as well as representative images ofhe staining of the liver metastatic lesions scale bar μm student™s ttest ˆ—p ˆ—ˆ—p ˆ—ˆ—ˆ—p 0c of immunology researchse erocs tnemhcirnewnt signaling pathwaynes “p mpc1 highmpc1 lowoitar pof pot evitaler𝛽cateninlamin ac𝛽cateningapdhmpc1alovosw480““csuelcunlatot icanmalnnetac𝛽iŽŽŽŽlovoncmpc1bŽsw480Žlovosw480ncmpc1d𝛽catenindapimerge𝛽catenindapimerge𝛽catenincpmcnovolnoisserpxe evitaleregnahc doflelovoŽŽŽŽlianscymŽpmmncmpc1nirehdacehcpmcnwsfsw480ŽŽŽlianscymŽŽpmmncmpc1nirehdaceifigure continuedcmcnhscpmhsmc38gŽŽlianscymŽŽpmmŽnirehdaceshncshmpc1jnoisserpxe evitaleregnahc doflnoisserpxe evitaleregnahc dofl 0c of immunology researchmmp7ecadherinsnail1myccmcnhscpmhscmcnhscpmhscmcnhscpmhscmcnhscpmhsklmnfigure decreased mpc1 activates the wntcatenin pathway by promoting nuclear translocation of catenin a gsea analysis ofmpc1 expression in crc using the tcga dataset nes normalized enrichment score b luciferase reporter gene assay of crc cellstreated with mpc1 overexpression or not c the expression of total catenin and nuclear catenin was detected in control and mpc1overexpression crc cells respectively gapdh and lamin ac were used as the loading control of total and nuclear protein respectivelyd the gray value analysis of nuclear catenin in mpc1overexpression cells and control cells e f mpc1 overexpression could inhibitthe nuclear translocation of catenin in crc cells scale bar μm g ihc staining of catenin in mouse liver metastatic lesionsinoculated with mc38 cells treatment with shmpc1 or control vector scale bar μm h i relative mrna expression level of catenin target genes in crc cells with mpc1 overexpression or control vector j relative mrna expression level of catenin targetgenes in mc38 cells with shmpc1 or control vector k“n relative protein expression level of catenin target genes in mouse livertissue detected by ihc scale bar μm student™s ttest ˆ—p ˆ—ˆ—p we suspect that mpc1 is involved in bowel inflammation totumorigenesis and more studies need to be devised to illustrate this processfollowing in the analysis between the clinical significance and mpc1 expression in crc we found that mpc1expression was especially correlated with metastasis inspiredby this we detected the mpc1 expression pattern in normaltissue primary crc and metastasis crc by geo datasetsand patients™ tissue all results revealed that gradually downregulated mpc1 was in patients with an increase in metastasis ability survival analysis indicated that worse outcome waspresented in patients with lower mpc1 expression especiallyin patients with metastasis additionally function assays verified that mpc1 overexpression could attenuate the migration and invasion capacities of crc cells in vitro andmpc1 knockdown could enhance the metastasis capacityin vivo and existing studies have revealed that the mpc1was participated in metastatic dissemination of pgc1αtransduced cholangiocarcinoma through elevating reactiveoxygen species ros production besides mpc inhibitor uk5099 treatment could trigger strong invasive capacitythrough blocking pyruvate translocation into the mitochondria so as to attenuate mitochondrial oxidative phosphorylation and trigger aerobic glycolysis these phenomenatogether with our results indicate that mpc1 could act as atumor suppressor through inhibiting tumor metastasis andexisting studies have shown that mpc1 could alter the maintenance and fate of stem cells through regulating cancermetabolism in crc however the relationship betweenthe metabolism and tumor metastasis regulated by mpc1was not being mentioned thus further evidence should bereceived to confirm thissubsequently the underlying mechanism of mpc1 inregulating metastasis was explored for this purpose gseaanalysis was performed the results indicated that mpc1was involved in the wntcatenin signaling the next seriesof experiments also confirmed that mpc1 could mediate thewntcatenin pathway by redistribution of catenin previous reports showed that cytoplasmic catenin phosphorylated in nterminally localized to sites of cellcell contact isassociated with ecadherin and was required for intact cellcell adhesions without any change detected in the levels oftotal catenin [“] simultaneously cell“cell adhesionbased on cadherin binding with catenin limited wnt signals in addition catenin was reported to interact withusp9x to inhibit the degradation of catenin through thedeubiquitination of catenin in breast cancer a constitutive irs1 and catenin protein interaction activated mycexpression in acute lymphoblastic leukemia cells inhcc catenin was reported to interact with yap1 to leadto rapid tumorigenesis hence it is reasonable to guessthat accumulated cytoplasmic catenin maybe crosstalkwith other genes or involved in other biological processeswhat is more some downstream target genes of cateninsuch as mmp7 ecadherin snail1 and myc were changedin expression as known to us mmp7 is a member of theproteolytic enzyme family which promotes the invasionand metastasis of tumor cells by degrading the basementmembrane and extracellular matrix and previous studies had evidenced for involvement of mmp7 activation incolorectal cancer liver metastases [ ] ecadherin andsnail1 were considered as the epithelialmesenchymal transition emt marker which was involved in metastasis ofmalignant tumor moreover ecadherin was reportedto be involved in cellcell junction to regulate cancer invasionand metastasis [ ] and the gsea analysis also revealedthat mpc1 could aï¬ect the cellcell contacts data notshown as described previously mmp7 could facilitatemorphological transition by cleaving ecadherin thecommunication between the cells is disrupted when ecadherin was shredded leading to destructed cell adhesionand induction of emt followed by increased cell migration inspired by this we assumed that mpc1 could mediatetumor cell motility through aï¬ecting mmp7 activity cellcell 0c of immunology researchcontacts and emt however further studies need to be performed to clarify the detailed underlying mechanisms conclusionsin conclusion we firstly demonstrated that decreased mpc1was closely correlated with patient™s metastasis as well as ledto poor outcome moreover mpc1driven nuclear translocation of catenin contributed to crc cell motility thismeans that mpc1 has the potential to be a diagnostic biomarker and therapeutic target for metastasis patientsabbreviationscrc colorectal carcinomampc mitochondrial pyruvate carrieremt epithelialmesenchymal transitiongsea gene set enrichment analysisdata availabilitythe data used to support the findings of this study are available from the corresponding author upon requestconflicts of interestall authors declare no conflicts of interestauthors™ contributionsyahui wang and guangang tian conceived and designedthe study chunjie xu and kaixia zhou obtained and anized the data guangang tian analyzed the data zhigangzhang and jianren gu contributed reagentsmaterialsanalysis tools xueli zhang and guangang tian wrote themanuscript guangang tian chunjie xu and kaixiazhou contributed equally to this workacknowledgmentsthis work was supported by the national natural sciencefoundation of china no to zhigang zhangand the natural science foundation of shanghai no18zr1436900 to xuelili zhangsupplementary materialsœsupplementary figure expression analysis of mpc1 innormal ibd and tumor tissues using the gse4183 datasetoneway anova was used to analyze the statistical diï¬erences between ibd adenoma and crc tissues ns no significance student™s ttest ˆ—ˆ—ˆ—p ˆ—ˆ—p ˆ—p supplementary materialsreferences h brody œcolorectal cancer nature vol no r l siegel k d miller s a fedewa et al œcolorectal cancerstatistics  ca a cancer for clinicians vol no pp “ a j rauckhorst and e b taylor œmitochondrial pyruvatecarrier function and cancer metabolism current opinion ingenetics development vol pp “ m g vander heiden l c cantley and c b thompsonœunderstanding the warburg eï¬ect the metabolic requirements of cell proliferation science vol no pp “ s herzig e raemy s montessuit et al œidentification andfunctional expression of the mitochondrial pyruvate carrierscience vol no pp “ c yang b ko c t hensley et al œglutamine oxidationmaintains the tca cycle and cell survival during impairedmitochondrial pyruvate transport molecular cell vol no pp “ t bender g pena and j c martinou œregulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes the embo vol no pp “ a g ramstead j a wallace sh lee et al œmitochondrialpyruvate carrier promotes peripheral t cell homeostasisthrough metabolic regulation of thymic development cellreports vol no pp “2899e6 x zhou z j xiong s m xiao et al œoverexpression ofmpc1 inhibits the proliferation migration invasion and stemcelllike properties of gastric cancer cells oncotargets andtherapy vol pp “ d li c wang p ma et al œpgc1α promotes cholangiocarcinoma metastasis by upregulating pdha1 and mpc1 expression to reverse the warburg eï¬ect cell death diseasevol no j c schell k a olson l jiang et al œa role for the mitochondrial pyruvate carrier as a repressor of the warburg eï¬ectand colon cancer cell growth molecular cell vol no pp “ y li x li q kan et al œmitochondrial pyruvate carrierfunction is negatively linked to warburg phenotype in vitroand malignant features in esophageal squamous cell carcinomas oncotarget vol no pp “ h zou q chen a zhang et al œmpc1 deficiency accelerateslung adenocarcinoma progression through the stat3 pathway cell death disease vol no l wang m xu j qin et al œmpc1 a key gene in cancermetabolism is regulated by couptfii in human prostatecancer oncotarget vol no pp “ y zhong x li d yu et al œapplication of mitochondrialpyruvate carrier blocker uk5099 creates metabolic reprogramand greater stemlike properties in lncap prostate cancer cellsin vitro oncotarget vol no pp “ x p tang q chen y li et al œmitochondrial pyruvatecarrier functions as a tumor suppressor and predicts theprognosis of human renal cell carcinoma laboratory investigation vol no pp “ g a tian c c zhu x x zhang et al œccbe1 promotesgist developmentthrough enhancing angiogenesis andmediating resistance to imatinib scientific reports vol no article c xu g tian c jiang et al œnptx2 promotes colorectalcancer growth and live Answer:

Colon_Cancer

" recent impressive advances in cancer immunotherapy have been largely derived from cellular immunity the role of humoral immunity in carcinogenesis has been less understood based on our previous observations we hypothesize that an immunoglobulin subtype igg4 plays an essential role in cancer immune evasionmethods the distribution abundance actions properties and possible mechanisms of igg4 were investigated with human cancer samples and animal tumor models with an extensive array of techniques both in vitro and in vivoresults in a cohort of patients with esophageal cancer we found that igg4 containing b lymphocytes and igg4 concentration were significantly increased in cancer tissue and igg4 concentrations increased in serum of patients with cancer both were positively related to increased cancer malignancy and poor prognoses that is more igg4 appeared to associate with more aggressive cancer growth we further found that igg4 regardless of its antigen specificity inhibited the classic immune reactions of antibody dependent cell mediated cytotoxicity antibody dependent cellular phagocytosis and complement dependent cytotoxicity against cancer cells in vitro and these effects were obtained through its fc fragment reacting to the fc fragments of cancer specific igg1 that has been bound to cancer antigens we also found that igg4 competed with igg1 in reacting to fc receptors of immune effector cells therefore locally increased igg4 in cancer microenvironment should inhibit antibody mediated anticancer responses and help cancer to evade local immune attack and indirectly promote cancer growth this hypothesis was verified in three different immune potent mouse models we found that local application of igg4 significantly accelerated growth of inoculated breast and colorectal cancers and carcinogen induced skin papilloma we also tested the antibody drug for cancer immunotherapy nivolumab which was igg4 in nature with a stabilizing s228p mutation and found that it significantly promoted cancer growth in mice this may provide an explanation to the newly appeared hyperprogressive disease sometimes associated with cancer immunotherapy there appears to be a previously unrecognized immune evasion mechanism with igg4 playing an essential role in cancer microenvironment with implications in cancer diagnosis and immunotherapyintroductionwhile new immune therapy for cancer focuses mostly on manipulating cellular immunity humoral immunity also holds great promise for cancer therapy1 igg4 is a unique antibody that has the lowest concentration among igg subtypes in healthy individuals and its function has not been well understood3“ igg4 was regarded as a ˜blocking antibody™ because of its reduced ability to trigger effector immune reactions6 therefore whatever molecules igg4 reacts to the subsequent immune reaction was subdued8 igg4 has a unique structure of fab arm exchange fae in which the two heavy and light chains of two different antibodies with different specificities are exchanged resulting in an asymmetric bispecific antibody with reduced ability to bind to antigen and to form immune complexes9 another unique feature of igg4 is that it can react to other iggs via its fc fragment and the significance of this property has not been well understood evidence suggests that fae and fc fc reactivity may involve the same molecular structure on igg4 molecule10 davies et al11 resolved the crystal structure of igg4 fc fragment revealing a unique molecular conformation supporting its fc binding property recent interests in igg4 related diseases unveiled a wide range of pathologies with a common phenomenon of often increased igg4 concentration in the serum and igg4 postive plasma cells in the affected ans accompanied by local inflammation and fibrosis but its pathogenic mechanism is still poorly understood13“to cite wang a0h xu a0q zhao a0c et a0al an immune evasion mechanism with igg4 playing an essential role in cancer and implication for immunotherapy for immunotherapy of cancer 20208e000661 101136jitc2020000661 –º additional material is published online only to view please visit the online http dx jitc hw qx cz and zz are joint first authorsaccepted july authors or their employers re use permitted under cc by nc no commercial re use see rights and permissions published by bmjfor numbered affiliations see end of correspondence toprofessor jiang gu qq comwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access the possible functions of igg4 in cancer and also in the immune system have not been well elucidated increases of igg4 positive plasma cells were reported in gastrocarcinoma16 extrahepatic cholangiocarcinoma17 and melanoma18 the above studies were performed on limited number of cases without convincing explanation of mechanism or significance wu 19 reported that serum igg4igg ratio could predict recurrence of hepatocellular carcinoma after surgery the most extensive study on igg4 and cancer was performed by karagiannis 20 who investigated the possible effect of cancer specific igg4 in inhibiting cancer immunity in melanomas and suggested competition between cancer specific igg4 and igg1 in binding to cancer antigens as the cause for the inhibition a recent report raised the concept of cancer educated b cells and toxic igg produced by these cells in facilitating lymph node metastasis for breast cancer in a mouse model21 we performed a multidimensional investigation of igg4 in a wide array of patients with cancer and tissues with both in vitro and in vivo experiments extensive new evidence led us to hypothesize that there is a potent humoral immune editing mechanism in cancer microenvironment with igg4 playing an essential role we propose that fc fc reaction could be the basic mechanism of this immune inhibition we validated this in three immune potent animal models this property was also found applicable to cancer immunotherapy drug nivolumab which was igg4 in nature our study points to a so far little appreciated mechanism of immune evasion in cancermaterials and methodskey resourcesdetailed information about key resources including antibodies biological samples chemicals assay kits cell lines and software are shown in online supplementary table experimental model and subject detailspatients and healthy volunteerswe collected tissue and blood samples from over patients with cancer in shantou university affiliated tumor hospital and the east guangdong provincial pathological consultation center details of the human samples are shown in online supplementary table immunohistochemistrydetails of the protocols and the antibodies are shown in online supplementary datasds techniquethe expressions of igg1 igg2 igg3 and igg4 in esophageal cancer were detected at the histological level the stain decolorize stain sds technology22 was performed on the same slide sequentially with four different antibodies to demonstrate the four antigens the distribution pattern abundance and relationship of the four antigens were processed with an image software photoshop to achieve an integrated figure the proximity of different cell types on the tissue sections was measured with an image analyzing software image pro plus v60 details of the protocol are presented in the online supplementary dataimmunofluorescence double stainingtwo antibodies from different species were incubated on the same tissue section primary antibodies were detected with goat antimouse 555antirabbit igg or goat antimouse 488antirabbit igg alexa fluor life sciences and immunoreactivity was visualized with a fluorescence microscope antigens were detected and demonstrated with two fluorescence signals in red and green and the blue signal of ' diamidino2 phenylindole dapi as for cell nuclei images were acquired with the evos fl fluorescence microscope life technologies usaigg4 immunohistochemistryto verify that igg4 could react to igg1 that had been immobilized to tissue sections we used human pancreas and brain and antibodies to insulin glucagon and neurofilament as models detailed protocol of this experiment is shown in the online supplementary dataimmunoglobulin preparationsfab and fc fragments of igg igg1 and igg4 were prepared with papain digestion in the presence of cysteine iggs were cleaved at a position above the hinge region by papain at °c for hours after purification with protein a affinity chromatography pure fab and fc fragments were isolated with elution buffer igg fc fragment was washed down from protein a column after centrifugation and concentration measurement igg preparations were collected and stored at °c before usewestern blotigg subclasses were resolved on sodium dodecyl sulfate polyacrylamide gels under reducing conditions and then transferred onto nitrocellulose membranes ge healthcare life sciences the membranes were blocked for hour with bovine serum albumin bsa in tris buffered saline with tween20 ph and then incubated with primary antibody biotin labeling kit anaspec overnight at °c it was then incubated with secondary antibody for hour at room temperature finally reaction density was measured with odyssey western at nm and nm detection channelserum igg4 and igg assessmentsera samples collected from patients with esophageal cancer and healthy volunteers were sent to golden field medical test company guangzhou china and roche immune turbidimetry method was used to wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cmeasure serum igg4 and total igg concentrations all serum samples were stored at ˆ’°c freezer immediately before analysis all quantitative data were treated statisticallymeasurement of igg4 concentrations in tumor and tumoradjacent normal tissues with elisaconcentrations of igg4 in pairs of esophageal cancer and adjacent normal tissues cm from the edge of the cancer mass were measured with elisa detailed protocol of this experiment is shown in the online supplementary datacell culture for fc receptor binding assaysu937 cell line was bought from procell life science technology china cl0239 and cells were cultured in rpmi gibco c22400500bt supplemented with fetal bovine serum gibco “ and uml penicillin streptomycin gibco “ at ×106ml in ml cell culture bottleprotein preparationsdetails of the protocols for protein preparation separation and papain digestion are shown in online supplementary datafluorescence activating cell sorter facs for fc receptor assaysdetails of the relevant protocols are shown in online supplementary dataadcc adcp and cdc teststhe classic antibody dependent cell mediated cytotoxicity adcc antibody dependent cellular phagocytosis adcp and complement dependent cytotoxicity cdc tests were performed based on previously reported protocols23“ non specific igg4 instead of cancer specific igg4 was used to inhibit these reactions igg1 was used as control details of the protocols are shown in online supplementary dataanimal modelsbreast cancer and colon cancer modelsbalbc mice were obtained from vital river technical beijing china mice aged between and weeks and weighed ± g were used in all experiments all mice were inoculated with 4t1 mouse breast cancer cells or ct26 mouse colon cancer cells subcutaneously under the left forearm × 4t1 cells per mouse to build cancer models the mice were divided into different groups and were treated with igg1 or igg4 derived from intravenous igg ivig ivig without igg4 nivolumab and fc of nivolumab respectively details of the protocols are shown in online supplementary datacarcinogeninduced skin tumor modelwe employed a well established carcinogen induced skin tumor model to study the effect of igg4 and ivig without igg4 in comparison with controls phosphate buffered open accesssaline pbs detailed protocol is shown in online supplementary dataquantification and statistical analysisdata were analyzed in graphpad prism all reported p values were derived from two sided comparisons with values of less than considered to be statistically significantresultsigg4 was significantly increased in the serum of patients with cancer and this increase was related to cancer stage and patient prognosiswe first measured the concentrations of igg1 igg2 igg3 and igg4 in sera of patients with esophageal cancer n82 igg4 was significantly increased in patients with cancer in comparison with normal healthy subjects n70 the concentration of igg4 was increased from about to the ratio of igg4iggtotal was also significantly increased the statistical significance of both reached p00001 the increase of igg4 was also positively correlated to the stages of cancer with late stage cancers having more obvious increases higher igg4 serum concentrations were associated with worse prognosis figure 1a“digg4containing lymphocytes and igg4 concentration were significantly increased in cancer microenvironment and this increase was associated with cancer cell infiltrationigg4 positive lymphocytes were significantly increased in cancer microenvironment in comparison with tissue more distant to the cancer mass igg4 positive lymphocytes were barely detectable in tumor adjacent normal tissue figure 1h on the same tissue sections employing the sds technique22 to simultaneously visualize the four subtypes of igg with four distinct colors we found that one plasma cell only contained one igg subtype that is igg1 igg2 igg3 and igg4 were all contained in their own plasma cell populations separately figure 1i the marked increase of igg4 containing plasma cells in comparison with other subtypes was clearly visualized on cancer tissue sections igg4 positive plasma cells appeared to accumulate more in tissues with extensive cancer cell infiltration than in other areas figure 1eh in addition igg4 positive cells are often in close proximity to cells containing igg1 and igg2 but not to igg3 this phenomenon was not seen among other igg subtypes apart from igg4 quantitative data of the proximity of different cell types are presented in online supplementary figure s2 with the multiple immunostaining method we also demonstrated that igg1 containing and igg4 containing lymphocytes were distinct from cd3 positive t lymphocytes in the same region of the cancer tissue figure 1j in addition we measured the concentrations of igg4 in cancer tissue and cancer adjacent normal tissue n46 pairs the average concentration of igg4 in cancer tissue was about four times higher than that in the adjacent wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access figure significant increase of igg4 and igg4iggtotal in serum and igg4 positive b lymphocytes in esophageal cancer a igg4 in serum of esophageal squamous cell cancer escc n82 was significantly higher when compared with healthy controls n70 p00001 b igg4iggtotal ratio in escc n82 was significantly higher than that in matching healthy adults n70 p00001 c igg4 in stage …³ n18 was significantly higher than those in stages …° and …± n16 p001 d igg4iggtotal in serum of stage iv escc n18 was significantly higher than those in stages …° and …± n16 p005 e scatter diagram of igg4 positive cell numbers in cancer cancer adjacent normal tissue adjacent and normal tissues igg4 positive lymphocytes in and around the esophageal cancer mass n110 are significantly more abundant than those in the adjacent normal tissue n60 and normal lymphoid tissues n63 p0001 for both increases of igg4 positive lymphocytes were most abundant in areas of cancer cell proliferation f the increase of igg4 positive cell numbers was related to the prognoses of the patients more igg4 positive cells were associated with worse outcome p005 g igg4 concentration in cancer tissue n46 was significantly higher than that in adjacent normal tissue n46 p001 h immunohistochemistry of igg4 in esophageal cancer tissues from left to right are igg4 in cancer tissues cancer adjacent tissue and normal lymphoid tissue tonsil it clearly demonstrates that igg4 positive lymphocytes red were markedly increased left in comparison with normal lymphoid tissue right and with tumor adjacent normal tissue middle scale bar µm i demonstration of four subpopulations of igg containing plasma cells with multiple immunostaining sds method in cancer each subclass has its own distribution pattern and one plasma cell only produces one subclass of igg igg1 yellow igg2 green igg3 purple igg4 red j on the same tissue section a triple immunostaining was performed with the sds method to demonstrate the distribution and relationship among cd3 positive t cells yellow igg1 positive b cells greens and igg4 positive b cells red each cell type has its own distribution and no overlap between different cell types is observed sds stain decolorize stainwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cnormal tissue and the difference between these two groups was statistically significant p001 figure 1gigg1 extracted from patients with cancer reacted to cancer cells of the same patients but igg4 extracted from the same patients did notwe extracted igg1 and igg4 from the serum of patients with esophageal cancer with breast cancer and with colon cancer with respective specific antibody columns we then labeled the antibodies with biotin and tested the reactivity of these extracted antibodies to cancer tissue sections of the same patients in all cases igg1 reacted to cancer cells from the same patients but igg4 did not figure 2a it appeared that the increased igg4 in cancer microenvironment and in the patient™s serum was not reactive to cancer antigens while igg1 extracted from the same patients reacted to the cancer antigensigg4 reacted to cancerspecific igg1 that was bound to cancer cellsalthough igg4 extracted from patients with cancer did not react to cancer antigen it did react to cancer specific igg1 that was bound to cancer antigen on tissue sections as shown in figure 2b when cancer specific igg1 that was not labeled with biotin was applied to cancer tissue sections followed by biotin labeled igg4 the cancer cells became positive while when the same biotin labeled igg4 was applied to the same cancer tissue section without prior application of igg1 it did not react this reaction of igg4 to cancer specific igg1 was not via the antigen specific variable region of igg4 as such igg4 was neither specifically against igg1 nor was it specifically against cancer antigen with its antigen recognizing fab variable region as shown in figure 2a the only explanation was that igg4 reacted to igg1 through its fc region this was validated by subsequent experiments with western blot analysis as shown in figure 3a“ein western blot igg4 was found to react to other iggs igg1 igg2 igg3 and igg4 via an fcfc mechanism and this reaction was across species but igg4 did not react to other ig subtypes igm ige iga or igdto test if and how igg4 could react to igg1 we performed western blot with igg4 from different sources extracted from patients with cancer from normal adults and commercially purchased igg4 was found to react to igg1 igg2 igg3 and igg4 at the molecular weight mw of about kd and this reaction was not seen when igg1 igg2 or igg3 was used as the antibody and igg4 as the target molecule running on the gel the above phenomenon was observed in western blot of both reducing and non reducing conditions figure 3a online supplementary figure s3 however human igg4 did not react to human igm iga igd or ige online supplementary figure s4 nevertheless we found that this reaction was across species that is human igg4 reacted to iggs of human mouse rabbit and goat online supplementary figure s5 we open accessigg4 extracted from a patient with cancer reacted figure to cancer bound igg1 and blocked antibody mediated cancer immunity a upper panel these photos serve as an example of the reactivity of igg1 and igg4 extracted from patients with cancer igg1 from the serum of a patient with breast cancer was labeled with biotin and stained a frozen cancer tissue section of the same patient cancer cells were positively stained by igg1 left the cancer cells were confirmed by their characteristic histopathology of he staining middle igg4 from serum of the same patient labeled with biotin and applied on the same cancer on a consecutive section was completely negative right lower panel another breast cancer positively stained by igg1 from the patient™s serum left the cancer cells were identified by positive immunostaining of cytokeratin ck on a consecutive section middle igg4 from the same patient was not reactive to the same cancer on a consecutive section right b the upper panel illustrates the principle of the experimental reactions and the middle and lower panels show staining results from two patients with breast cancer left igg1 from a patient with cancer positively reacted to frozen cancer tissue of the same patient brown cells middle igg4 from the same patient with cancer applied to consecutive sections but did not react to the same cancer right however when unlabeled igg1 was applied to the same cancer tissue section followed by biotin labeled igg4 the cancer cells were positively stained brown cellsfurther digested human igg4 and igg1 into fab and fc fragments with papain it was found that it was the fc fragment of igg4 reacting to fc of igg1 figure 3b“e this reaction was easy to occur as only min incubation wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access igg4 reacted to igg1 in western blot and tissue figure sections in an fc fc fashion a in western blot non cancer specific igg4 from a patient with breast cancer reacted to igg1 and igg4 from the same patient with cancer right panel arrows however when igg1 and igg4 were run on the gel and biotin labeled igg1 was used as the primary antibody no band was seen left panel these are the same antibodies used in figure a02ab providing support to explain the reaction between igg4 and igg1 seen on cancer tissue b western blot demonstrated that igg4 reacted with igg1 igg2 igg3 and igg4 c igg4 reacted with igg fc fragment but not with fab arm d igg4 reacted with igg1 fc fragment but not with fab arm e biotin labeled igg4 fc fragment reacted to igg1 and igg4 fc fragments but not with igg1 or igg4 fab biotin labeled igg4 fab did not react to igg1 or igg4 fc or fab these results demonstrate that it is the fc region of igg4 that reacted to fc of igg1resulted in a clear band therefore the positive reaction obtained by sequential applications with cancer specific igg1 followed by non cancer specific igg4 on cancer tissue figure 2b had to take place between the fc of igg4 and the fc of igg1 as shown in figure the fcfc reaction between igg4 and igg1 bound to tissue sections was further tested and validated with a number of antibodies and tissue types apart from cancerfollowing the same logic we tested the reactivity between the fc fragments of igg4 and igg1 already demonstrated in western blot on tissue sections we used igg1 primary antibodies to insulin and glucagon in normal human pancreas and antibody to neurofilament in human brain for this test the same principle was established with these normal tissues detailed results and figures are shown in online supplementary figure s6igg4 competed with igg1 to bind to fc receptors of pbmc and macrophageswe performed immunoglobulin and fc receptor binding assays with peripheral blood monocytes pbmc and with a human monocyte cell line u937 procell life science technology china cl0239 igg1 and igg4 were extracted from the serum of patients with cancer and pbmcs were isolated from the same human subjects the extracted and purchased igg1 and igg4 were labeled with biotin or fitc in the igg1 and igg4 binding assay we found that igg1 and igg4 competed with one another in binding to pbmc and this reaction could be completely blocked by fc receptor blocker this competition was concentration dependent that is as the concentration of igg4 increased more igg1 bound to pbmc was replaced the reverse was also true that is igg1 could also replace igg4 in this competition assay this phenomenon was demonstrated on cytospin slides of pbmc preparation online supplementary figure s7in addition flow cytometry was performed to examine the binding properties of igg1 and igg4 to fc receptors of monocytes the ability of igg1 and igg4 to bind to all three subtypes of fc gamma receptor fcγr”fcγr…° cd64 fcγr…± cd32 and fcγr…² cd16”was examined with corresponding antibodies we found that igg4 could compete with igg1 in binding to the fc receptor of monocytes u937 we further found that the binding force of igg1 was about twice as strong as that of igg4 for individual receptor subtypes igg1 could bind to all three receptor subtypes that is fcγr…° cd64 fcγr…± cd32 fcγr…² cd16 in contrast igg4 could only bind to fcγr…° cd64 although their binding sites were different igg4 could completely block igg1 we also found that it was necessary for a relatively high concentration of igg4 to be present in the solution in order to compete with igg1 in binding to fc receptors online supplementary figure s8igg4 inhibited the classic immune reactions of adcc adcp and cdc mediated via cancerspecific igg1 and effector immune cellscomplementswe first verified that non cancer specific igg4 indeed reacted to igg1 cetuximab used in adcc adcp and cdc figure 4a we then found that igg4 inhibited adcc elicited cytotoxicity with cancer specific igg1 antibody and pbmc figure 4b the igg4 used in our test was not directed against cancer antigen or to lymphocytes non cancer specific igg1 could also inhibit adcc but to a much lesser extent also reached statistical significance we obtained evidence to show that inhibition of adcc appeared to take place at the site of the cancer specific antibody that is igg4 reacted to the igg1 antibody bound to cancer cells figure 2b and the site of immune effector fc receptors the latter effect could wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen accessfigure non cancer specific igg4 inhibited classic adcc adcp and cdc reactions against cancer but had no direct effect on cancer cell growth a on western blot the chimeric antibody cetuximab igg1 against egfr was run on the gel and igg4 and igg1 at concentrations of and µgml were used as the primary antibodies igg4 reacted to cetuximab at a concentration dependent manner but igg1 did not react b left in a classic adcc experiment cetuximab igg1 was incubated with an egfr expressing lung cancer cell line a549 and then with pbmc from normal healthy adult cancer cell activity was significantly reduced n12 non cancer specific igg1 and hsa were used as controls showing that they had no direct effect on the cancer cells n12 middle when non cancer specific igg4 was added to the mixture the effect of cetuximab was significantly reversed demonstrating an inhibitory effect of igg4 in adcc n12 non cancer specific igg1 had a much smaller but also significant effect in inhibiting adcc action n12 right when fc receptor blocker was incubated with pbmc the effect of cytotoxicity was blocked c“e adcp was performed with a lung cancer cell line a549 expressing egfr as the targets human peripheral monocyte derived macrophages as the effector cells and the antibody cetuximab igg1 against egfr as the mediating antibody the tumor cells were stained with cfda se fluorescence probes green macrophages derived from pbmc were stained with dii fluorescent probes orange blue fluorescence is the nuclei stained with dapi d higher magnification of c the orange colored macrophages were in close contact with green tumor cells tumor debris ingested by macrophages appeared yellow in the cytoplasm of macrophages e bar chart showing the effect of adcp and its inhibition by igg4 f left in µgml rituximab mediated adcp model giemsa staining results of phagocytosis of raji cells by macrophages after the addition of µgml igg1 and igg4 respectively right igg4 significantly inhibited rituximab mediated adcp in phagocytosis by macrophage but igg1 could not inhibit the adcp effect scale bar30 µm p005 p001 p0001 g in a classic cdc experiment cetuximab anti egfr antibody was incubated with an egfr expressing lung cancer cell line a549 and then with complement co from serum of a normal healthy adult the cancer cell activity was significantly reduced h when non cancer specific igg4 was added to the mixture in the above cdc experiment the effect of cetuximab was significantly reversed i igg4 and igg1 were incubated with kyse150 for hours and no effect on cell growth was found adcc antibody dependent cell mediated cytotoxicity adcp antibody dependent cellular phagocytosis cdc complement dependent cytotoxicity cfse da carboxyfluorescein diacetate succinimidyl ester dapi ' diamidino2 phenylindole egfr epidermal growth factor receptor hsa human serum albumin pbmc peripheral blood mononuclear cellwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access be abolished with the addition of fc receptor blocker human trustain fcx biolegend china to the pbmcwe also performed an adcp experiment employing human monocyte derived macrophages and esophageal cancer cells cetuximab igg1 was used as the mediating antibody this was performed employing a coculture and cell counting method and fitc labeled antibody flow cytometry two models were employed and both methods showed that non cancer specific igg4 was able to reduce the effect of adcp mediated by cancer specific igg1 figure 4c“fin a classic cdc assay we used cancer cell line a549 atcc usa c4215 as the target cancer cells cetuximab as the cancer specific igg1 mediating antibody and human plasma as the source of complements and demonstrated the destructive effect on cancer cells we then used non cancer specific igg4 or igg1 to inhibit the effect we found that the cdc effect was partially inhibited by non cancer specific igg4 but not by igg1 figure 4ghfor comparison we added igg4 or igg1 at various concentrations in cancer cell culture for different periods of time and found no direct effect of these proteins on cancer cell growth figure 4iigg4 including nivolumab significantly accelerated breast cancer cell and colon cancer cell growth in two immune potent mouse models in vivothe above results point to a mechanism that igg4 plays an important role in local immune evasion by blocking immune responses mediated by cancer specific igg antibodies to further examine this mechanism mediated by such antibodies we performed in vivo studies to verify this hypothesis with immune competent mouse models in one model we injected non cancer specific igg4 into a location where breast cancer cells were inoculated subcutaneously in this group of mice cancer cell growth was significantly increased resulting in a much larger cancer mass by days in comparison with other groups injections of pbs or igg without igg4 figure 5ab as there is no direct effect of igg4 on cancer cell growth figure 4i these results unequivocally confirmed that igg4 can inhibit local immune reaction and thereby promote cancer growth in vivo through immune evasionin a separate but similar experiment of a colon cancer mouse model we injected antibody to programmed cell death1 pd1 nivolumab which is a widely used drug in cancer immune therapy and is also an igg4 isotype with s228p mutation which replaces a serine residu

cancer7509

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " recent impressive advances in cancer immunotherapy have been largely derived from cellular immunity the role of humoral immunity in carcinogenesis has been less understood based on our previous observations we hypothesize that an immunoglobulin subtype igg4 plays an essential role in cancer immune evasionmethods the distribution abundance actions properties and possible mechanisms of igg4 were investigated with human cancer samples and animal tumor models with an extensive array of techniques both in vitro and in vivoresults in a cohort of patients with esophageal cancer we found that igg4 containing b lymphocytes and igg4 concentration were significantly increased in cancer tissue and igg4 concentrations increased in serum of patients with cancer both were positively related to increased cancer malignancy and poor prognoses that is more igg4 appeared to associate with more aggressive cancer growth we further found that igg4 regardless of its antigen specificity inhibited the classic immune reactions of antibody dependent cell mediated cytotoxicity antibody dependent cellular phagocytosis and complement dependent cytotoxicity against cancer cells in vitro and these effects were obtained through its fc fragment reacting to the fc fragments of cancer specific igg1 that has been bound to cancer antigens we also found that igg4 competed with igg1 in reacting to fc receptors of immune effector cells therefore locally increased igg4 in cancer microenvironment should inhibit antibody mediated anticancer responses and help cancer to evade local immune attack and indirectly promote cancer growth this hypothesis was verified in three different immune potent mouse models we found that local application of igg4 significantly accelerated growth of inoculated breast and colorectal cancers and carcinogen induced skin papilloma we also tested the antibody drug for cancer immunotherapy nivolumab which was igg4 in nature with a stabilizing s228p mutation and found that it significantly promoted cancer growth in mice this may provide an explanation to the newly appeared hyperprogressive disease sometimes associated with cancer immunotherapy there appears to be a previously unrecognized immune evasion mechanism with igg4 playing an essential role in cancer microenvironment with implications in cancer diagnosis and immunotherapyintroductionwhile new immune therapy for cancer focuses mostly on manipulating cellular immunity humoral immunity also holds great promise for cancer therapy1 igg4 is a unique antibody that has the lowest concentration among igg subtypes in healthy individuals and its function has not been well understood3“ igg4 was regarded as a ˜blocking antibody™ because of its reduced ability to trigger effector immune reactions6 therefore whatever molecules igg4 reacts to the subsequent immune reaction was subdued8 igg4 has a unique structure of fab arm exchange fae in which the two heavy and light chains of two different antibodies with different specificities are exchanged resulting in an asymmetric bispecific antibody with reduced ability to bind to antigen and to form immune complexes9 another unique feature of igg4 is that it can react to other iggs via its fc fragment and the significance of this property has not been well understood evidence suggests that fae and fc fc reactivity may involve the same molecular structure on igg4 molecule10 davies et al11 resolved the crystal structure of igg4 fc fragment revealing a unique molecular conformation supporting its fc binding property recent interests in igg4 related diseases unveiled a wide range of pathologies with a common phenomenon of often increased igg4 concentration in the serum and igg4 postive plasma cells in the affected ans accompanied by local inflammation and fibrosis but its pathogenic mechanism is still poorly understood13“to cite wang a0h xu a0q zhao a0c et a0al an immune evasion mechanism with igg4 playing an essential role in cancer and implication for immunotherapy for immunotherapy of cancer 20208e000661 101136jitc2020000661 –º additional material is published online only to view please visit the online http dx jitc hw qx cz and zz are joint first authorsaccepted july authors or their employers re use permitted under cc by nc no commercial re use see rights and permissions published by bmjfor numbered affiliations see end of correspondence toprofessor jiang gu qq comwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access the possible functions of igg4 in cancer and also in the immune system have not been well elucidated increases of igg4 positive plasma cells were reported in gastrocarcinoma16 extrahepatic cholangiocarcinoma17 and melanoma18 the above studies were performed on limited number of cases without convincing explanation of mechanism or significance wu 19 reported that serum igg4igg ratio could predict recurrence of hepatocellular carcinoma after surgery the most extensive study on igg4 and cancer was performed by karagiannis 20 who investigated the possible effect of cancer specific igg4 in inhibiting cancer immunity in melanomas and suggested competition between cancer specific igg4 and igg1 in binding to cancer antigens as the cause for the inhibition a recent report raised the concept of cancer educated b cells and toxic igg produced by these cells in facilitating lymph node metastasis for breast cancer in a mouse model21 we performed a multidimensional investigation of igg4 in a wide array of patients with cancer and tissues with both in vitro and in vivo experiments extensive new evidence led us to hypothesize that there is a potent humoral immune editing mechanism in cancer microenvironment with igg4 playing an essential role we propose that fc fc reaction could be the basic mechanism of this immune inhibition we validated this in three immune potent animal models this property was also found applicable to cancer immunotherapy drug nivolumab which was igg4 in nature our study points to a so far little appreciated mechanism of immune evasion in cancermaterials and methodskey resourcesdetailed information about key resources including antibodies biological samples chemicals assay kits cell lines and software are shown in online supplementary table experimental model and subject detailspatients and healthy volunteerswe collected tissue and blood samples from over patients with cancer in shantou university affiliated tumor hospital and the east guangdong provincial pathological consultation center details of the human samples are shown in online supplementary table immunohistochemistrydetails of the protocols and the antibodies are shown in online supplementary datasds techniquethe expressions of igg1 igg2 igg3 and igg4 in esophageal cancer were detected at the histological level the stain decolorize stain sds technology22 was performed on the same slide sequentially with four different antibodies to demonstrate the four antigens the distribution pattern abundance and relationship of the four antigens were processed with an image software photoshop to achieve an integrated figure the proximity of different cell types on the tissue sections was measured with an image analyzing software image pro plus v60 details of the protocol are presented in the online supplementary dataimmunofluorescence double stainingtwo antibodies from different species were incubated on the same tissue section primary antibodies were detected with goat antimouse 555antirabbit igg or goat antimouse 488antirabbit igg alexa fluor life sciences and immunoreactivity was visualized with a fluorescence microscope antigens were detected and demonstrated with two fluorescence signals in red and green and the blue signal of ' diamidino2 phenylindole dapi as for cell nuclei images were acquired with the evos fl fluorescence microscope life technologies usaigg4 immunohistochemistryto verify that igg4 could react to igg1 that had been immobilized to tissue sections we used human pancreas and brain and antibodies to insulin glucagon and neurofilament as models detailed protocol of this experiment is shown in the online supplementary dataimmunoglobulin preparationsfab and fc fragments of igg igg1 and igg4 were prepared with papain digestion in the presence of cysteine iggs were cleaved at a position above the hinge region by papain at °c for hours after purification with protein a affinity chromatography pure fab and fc fragments were isolated with elution buffer igg fc fragment was washed down from protein a column after centrifugation and concentration measurement igg preparations were collected and stored at °c before usewestern blotigg subclasses were resolved on sodium dodecyl sulfate polyacrylamide gels under reducing conditions and then transferred onto nitrocellulose membranes ge healthcare life sciences the membranes were blocked for hour with bovine serum albumin bsa in tris buffered saline with tween20 ph and then incubated with primary antibody biotin labeling kit anaspec overnight at °c it was then incubated with secondary antibody for hour at room temperature finally reaction density was measured with odyssey western at nm and nm detection channelserum igg4 and igg assessmentsera samples collected from patients with esophageal cancer and healthy volunteers were sent to golden field medical test company guangzhou china and roche immune turbidimetry method was used to wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cmeasure serum igg4 and total igg concentrations all serum samples were stored at ˆ’°c freezer immediately before analysis all quantitative data were treated statisticallymeasurement of igg4 concentrations in tumor and tumoradjacent normal tissues with elisaconcentrations of igg4 in pairs of esophageal cancer and adjacent normal tissues cm from the edge of the cancer mass were measured with elisa detailed protocol of this experiment is shown in the online supplementary datacell culture for fc receptor binding assaysu937 cell line was bought from procell life science technology china cl0239 and cells were cultured in rpmi gibco c22400500bt supplemented with fetal bovine serum gibco “ and uml penicillin streptomycin gibco “ at ×106ml in ml cell culture bottleprotein preparationsdetails of the protocols for protein preparation separation and papain digestion are shown in online supplementary datafluorescence activating cell sorter facs for fc receptor assaysdetails of the relevant protocols are shown in online supplementary dataadcc adcp and cdc teststhe classic antibody dependent cell mediated cytotoxicity adcc antibody dependent cellular phagocytosis adcp and complement dependent cytotoxicity cdc tests were performed based on previously reported protocols23“ non specific igg4 instead of cancer specific igg4 was used to inhibit these reactions igg1 was used as control details of the protocols are shown in online supplementary dataanimal modelsbreast cancer and colon cancer modelsbalbc mice were obtained from vital river technical beijing china mice aged between and weeks and weighed ± g were used in all experiments all mice were inoculated with 4t1 mouse breast cancer cells or ct26 mouse colon cancer cells subcutaneously under the left forearm × 4t1 cells per mouse to build cancer models the mice were divided into different groups and were treated with igg1 or igg4 derived from intravenous igg ivig ivig without igg4 nivolumab and fc of nivolumab respectively details of the protocols are shown in online supplementary datacarcinogeninduced skin tumor modelwe employed a well established carcinogen induced skin tumor model to study the effect of igg4 and ivig without igg4 in comparison with controls phosphate buffered open accesssaline pbs detailed protocol is shown in online supplementary dataquantification and statistical analysisdata were analyzed in graphpad prism all reported p values were derived from two sided comparisons with values of less than considered to be statistically significantresultsigg4 was significantly increased in the serum of patients with cancer and this increase was related to cancer stage and patient prognosiswe first measured the concentrations of igg1 igg2 igg3 and igg4 in sera of patients with esophageal cancer n82 igg4 was significantly increased in patients with cancer in comparison with normal healthy subjects n70 the concentration of igg4 was increased from about to the ratio of igg4iggtotal was also significantly increased the statistical significance of both reached p00001 the increase of igg4 was also positively correlated to the stages of cancer with late stage cancers having more obvious increases higher igg4 serum concentrations were associated with worse prognosis figure 1a“digg4containing lymphocytes and igg4 concentration were significantly increased in cancer microenvironment and this increase was associated with cancer cell infiltrationigg4 positive lymphocytes were significantly increased in cancer microenvironment in comparison with tissue more distant to the cancer mass igg4 positive lymphocytes were barely detectable in tumor adjacent normal tissue figure 1h on the same tissue sections employing the sds technique22 to simultaneously visualize the four subtypes of igg with four distinct colors we found that one plasma cell only contained one igg subtype that is igg1 igg2 igg3 and igg4 were all contained in their own plasma cell populations separately figure 1i the marked increase of igg4 containing plasma cells in comparison with other subtypes was clearly visualized on cancer tissue sections igg4 positive plasma cells appeared to accumulate more in tissues with extensive cancer cell infiltration than in other areas figure 1eh in addition igg4 positive cells are often in close proximity to cells containing igg1 and igg2 but not to igg3 this phenomenon was not seen among other igg subtypes apart from igg4 quantitative data of the proximity of different cell types are presented in online supplementary figure s2 with the multiple immunostaining method we also demonstrated that igg1 containing and igg4 containing lymphocytes were distinct from cd3 positive t lymphocytes in the same region of the cancer tissue figure 1j in addition we measured the concentrations of igg4 in cancer tissue and cancer adjacent normal tissue n46 pairs the average concentration of igg4 in cancer tissue was about four times higher than that in the adjacent wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access figure significant increase of igg4 and igg4iggtotal in serum and igg4 positive b lymphocytes in esophageal cancer a igg4 in serum of esophageal squamous cell cancer escc n82 was significantly higher when compared with healthy controls n70 p00001 b igg4iggtotal ratio in escc n82 was significantly higher than that in matching healthy adults n70 p00001 c igg4 in stage …³ n18 was significantly higher than those in stages …° and …± n16 p001 d igg4iggtotal in serum of stage iv escc n18 was significantly higher than those in stages …° and …± n16 p005 e scatter diagram of igg4 positive cell numbers in cancer cancer adjacent normal tissue adjacent and normal tissues igg4 positive lymphocytes in and around the esophageal cancer mass n110 are significantly more abundant than those in the adjacent normal tissue n60 and normal lymphoid tissues n63 p0001 for both increases of igg4 positive lymphocytes were most abundant in areas of cancer cell proliferation f the increase of igg4 positive cell numbers was related to the prognoses of the patients more igg4 positive cells were associated with worse outcome p005 g igg4 concentration in cancer tissue n46 was significantly higher than that in adjacent normal tissue n46 p001 h immunohistochemistry of igg4 in esophageal cancer tissues from left to right are igg4 in cancer tissues cancer adjacent tissue and normal lymphoid tissue tonsil it clearly demonstrates that igg4 positive lymphocytes red were markedly increased left in comparison with normal lymphoid tissue right and with tumor adjacent normal tissue middle scale bar µm i demonstration of four subpopulations of igg containing plasma cells with multiple immunostaining sds method in cancer each subclass has its own distribution pattern and one plasma cell only produces one subclass of igg igg1 yellow igg2 green igg3 purple igg4 red j on the same tissue section a triple immunostaining was performed with the sds method to demonstrate the distribution and relationship among cd3 positive t cells yellow igg1 positive b cells greens and igg4 positive b cells red each cell type has its own distribution and no overlap between different cell types is observed sds stain decolorize stainwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cnormal tissue and the difference between these two groups was statistically significant p001 figure 1gigg1 extracted from patients with cancer reacted to cancer cells of the same patients but igg4 extracted from the same patients did notwe extracted igg1 and igg4 from the serum of patients with esophageal cancer with breast cancer and with colon cancer with respective specific antibody columns we then labeled the antibodies with biotin and tested the reactivity of these extracted antibodies to cancer tissue sections of the same patients in all cases igg1 reacted to cancer cells from the same patients but igg4 did not figure 2a it appeared that the increased igg4 in cancer microenvironment and in the patient™s serum was not reactive to cancer antigens while igg1 extracted from the same patients reacted to the cancer antigensigg4 reacted to cancerspecific igg1 that was bound to cancer cellsalthough igg4 extracted from patients with cancer did not react to cancer antigen it did react to cancer specific igg1 that was bound to cancer antigen on tissue sections as shown in figure 2b when cancer specific igg1 that was not labeled with biotin was applied to cancer tissue sections followed by biotin labeled igg4 the cancer cells became positive while when the same biotin labeled igg4 was applied to the same cancer tissue section without prior application of igg1 it did not react this reaction of igg4 to cancer specific igg1 was not via the antigen specific variable region of igg4 as such igg4 was neither specifically against igg1 nor was it specifically against cancer antigen with its antigen recognizing fab variable region as shown in figure 2a the only explanation was that igg4 reacted to igg1 through its fc region this was validated by subsequent experiments with western blot analysis as shown in figure 3a“ein western blot igg4 was found to react to other iggs igg1 igg2 igg3 and igg4 via an fcfc mechanism and this reaction was across species but igg4 did not react to other ig subtypes igm ige iga or igdto test if and how igg4 could react to igg1 we performed western blot with igg4 from different sources extracted from patients with cancer from normal adults and commercially purchased igg4 was found to react to igg1 igg2 igg3 and igg4 at the molecular weight mw of about kd and this reaction was not seen when igg1 igg2 or igg3 was used as the antibody and igg4 as the target molecule running on the gel the above phenomenon was observed in western blot of both reducing and non reducing conditions figure 3a online supplementary figure s3 however human igg4 did not react to human igm iga igd or ige online supplementary figure s4 nevertheless we found that this reaction was across species that is human igg4 reacted to iggs of human mouse rabbit and goat online supplementary figure s5 we open accessigg4 extracted from a patient with cancer reacted figure to cancer bound igg1 and blocked antibody mediated cancer immunity a upper panel these photos serve as an example of the reactivity of igg1 and igg4 extracted from patients with cancer igg1 from the serum of a patient with breast cancer was labeled with biotin and stained a frozen cancer tissue section of the same patient cancer cells were positively stained by igg1 left the cancer cells were confirmed by their characteristic histopathology of he staining middle igg4 from serum of the same patient labeled with biotin and applied on the same cancer on a consecutive section was completely negative right lower panel another breast cancer positively stained by igg1 from the patient™s serum left the cancer cells were identified by positive immunostaining of cytokeratin ck on a consecutive section middle igg4 from the same patient was not reactive to the same cancer on a consecutive section right b the upper panel illustrates the principle of the experimental reactions and the middle and lower panels show staining results from two patients with breast cancer left igg1 from a patient with cancer positively reacted to frozen cancer tissue of the same patient brown cells middle igg4 from the same patient with cancer applied to consecutive sections but did not react to the same cancer right however when unlabeled igg1 was applied to the same cancer tissue section followed by biotin labeled igg4 the cancer cells were positively stained brown cellsfurther digested human igg4 and igg1 into fab and fc fragments with papain it was found that it was the fc fragment of igg4 reacting to fc of igg1 figure 3b“e this reaction was easy to occur as only min incubation wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access igg4 reacted to igg1 in western blot and tissue figure sections in an fc fc fashion a in western blot non cancer specific igg4 from a patient with breast cancer reacted to igg1 and igg4 from the same patient with cancer right panel arrows however when igg1 and igg4 were run on the gel and biotin labeled igg1 was used as the primary antibody no band was seen left panel these are the same antibodies used in figure a02ab providing support to explain the reaction between igg4 and igg1 seen on cancer tissue b western blot demonstrated that igg4 reacted with igg1 igg2 igg3 and igg4 c igg4 reacted with igg fc fragment but not with fab arm d igg4 reacted with igg1 fc fragment but not with fab arm e biotin labeled igg4 fc fragment reacted to igg1 and igg4 fc fragments but not with igg1 or igg4 fab biotin labeled igg4 fab did not react to igg1 or igg4 fc or fab these results demonstrate that it is the fc region of igg4 that reacted to fc of igg1resulted in a clear band therefore the positive reaction obtained by sequential applications with cancer specific igg1 followed by non cancer specific igg4 on cancer tissue figure 2b had to take place between the fc of igg4 and the fc of igg1 as shown in figure the fcfc reaction between igg4 and igg1 bound to tissue sections was further tested and validated with a number of antibodies and tissue types apart from cancerfollowing the same logic we tested the reactivity between the fc fragments of igg4 and igg1 already demonstrated in western blot on tissue sections we used igg1 primary antibodies to insulin and glucagon in normal human pancreas and antibody to neurofilament in human brain for this test the same principle was established with these normal tissues detailed results and figures are shown in online supplementary figure s6igg4 competed with igg1 to bind to fc receptors of pbmc and macrophageswe performed immunoglobulin and fc receptor binding assays with peripheral blood monocytes pbmc and with a human monocyte cell line u937 procell life science technology china cl0239 igg1 and igg4 were extracted from the serum of patients with cancer and pbmcs were isolated from the same human subjects the extracted and purchased igg1 and igg4 were labeled with biotin or fitc in the igg1 and igg4 binding assay we found that igg1 and igg4 competed with one another in binding to pbmc and this reaction could be completely blocked by fc receptor blocker this competition was concentration dependent that is as the concentration of igg4 increased more igg1 bound to pbmc was replaced the reverse was also true that is igg1 could also replace igg4 in this competition assay this phenomenon was demonstrated on cytospin slides of pbmc preparation online supplementary figure s7in addition flow cytometry was performed to examine the binding properties of igg1 and igg4 to fc receptors of monocytes the ability of igg1 and igg4 to bind to all three subtypes of fc gamma receptor fcγr”fcγr…° cd64 fcγr…± cd32 and fcγr…² cd16”was examined with corresponding antibodies we found that igg4 could compete with igg1 in binding to the fc receptor of monocytes u937 we further found that the binding force of igg1 was about twice as strong as that of igg4 for individual receptor subtypes igg1 could bind to all three receptor subtypes that is fcγr…° cd64 fcγr…± cd32 fcγr…² cd16 in contrast igg4 could only bind to fcγr…° cd64 although their binding sites were different igg4 could completely block igg1 we also found that it was necessary for a relatively high concentration of igg4 to be present in the solution in order to compete with igg1 in binding to fc receptors online supplementary figure s8igg4 inhibited the classic immune reactions of adcc adcp and cdc mediated via cancerspecific igg1 and effector immune cellscomplementswe first verified that non cancer specific igg4 indeed reacted to igg1 cetuximab used in adcc adcp and cdc figure 4a we then found that igg4 inhibited adcc elicited cytotoxicity with cancer specific igg1 antibody and pbmc figure 4b the igg4 used in our test was not directed against cancer antigen or to lymphocytes non cancer specific igg1 could also inhibit adcc but to a much lesser extent also reached statistical significance we obtained evidence to show that inhibition of adcc appeared to take place at the site of the cancer specific antibody that is igg4 reacted to the igg1 antibody bound to cancer cells figure 2b and the site of immune effector fc receptors the latter effect could wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen accessfigure non cancer specific igg4 inhibited classic adcc adcp and cdc reactions against cancer but had no direct effect on cancer cell growth a on western blot the chimeric antibody cetuximab igg1 against egfr was run on the gel and igg4 and igg1 at concentrations of and µgml were used as the primary antibodies igg4 reacted to cetuximab at a concentration dependent manner but igg1 did not react b left in a classic adcc experiment cetuximab igg1 was incubated with an egfr expressing lung cancer cell line a549 and then with pbmc from normal healthy adult cancer cell activity was significantly reduced n12 non cancer specific igg1 and hsa were used as controls showing that they had no direct effect on the cancer cells n12 middle when non cancer specific igg4 was added to the mixture the effect of cetuximab was significantly reversed demonstrating an inhibitory effect of igg4 in adcc n12 non cancer specific igg1 had a much smaller but also significant effect in inhibiting adcc action n12 right when fc receptor blocker was incubated with pbmc the effect of cytotoxicity was blocked c“e adcp was performed with a lung cancer cell line a549 expressing egfr as the targets human peripheral monocyte derived macrophages as the effector cells and the antibody cetuximab igg1 against egfr as the mediating antibody the tumor cells were stained with cfda se fluorescence probes green macrophages derived from pbmc were stained with dii fluorescent probes orange blue fluorescence is the nuclei stained with dapi d higher magnification of c the orange colored macrophages were in close contact with green tumor cells tumor debris ingested by macrophages appeared yellow in the cytoplasm of macrophages e bar chart showing the effect of adcp and its inhibition by igg4 f left in µgml rituximab mediated adcp model giemsa staining results of phagocytosis of raji cells by macrophages after the addition of µgml igg1 and igg4 respectively right igg4 significantly inhibited rituximab mediated adcp in phagocytosis by macrophage but igg1 could not inhibit the adcp effect scale bar30 µm p005 p001 p0001 g in a classic cdc experiment cetuximab anti egfr antibody was incubated with an egfr expressing lung cancer cell line a549 and then with complement co from serum of a normal healthy adult the cancer cell activity was significantly reduced h when non cancer specific igg4 was added to the mixture in the above cdc experiment the effect of cetuximab was significantly reversed i igg4 and igg1 were incubated with kyse150 for hours and no effect on cell growth was found adcc antibody dependent cell mediated cytotoxicity adcp antibody dependent cellular phagocytosis cdc complement dependent cytotoxicity cfse da carboxyfluorescein diacetate succinimidyl ester dapi ' diamidino2 phenylindole egfr epidermal growth factor receptor hsa human serum albumin pbmc peripheral blood mononuclear cellwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access be abolished with the addition of fc receptor blocker human trustain fcx biolegend china to the pbmcwe also performed an adcp experiment employing human monocyte derived macrophages and esophageal cancer cells cetuximab igg1 was used as the mediating antibody this was performed employing a coculture and cell counting method and fitc labeled antibody flow cytometry two models were employed and both methods showed that non cancer specific igg4 was able to reduce the effect of adcp mediated by cancer specific igg1 figure 4c“fin a classic cdc assay we used cancer cell line a549 atcc usa c4215 as the target cancer cells cetuximab as the cancer specific igg1 mediating antibody and human plasma as the source of complements and demonstrated the destructive effect on cancer cells we then used non cancer specific igg4 or igg1 to inhibit the effect we found that the cdc effect was partially inhibited by non cancer specific igg4 but not by igg1 figure 4ghfor comparison we added igg4 or igg1 at various concentrations in cancer cell culture for different periods of time and found no direct effect of these proteins on cancer cell growth figure 4iigg4 including nivolumab significantly accelerated breast cancer cell and colon cancer cell growth in two immune potent mouse models in vivothe above results point to a mechanism that igg4 plays an important role in local immune evasion by blocking immune responses mediated by cancer specific igg antibodies to further examine this mechanism mediated by such antibodies we performed in vivo studies to verify this hypothesis with immune competent mouse models in one model we injected non cancer specific igg4 into a location where breast cancer cells were inoculated subcutaneously in this group of mice cancer cell growth was significantly increased resulting in a much larger cancer mass by days in comparison with other groups injections of pbs or igg without igg4 figure 5ab as there is no direct effect of igg4 on cancer cell growth figure 4i these results unequivocally confirmed that igg4 can inhibit local immune reaction and thereby promote cancer growth in vivo through immune evasionin a separate but similar experiment of a colon cancer mouse model we injected antibody to programmed cell death1 pd1 nivolumab which is a widely used drug in cancer immune therapy and is also an igg4 isotype with s228p mutation which replaces a serine residu Answer:

Colon_Cancer

" tumor associated macrophages tam constitute the most abundant immune cells in the tumor stroma initiating pro inflammatory m1 or immunosuppressive m2 responses depending on their polarization status advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patientsmethods to test the effects of radiotherapy on tam we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry we furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short course radiotherapy and compared findings to non pretreated rectal cancer using an immunostaining approach anotypic assays ota consisting of macrophages cancer associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophagesresults we demonstrate that short course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of tam towards an m1 like pro inflammatory phenotype in addition ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for t cell activation in ota we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles ev derived from irradiated tumor cells we identified high mobility group box in ev from irradiated tumor cells as a potential effector signal in that crosstalks our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro inflammation our data indicate that clinically applied short term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategiesintroductionsince the introduction of immune checkpoint blockade immunotherapy has become an attractive therapeutic option in cancer1“ irradiation used as standard therapy in a number of solid malignancies induces immunogenic cell death icd by the release of damage associated pattern damp4 studies based on murine models indicated that irradiation induced dna damage of tumor cells elicited an antitumor immune response5 it was shown that alterations of the infiltrating immune cells by irradiation might be augmented when combined with immune modulating drugs6“ a detailed understanding of the impact of radiotherapy on the immune system in humans should allow application of radiotherapy as a part of novel immunotherapeutic concepts however little is known about the detailed regulation of the immunogenic effect of irradiation in clinical settingsmacrophages are one of the most abundant immune cell subsets in tumor tissue9 and play a key role in the cancer microenvironment11 tumor associated macrophages tam are functionally diverse13 and display high plasticity upon immunological stimuli14 the concept of m1 and m2 macrophages was introduced to describe the heterogeneity of this cell subset16 m1 like macrophages possess the capacity to clear infections in support of a t helper type th1 immune response17 whereas m2 like macrophages respond in general to th2 cytokines and are strongly enriched in the tumor microenvironment18 the concept of m1m2 has been challenged and is seen as an oversimplified approach to the phenotype of macrophages but can be viewed as a linear scale on which m1 and m2 present two extremes19 tam recognize damp and respond by producing a variety of cytokines and growth factors to promote innate and stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access adaptive immunity9 in response to these signals macrophages are able to undergo reprogramming with enhanced antitumor features making them an attractive target for anticancer therapies22 there are conflicting results with respect to the effect of irradiation on the macrophage phenotype klug et al demonstrated in a murine model of breast cancer and human pancreatic cancer that low dose irradiation gy induced repolarization of m2 like macrophages to m1 like macrophages via induction of nitric oxide synthase inos24 moreover expression of inos correlated with vessel normalization and an influx of cd8 t cells suggesting a tumor ablative as well as pro inflammatory effect of repolarized macrophages upregulation of inos was also observed in murine prostate cancer which has been irradiated with up to gy25 similarly agonists of the toll like receptor further stimulated the m1 phenotype of macrophages and enhanced the antitumor effects of irradiation in a murine model of breast cancer26 other studies suggested that irradiation of tumors was associated with a more immunosuppressive phenotype of tam27“ jones found that the depletion of macrophages by an anti csf antibody greatly increased tumor ablation upon irradiation gy in murine tumors generated from a colorectal and a pancreatic cell line obviously the effect of irradiation appears to depend on the model irradiation dose tissue as well as on the investigated time point after irradiation however despite the controversial reports an important role for tam in response to radiotherapy seems evidentmore recently extracellular vesicles ev have attracted attention in mediating signals to immune cells ev are rich in molecular cargos and are emerging as critical messengers in the cell to cell crosstalk they contain a variety of small signaling molecules which can be transferred to other cell types to modulate cell functions30“ thus we hypothesized that ev might be involved in macrophages regulation in the tumor microenvironment in irradiated cells using a clinically relevant approach we show that short course irradiation increased the proportion of m1 like tam in human rectal cancer tissue primary short term cultures and anotypic tumor assays ota consisting of tumor cell lines macrophages and cancer associated fibroblasts allowed to further dissect irradiation induced changes in colorectal tam using irradiated tumor cell lines we demonstrate that ev are able to mediate irradiation induced repolarization of macrophagesmethodspatients and tissue materialpatients with clinical t3 rectal cancer patients characteristics online supplementary table received neoadjuvant hyperfractionated radiotherapy over the course of days within the frame of a controlled clinical study34 as a control group we used historical surgical tumor specimen of non pretreated rectal cancer lesions of patients from the same institution with no history of irradiation therapy or cytoablative treatment for immunofluorescence and immunohistochemical stainings surgical paraffin embedded specimen of the cancer lesions were cut in µm sections and mounted on slides for ex vivo cultures and subsequent flow cytometric stainings rectal cancer tissue was obtained from patients with histologically verified rectal cancer with no history of irradiation therapy or cytoablative treatment studies involving patient material were performed according to the declaration of helsinki and approved by the local ethics committee of the medical university of vienna for ota hct116 ccl147 and dld1 ccl221 were purchased from atcc peripheral blood mononuclear cells for macrophage differentiation were isolated from healthy blood donorsprimary ex vivo cultures of leucocytes and flow cytometrytissue samples of non irradiated rectal cancer were minced resuspended in rpmi medium with fetal bovine serum and plated in a mm petri dish irradiation protocol was applied as indicated below after hour of incubation °c co2 a single cell suspension from cancer tissue was prepared briefly tissue was digested with collagenase iv unitsml and dnase i mgml for hour min in rpmi supplemented with fetal bovine serum and hepes buffer solution at °c afterwards cell suspension was rinsed through a µm mesh and leucocytes were isolated by density gradient centrifugation using ficoll gradients for flow cytometry analysis cells were stained with fluorescence antibodies listed in the online supplementary table livedead discrimination was performed with fixable viability dye biolegend samples were acquired on a facsaria iii bd and analyzed with flowjo software v1061irradiation protocola theratron mds nordion radiotherapy unit with a cobalt60 source was used for γirradiation of ota ex vivo cultures of rectal cancer tissue monocyte derived macrophages and cancer cell lines tissue samples of rectal cancer were minced and cultivated petri dishes with cancer tissue were irradiated with × gy after hours of incubation a single cell suspension was prepared ota and cancer cell lines hct116 and dld1 were irradiated with × gy or × gy and subsequently cultivated over the course of hours monocyte derived macrophages were irradiated with gy or gy a negative control gy was always transported to the radiotherapy unit but was not exposed to γirradiationex vivo phagocytosis assaymononuclear cells isolated of rectal cancer lesions were spun down at g for min at °c for ex vivo escherichia coli e coli phagocytosis assay irradiated and non irradiated leucocytes were resuspended in µl phrodo red e coli bioparticles thermo fisher scientific as per the manufacturer™s instruction a same set of cells were resuspended in phosphate buffered saline pbs as controls after hours incubation at °c stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessleucocytes were washed and stained for flow cytometry tam cd11b cd14 hla dr viable cd45 cells that were pe high were considered to be phagocytosing for ex vivo tumor cell phagocytosis assay tam were isolated via fluorescence activated cell sorting facs cd11b cd14 hla dr viable cd45 cells from healthy colon mucosa or colorectal cancer lesions and incubated overnight with ev isolated from irradiated × gy and non irradiated gy dld1 cells evtam after washing dld1 were labeled with carboxyfluorescein diacetate succinimidyl ester cfse thermo fisher µm and cocultured with tam at an effector to target cell ratio of the proportion of cfse tam was assessed by flow cytometry after hours of incubation at °cat the air liquid interface in dmem supplemented with egm for seeding of tumor cells nylon discs with collagen cell cylinders were transferred into well plates five hundred microlitres of a tumor cell suspension with × cellsml hct116 or dld1 were added to each well after hours the tumor cells attached to the surface of the collagen gels and the nylon meshes with the tumor cell colonized collagen cell cylinders were put back onto the metal grids and incubated for days with media changes every days prior to further experiments for further assessment ota were embedded in optimal cutting temperature media snap frozen in liquid nitrogen and stored at “°c until processing or fixed and embedded in paraffinimmunofluorescence and immunohistochemical stainingdirectly and indirectly labeled monoclonal antibodies as listed in online supplementary table and dapi as a nuclear marker were used an isotype was used as negative control in brief after incubation with the primary antibodies overnight at °c an appropriate secondary fluorescence labeled antibody was applied for min at room temperature followed by staining of dapi nuclear marker for immunohistochemical staining antibodies were mixed with bovine serum albumin in pbs and applied overnight to sections in a humid chamber at °c for visualization of the cells aec was used as chromogen sections were counter stained with hematoxylin for evaluation of staining results images of whole tissue sections one section per patient were acquired using a z1 axio observer microscope equipped with a ld plan neofluar × objective zeiss for leucocyte evaluation the tumor normal interface mm on tumor and normal zone was defined as regions of interests roi for γh2ax staining only tumor tissue was selected as roi roi were automatically quantified using tissuefaxstissuequest image analysis software tissuegnostics gmbhpreparation of otaota were set up as previously described35 briefly cancer associated fibroblasts caf were isolated and cultured from fresh samples of colon adenocarcinomas for the preparation of macrophages monocytes were isolated using the easysep direct human monocyte isolation kit stemcell technologies according to the manufacturer™s instructions × caf and monocytes per ota were mixed and pelleted by centrifugation at g for min for the collagen gel preparation all steps were performed on ice collagen solutions were prepared by mixing mgml of collagen i rat tail mgml becton dickinson and the fibroblastmonocyte suspension in dulbecco's modified eagle medium dmem supplemented with endothelial cell growth medium mv2 egm promocell a total of µl each of the collagen cell suspension were transferred into silicone gel casting devices after polymerization of the collagen solution the gels were lifted up on nylon mesh discs and transferred onto metal grids in well plates and cultured macrophage differentiationpbmc were obtained by ficoll plaque density gradient centrifugation pbmc were seeded at a concentration of ×106well well plates in rpmi medium monocytes were isolated using the ability of monocytes to adhere to non tissue culture treated plastic culture dishes attached cells were cultivated in rpmi medium with glutamax thermo fisher scientific supplemented with ngml macrophage colony stimulating factor m csf thermo fisher scientific fetal bovine serum uml penicillin uml streptomycin and µgml fungizone in a humidified atmosphere at °c cells were cultivated for days with two medium changes to obtain m1 lpsifnγ polarized macrophages cells were stimulated with ngml lipopolysaccharide lps sigma aldrich and ngml interferonγ ifnγ thermo fisher scientific for hours m2 il4il13 polarized macrophages were generated using ngml interleukin il4 strathmann and ngml il13 biolegendev preparation and characterizationev were isolated by differential centrifugation hct116 and dld1 cells were cultured in mccoy™s 5a medium supplemented with exosome depleted fetal calf serum briefly cancer cell line culture medium was centrifuged at g for min to pellet cells supernatant was transferred to new falcon tubes and then centrifuged at g for min to pellet dead cells and apoptotic bodies rotanta 460rc hettich supernatant was transferred to the high speed centrifugation tubes and large ev were removed by ultracentrifugation at g for min sorvall evolution rc thermo fisher scientific after filtration of the supernatant through a µm syringe filter small ev were pelleted by ultracentrifugation at g for min rotor t1250 suspended in ice cold pbs and collected after another ultracentrifugation at g for min rotor type sorvall wx ultra thermo fisher scientific the ev pellet was resuspended in cold pbs for further use ev count was determined using a nanoparticle tracking analyzer zetaview particlemetrix results were analyzed with zetaview software ev were added to macrophages at a stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access concentration of ×× cells we have submitted all relevant data of our experiments to the ev track knowledgebase ev track id ev20006537western blotcells were scraped from cell culture plates washed in ice cold pbs and lysed in × ripa buffer mm tris“hcl ph mm nacl np40 sodium deoxycholate sds containing × halt protease inhibitor cocktail thermo fisher scientific ev suspensions were mixed with × ripa buffer containing × halt protease inhibitor cocktail to obtain × concentration protein extracts were incubated at °c for min and centrifuged at g for min clear protein extract supernatants were quantified with the bca protein assay kit thermo fisher scientific one to five micrograms of protein were denatured with laemmli buffer loaded into each well of a acrylamide bisacrylamide gel containing sds and electrophoresed with magel for vhours proteins were transferred to a polyvinylidene fluoride membrane using the transblot turbo and the rta ready to assemble transfer kit bio rad after several hours of blocking pvdf membranes were incubated with primary antibodies online supplementary table at °c overnight after washing with tris buffered saline tween detergent membranes were incubated with the secondary antibody anti rabbit immunoglobulin g igg hrp linked antibody cell signaling technology or anti mouse igg hrp linked antibody cell signaling technology at room temperature for hour detection was done with clarity or clarity max western ecl substrate bio rad protein band intensities were quantified with imagequant tl ge healthcareelectron microscopyafter mounting cu mesh r22 holy carbon grids quantifoil with a tweezer into a leica gp leica microsystems grid plunger µl of ev sample solution were applied to grid™s front side and blotted for “ s grids were plunge frozen into liquid ethane for instant vitrification and transferred to a glacios cryo transmission microscope thermo fisher scientific equipped with a x feg images were recorded in low dose mode using the serialem software mastronarde with a falcon3 direct electron detector at magnifications of and with pixel sizes of and respectivelystatistical analysisstatistical analysis was performed using graphpad prism graphpad software statistical significance was determined by student's t test when comparing two groups the two way analysis of variance followed by tukey's multiple comparison test was used when comparing three or more groups significance was set at a p value of less than resultstam in rectal cancer polarize towards m1like phenotype upon irradiationwe first investigated the effect of irradiation on human tam in a primary ex vivo culture of rectal cancer specimen for this purpose minced tumor tissue was irradiated with gy and subsequently cultivated for hours before leucocyte isolation figure 1a the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer is shown in figure 1b tam as defined by cd14cd11bcd68hla dr accounted for over of all viable cd45 leucocytes in rectal cancer figure 1c with over cells per gram of tissue figure 1d there was no significant difference in percentage or absolute numbers of tam in non irradiated versus corresponding ex vivo irradiated tissue samples figure 1cd we then assessed the phenotype of treatment naïve and ex vivo irradiated tam using flow cytometry in naïve rectal cancer lesions tam were characterized by high expression of cd206 cd163 and cd64 and low levels of the chemokine receptor ccr7 indicating that the m2 like macrophage phenotype was present in untreated rectal cancer figure 1e this was also reflected in their cytokine pattern as more tam in untreated rectal cancer samples produced il10 il13 and il4 while few interferonγ ifnγ and tumor necrosis factor alpha tnfα producing tam were found in untreated samples in contrast ex vivo application of gy γirradiation to primary rectal cancer leucocytes resulted in a reduced presence of cd163 tam this phenotype correlated with enhanced levels of tnfα and inos tam as well as diminished detection of il10 and il13 tam hence low dose irradiation of rectal cancer tissue can polarize tam towards a pro inflammatory m1 like phenotype and may therefore directly contribute to antitumor activity by tnfα and inos productionlowdose irradiation reduces pd1 expression and enhances phagocytosis in tam to e colito further assess the distinct functional phenotype of tam on irradiation we investigated the ability of tam to phagocyte as an aspect of direct effector function and activation to model the phagocytic behavior of irradiated tam we incubated leucocytes derived from irradiated ex vivo tumor culture with ph sensitive pe labeled e coli particles in the phagosome the fluorescence of e coli particles increases as demonstrated in a representative example figure 2a phagocytosis by tam was significantly elevated upon irradiation with gy figure 2b demonstrating that the acquired shift towards m1 polarization due to irradiation was also functionally relevant as pd1 was previously shown to inhibit phagocytosis38 we next determined pd1 expression on irradiated ex vivo tumor culture derived tam we found variable pd1 expression on tam in non irradiated rectal cancer and observed a significant decrease of pd1 on tam derived from irradiated cultures figure 2c these stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0cchanges induced by irradiation can be instrumental for phagocytic activity of tamirradiation promotes antigen presentation and cytokines associated with th1 responseone of the hallmarks of m1 polarization of macrophages is the acquisition of antigen presenting features leading to efficient th1 response17 as a first step to determine whether irradiation may result in improved capability of tam to initiate immune responses we analyzed the expression of cd86 on tam derived from irradiated ex vivo tumor cultures irradiation correlated with a significant increase of cd86 on the cell surface of tam as compared with tam derived from non irradiated naïve cultures figure 2d we next investigated hla dr on non irradiated tam versus tam irradiated with gy we observed hla dr low expressing tam and high expressing tam clearly dividing them into two groups figure 2e in non irradiated samples more than of tam expressed high levels of hla dr while irradiation significantly increased the fraction of hla drhigh tam to over figure 2f we next investigated whether irradiation can induce the expression of il12 p70 and il23 p19 as markers for the initiation of adaptive immune responses whereas il23 p19 showed a tendency to be produced by tam on irradiation il12 p70 was significantly induced in irradiated tumor tissue compared with open accesstreatment naïve samples figure 2g together these results suggest that low dose irradiation influences tam polarization in rectal cancers by equipping the cells with an hla drhiil12hi il23hi cytokine profile this observation emphasizes that irradiated tam have a higher probability to participate in antitumor immune responses directly by phagocytosis and cytokine secretion as well as indirectly by exhibiting a broad armamentarium that potentially activates t cells as main players of antitumor adaptive immunityshortcourse irradiation of patients with rectal cancer increases the m1m2 ratio of tamto corroborate our ex vivo data with the in vivo situation on short course irradiation we examined tam polarization and function in rectal cancer patients treated by a routinely applied radiotherapy protocol we made use of a cohort of patients in clinical stage t3 rectal cancer who received neoadjuvant hyperfractionated short course radiotherapy two times gy per day over the course of days figure 3a surgery was performed “ days after radiotherapy in these patients as a control we used surgical specimen from treatment naïve clinical t3 rectal cancer patients importantly the indication for radiotherapy was not correlated with a more severe tumor progression compared with the treatment naïve cohort figure ex vivo γirradiation induces polarization of tumor associated macrophages a tissue samples of naïve rectal cancer were minced and irradiated after incubation a single cell suspension of cancer tissue was prepared b representative example of the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer c percentage of macrophages compared to total of viable cd45 cells in non irradiated and ex vivo irradiated rectal cancer lesions assessed by flow cytometry and presented as mean±sd n10 d mean numbers of macrophages in rectal cancer per gram in non irradiated and ex vivo irradiated rectal cancer presented as mean±sd n10 e expression of intracellular and extracellular markers in macrophages assessed by flow cytometry bars presented as mean percentage of indicated markers±sd of non irradiated and ex vivo irradiated macrophages n10 p005 p0001 p00001 by two tailed paired student™s t test gy gray tam tumor associated macrophages ifnγ interferonγ tnfα tumor necrosis factorα il interleukin inos inducible nitric oxide synthasestary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access irradiation increases phagocytosis and marker associated with antigen presentation and th1 response a figure representative histogram and flow cytometry plots indicating difference in pe fluorescence of non irradiated blue versus irradiated red tam in ex vivo phagocytosis assay total phagocytosis was analyzed by first gating on tam and then gating on pe cells b analysis of tam phagocytosis of phrodo e coli particles data presented as percentage of phagocytosis of non irradiated tam and corresponding irradiated tam gy n5 c percentage of tam positive for pd1 non irradiated versus ex vivo irradiated and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 d percentage of expression of cd86 in non irradiated versus ex vivo irradiated tam and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 e representative plots histogram and f expression of hla dr on tam of non irradiated and ex vivo irradiated rectal cancer data given as mean±sd n10 g expression of il12 p70 and il23 p19 data presented as mean percentage±sd of total tam n10 representative example of il12 p70 and il23 p19 expression on tam p005 p0001 by two tailed paired student™s t test th1 t helper type gy gray tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessfigure short course irradiation in patients modulates the immune infiltrate with induction of macrophage polarization a irradiation protocol of patients with neoadjuvant hyperfractionated × gy per day over the course of five days radiotherapy for clinical t3 rectal cancer surgery was performed the following week b quantitative in situ assessment of infiltrating macrophages cd68 t cells cd3cd56ˆ’ nk cells cd56cd3ˆ’ and nkt cells cd56cd3 in non irradiated n25 and irradiated rectal cancer n45 assessed by multicolor immunofluorescence staining data are given as absolute numbers of positive cells per mm2±sd c analysis of immunohistochemical staining for γh2ax as irradiation response in non irradiated n25 and irradiated tissue sections data are given as mean±sd d representative examples of immunohistochemical staining for γh2ax images below are magnified × e representative immunohistochemical staining of cd68 cells in non irradiated n25 and irradiated tissue sections n45 images below are magnified × f representative example of immunofluorescence multicolor staining of cd68 macrophages pe m1inos fitc m2 cd163 apc g quantitative in situ analysis of immunofluorescence staining of ratio of inos m1 to cd163 m2 tams cd68 macrophages presented as m1m2 in non irradiated n25 compared to irradiated sections n45 data are given as mean±sd h percentage of il10 cells to total cd68 cells data are presented as mean±sd i representative image of multicolor immunofluorescence staining of il10 in macrophages cd68 peil10 fitc of irradiated rectal cancer p0001 p00001 by two tailed unpaired student™s t test gy gray inos inducible nitric oxide synthase tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access since the stage in the tnm classification of malignant tumors and cancer differentiation of the two cohorts were comparable online supplementary table to assess the composition of the leucocytic infiltrate and the impact of radiotherapy we evaluated the distribution of macrophages t cells nk cells and nkt cells via immunofluorescence staining and automated image analysis irradiated tumor sections showed significantly less infiltration of cd68 cells which correlated with a tendency for a decrease of cd3 t cells while the amount of nk cells cd56 cd3ˆ’ or nkt cd56 cd3 cells did not show significant differences figure 3b immunostaining of phosphorylated γh2ax was used to confirm radiotherapy induced dna damage in analyzed tumor specimen irradiated tumors revealed a significantly higher amount of γh2ax positive cells than non irradiated tissue figure 3c γh2ax foci were primarily located in the nuclei of tumor cells rather than in the cells of the tumor infiltrating microenvironment figure 3d in both patient cohorts cd68 cells accumulated in regions surrounding tumor cell clusters figure 3eto evaluate the in vivo effect of irradiation on macrophage polarization and to correlate results with ex vivo irradiated tumor specimens we determined the ratio of m1 like tam to m2 like tam by staining for inos and cd163 in cd68 cells irradiation was associated with higher numbers of inos expressing tam while cd163 tam were drastically reduced resulting in a significant increase in the m1m2 ratio figure 3fg similar to the ex vivo cultures we observed a sixfold decrease of il10 tam in tumors from irradiated patients as compared with treatment naive tumors figure 3hi these data are corroborating hallmarks of our ex vivo irradiation protocols with macrophages becoming active players within the tumor microenvironment upon γirradiationirradiation of anotypic cultures promote macrophage activation towards an m1like phenotypeto be able to further dissect the irradiation induced findings in patients tissue sections and ex vivo cultures we used anotypic cultures which were established with colorectal cancer cell lines hct116 or dld1 primary caf and peripheral blood derived macrophages online supplementary figure a in collagen i gels in contrast to primary cultures ota allowed culture periods of up to days to mimic the clinical protocol of short course irradiation ota containing either hct116 or dld1 were exposed to irradiation with two times gy or two times gy over the course of hours compared with non irradiated controls figure 4ab frozen tissue sections of ota were assessed for macrophage marker using immunostaining and automated image analysis the number of macrophages in ota was comparable and not significantly different among the distinct conditions online supplementary figure b non irradiated gy ota harbored macrophages resembling an m2 like phenotype of cd11bcd68 macrophages as indicated by high expression of cd163 figure 4cd low expression of ccr7 figure 4e and modest expression of il10 figure 4fg upon irradiation of ota cd163 and il10 were reduced whereas the expression of the pro inflammatory marker ccr7 was elevated thus the marker profile of ota correlated with those of primary ex vivo tumor cultures and surgical specimen following neoadjuvant irradiation these observa

cancer7510

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " tumor associated macrophages tam constitute the most abundant immune cells in the tumor stroma initiating pro inflammatory m1 or immunosuppressive m2 responses depending on their polarization status advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patientsmethods to test the effects of radiotherapy on tam we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry we furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short course radiotherapy and compared findings to non pretreated rectal cancer using an immunostaining approach anotypic assays ota consisting of macrophages cancer associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophagesresults we demonstrate that short course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of tam towards an m1 like pro inflammatory phenotype in addition ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for t cell activation in ota we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles ev derived from irradiated tumor cells we identified high mobility group box in ev from irradiated tumor cells as a potential effector signal in that crosstalks our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro inflammation our data indicate that clinically applied short term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategiesintroductionsince the introduction of immune checkpoint blockade immunotherapy has become an attractive therapeutic option in cancer1“ irradiation used as standard therapy in a number of solid malignancies induces immunogenic cell death icd by the release of damage associated pattern damp4 studies based on murine models indicated that irradiation induced dna damage of tumor cells elicited an antitumor immune response5 it was shown that alterations of the infiltrating immune cells by irradiation might be augmented when combined with immune modulating drugs6“ a detailed understanding of the impact of radiotherapy on the immune system in humans should allow application of radiotherapy as a part of novel immunotherapeutic concepts however little is known about the detailed regulation of the immunogenic effect of irradiation in clinical settingsmacrophages are one of the most abundant immune cell subsets in tumor tissue9 and play a key role in the cancer microenvironment11 tumor associated macrophages tam are functionally diverse13 and display high plasticity upon immunological stimuli14 the concept of m1 and m2 macrophages was introduced to describe the heterogeneity of this cell subset16 m1 like macrophages possess the capacity to clear infections in support of a t helper type th1 immune response17 whereas m2 like macrophages respond in general to th2 cytokines and are strongly enriched in the tumor microenvironment18 the concept of m1m2 has been challenged and is seen as an oversimplified approach to the phenotype of macrophages but can be viewed as a linear scale on which m1 and m2 present two extremes19 tam recognize damp and respond by producing a variety of cytokines and growth factors to promote innate and stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access adaptive immunity9 in response to these signals macrophages are able to undergo reprogramming with enhanced antitumor features making them an attractive target for anticancer therapies22 there are conflicting results with respect to the effect of irradiation on the macrophage phenotype klug et al demonstrated in a murine model of breast cancer and human pancreatic cancer that low dose irradiation gy induced repolarization of m2 like macrophages to m1 like macrophages via induction of nitric oxide synthase inos24 moreover expression of inos correlated with vessel normalization and an influx of cd8 t cells suggesting a tumor ablative as well as pro inflammatory effect of repolarized macrophages upregulation of inos was also observed in murine prostate cancer which has been irradiated with up to gy25 similarly agonists of the toll like receptor further stimulated the m1 phenotype of macrophages and enhanced the antitumor effects of irradiation in a murine model of breast cancer26 other studies suggested that irradiation of tumors was associated with a more immunosuppressive phenotype of tam27“ jones found that the depletion of macrophages by an anti csf antibody greatly increased tumor ablation upon irradiation gy in murine tumors generated from a colorectal and a pancreatic cell line obviously the effect of irradiation appears to depend on the model irradiation dose tissue as well as on the investigated time point after irradiation however despite the controversial reports an important role for tam in response to radiotherapy seems evidentmore recently extracellular vesicles ev have attracted attention in mediating signals to immune cells ev are rich in molecular cargos and are emerging as critical messengers in the cell to cell crosstalk they contain a variety of small signaling molecules which can be transferred to other cell types to modulate cell functions30“ thus we hypothesized that ev might be involved in macrophages regulation in the tumor microenvironment in irradiated cells using a clinically relevant approach we show that short course irradiation increased the proportion of m1 like tam in human rectal cancer tissue primary short term cultures and anotypic tumor assays ota consisting of tumor cell lines macrophages and cancer associated fibroblasts allowed to further dissect irradiation induced changes in colorectal tam using irradiated tumor cell lines we demonstrate that ev are able to mediate irradiation induced repolarization of macrophagesmethodspatients and tissue materialpatients with clinical t3 rectal cancer patients characteristics online supplementary table received neoadjuvant hyperfractionated radiotherapy over the course of days within the frame of a controlled clinical study34 as a control group we used historical surgical tumor specimen of non pretreated rectal cancer lesions of patients from the same institution with no history of irradiation therapy or cytoablative treatment for immunofluorescence and immunohistochemical stainings surgical paraffin embedded specimen of the cancer lesions were cut in µm sections and mounted on slides for ex vivo cultures and subsequent flow cytometric stainings rectal cancer tissue was obtained from patients with histologically verified rectal cancer with no history of irradiation therapy or cytoablative treatment studies involving patient material were performed according to the declaration of helsinki and approved by the local ethics committee of the medical university of vienna for ota hct116 ccl147 and dld1 ccl221 were purchased from atcc peripheral blood mononuclear cells for macrophage differentiation were isolated from healthy blood donorsprimary ex vivo cultures of leucocytes and flow cytometrytissue samples of non irradiated rectal cancer were minced resuspended in rpmi medium with fetal bovine serum and plated in a mm petri dish irradiation protocol was applied as indicated below after hour of incubation °c co2 a single cell suspension from cancer tissue was prepared briefly tissue was digested with collagenase iv unitsml and dnase i mgml for hour min in rpmi supplemented with fetal bovine serum and hepes buffer solution at °c afterwards cell suspension was rinsed through a µm mesh and leucocytes were isolated by density gradient centrifugation using ficoll gradients for flow cytometry analysis cells were stained with fluorescence antibodies listed in the online supplementary table livedead discrimination was performed with fixable viability dye biolegend samples were acquired on a facsaria iii bd and analyzed with flowjo software v1061irradiation protocola theratron mds nordion radiotherapy unit with a cobalt60 source was used for γirradiation of ota ex vivo cultures of rectal cancer tissue monocyte derived macrophages and cancer cell lines tissue samples of rectal cancer were minced and cultivated petri dishes with cancer tissue were irradiated with × gy after hours of incubation a single cell suspension was prepared ota and cancer cell lines hct116 and dld1 were irradiated with × gy or × gy and subsequently cultivated over the course of hours monocyte derived macrophages were irradiated with gy or gy a negative control gy was always transported to the radiotherapy unit but was not exposed to γirradiationex vivo phagocytosis assaymononuclear cells isolated of rectal cancer lesions were spun down at g for min at °c for ex vivo escherichia coli e coli phagocytosis assay irradiated and non irradiated leucocytes were resuspended in µl phrodo red e coli bioparticles thermo fisher scientific as per the manufacturer™s instruction a same set of cells were resuspended in phosphate buffered saline pbs as controls after hours incubation at °c stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessleucocytes were washed and stained for flow cytometry tam cd11b cd14 hla dr viable cd45 cells that were pe high were considered to be phagocytosing for ex vivo tumor cell phagocytosis assay tam were isolated via fluorescence activated cell sorting facs cd11b cd14 hla dr viable cd45 cells from healthy colon mucosa or colorectal cancer lesions and incubated overnight with ev isolated from irradiated × gy and non irradiated gy dld1 cells evtam after washing dld1 were labeled with carboxyfluorescein diacetate succinimidyl ester cfse thermo fisher µm and cocultured with tam at an effector to target cell ratio of the proportion of cfse tam was assessed by flow cytometry after hours of incubation at °cat the air liquid interface in dmem supplemented with egm for seeding of tumor cells nylon discs with collagen cell cylinders were transferred into well plates five hundred microlitres of a tumor cell suspension with × cellsml hct116 or dld1 were added to each well after hours the tumor cells attached to the surface of the collagen gels and the nylon meshes with the tumor cell colonized collagen cell cylinders were put back onto the metal grids and incubated for days with media changes every days prior to further experiments for further assessment ota were embedded in optimal cutting temperature media snap frozen in liquid nitrogen and stored at “°c until processing or fixed and embedded in paraffinimmunofluorescence and immunohistochemical stainingdirectly and indirectly labeled monoclonal antibodies as listed in online supplementary table and dapi as a nuclear marker were used an isotype was used as negative control in brief after incubation with the primary antibodies overnight at °c an appropriate secondary fluorescence labeled antibody was applied for min at room temperature followed by staining of dapi nuclear marker for immunohistochemical staining antibodies were mixed with bovine serum albumin in pbs and applied overnight to sections in a humid chamber at °c for visualization of the cells aec was used as chromogen sections were counter stained with hematoxylin for evaluation of staining results images of whole tissue sections one section per patient were acquired using a z1 axio observer microscope equipped with a ld plan neofluar × objective zeiss for leucocyte evaluation the tumor normal interface mm on tumor and normal zone was defined as regions of interests roi for γh2ax staining only tumor tissue was selected as roi roi were automatically quantified using tissuefaxstissuequest image analysis software tissuegnostics gmbhpreparation of otaota were set up as previously described35 briefly cancer associated fibroblasts caf were isolated and cultured from fresh samples of colon adenocarcinomas for the preparation of macrophages monocytes were isolated using the easysep direct human monocyte isolation kit stemcell technologies according to the manufacturer™s instructions × caf and monocytes per ota were mixed and pelleted by centrifugation at g for min for the collagen gel preparation all steps were performed on ice collagen solutions were prepared by mixing mgml of collagen i rat tail mgml becton dickinson and the fibroblastmonocyte suspension in dulbecco's modified eagle medium dmem supplemented with endothelial cell growth medium mv2 egm promocell a total of µl each of the collagen cell suspension were transferred into silicone gel casting devices after polymerization of the collagen solution the gels were lifted up on nylon mesh discs and transferred onto metal grids in well plates and cultured macrophage differentiationpbmc were obtained by ficoll plaque density gradient centrifugation pbmc were seeded at a concentration of ×106well well plates in rpmi medium monocytes were isolated using the ability of monocytes to adhere to non tissue culture treated plastic culture dishes attached cells were cultivated in rpmi medium with glutamax thermo fisher scientific supplemented with ngml macrophage colony stimulating factor m csf thermo fisher scientific fetal bovine serum uml penicillin uml streptomycin and µgml fungizone in a humidified atmosphere at °c cells were cultivated for days with two medium changes to obtain m1 lpsifnγ polarized macrophages cells were stimulated with ngml lipopolysaccharide lps sigma aldrich and ngml interferonγ ifnγ thermo fisher scientific for hours m2 il4il13 polarized macrophages were generated using ngml interleukin il4 strathmann and ngml il13 biolegendev preparation and characterizationev were isolated by differential centrifugation hct116 and dld1 cells were cultured in mccoy™s 5a medium supplemented with exosome depleted fetal calf serum briefly cancer cell line culture medium was centrifuged at g for min to pellet cells supernatant was transferred to new falcon tubes and then centrifuged at g for min to pellet dead cells and apoptotic bodies rotanta 460rc hettich supernatant was transferred to the high speed centrifugation tubes and large ev were removed by ultracentrifugation at g for min sorvall evolution rc thermo fisher scientific after filtration of the supernatant through a µm syringe filter small ev were pelleted by ultracentrifugation at g for min rotor t1250 suspended in ice cold pbs and collected after another ultracentrifugation at g for min rotor type sorvall wx ultra thermo fisher scientific the ev pellet was resuspended in cold pbs for further use ev count was determined using a nanoparticle tracking analyzer zetaview particlemetrix results were analyzed with zetaview software ev were added to macrophages at a stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access concentration of ×× cells we have submitted all relevant data of our experiments to the ev track knowledgebase ev track id ev20006537western blotcells were scraped from cell culture plates washed in ice cold pbs and lysed in × ripa buffer mm tris“hcl ph mm nacl np40 sodium deoxycholate sds containing × halt protease inhibitor cocktail thermo fisher scientific ev suspensions were mixed with × ripa buffer containing × halt protease inhibitor cocktail to obtain × concentration protein extracts were incubated at °c for min and centrifuged at g for min clear protein extract supernatants were quantified with the bca protein assay kit thermo fisher scientific one to five micrograms of protein were denatured with laemmli buffer loaded into each well of a acrylamide bisacrylamide gel containing sds and electrophoresed with magel for vhours proteins were transferred to a polyvinylidene fluoride membrane using the transblot turbo and the rta ready to assemble transfer kit bio rad after several hours of blocking pvdf membranes were incubated with primary antibodies online supplementary table at °c overnight after washing with tris buffered saline tween detergent membranes were incubated with the secondary antibody anti rabbit immunoglobulin g igg hrp linked antibody cell signaling technology or anti mouse igg hrp linked antibody cell signaling technology at room temperature for hour detection was done with clarity or clarity max western ecl substrate bio rad protein band intensities were quantified with imagequant tl ge healthcareelectron microscopyafter mounting cu mesh r22 holy carbon grids quantifoil with a tweezer into a leica gp leica microsystems grid plunger µl of ev sample solution were applied to grid™s front side and blotted for “ s grids were plunge frozen into liquid ethane for instant vitrification and transferred to a glacios cryo transmission microscope thermo fisher scientific equipped with a x feg images were recorded in low dose mode using the serialem software mastronarde with a falcon3 direct electron detector at magnifications of and with pixel sizes of and respectivelystatistical analysisstatistical analysis was performed using graphpad prism graphpad software statistical significance was determined by student's t test when comparing two groups the two way analysis of variance followed by tukey's multiple comparison test was used when comparing three or more groups significance was set at a p value of less than resultstam in rectal cancer polarize towards m1like phenotype upon irradiationwe first investigated the effect of irradiation on human tam in a primary ex vivo culture of rectal cancer specimen for this purpose minced tumor tissue was irradiated with gy and subsequently cultivated for hours before leucocyte isolation figure 1a the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer is shown in figure 1b tam as defined by cd14cd11bcd68hla dr accounted for over of all viable cd45 leucocytes in rectal cancer figure 1c with over cells per gram of tissue figure 1d there was no significant difference in percentage or absolute numbers of tam in non irradiated versus corresponding ex vivo irradiated tissue samples figure 1cd we then assessed the phenotype of treatment naïve and ex vivo irradiated tam using flow cytometry in naïve rectal cancer lesions tam were characterized by high expression of cd206 cd163 and cd64 and low levels of the chemokine receptor ccr7 indicating that the m2 like macrophage phenotype was present in untreated rectal cancer figure 1e this was also reflected in their cytokine pattern as more tam in untreated rectal cancer samples produced il10 il13 and il4 while few interferonγ ifnγ and tumor necrosis factor alpha tnfα producing tam were found in untreated samples in contrast ex vivo application of gy γirradiation to primary rectal cancer leucocytes resulted in a reduced presence of cd163 tam this phenotype correlated with enhanced levels of tnfα and inos tam as well as diminished detection of il10 and il13 tam hence low dose irradiation of rectal cancer tissue can polarize tam towards a pro inflammatory m1 like phenotype and may therefore directly contribute to antitumor activity by tnfα and inos productionlowdose irradiation reduces pd1 expression and enhances phagocytosis in tam to e colito further assess the distinct functional phenotype of tam on irradiation we investigated the ability of tam to phagocyte as an aspect of direct effector function and activation to model the phagocytic behavior of irradiated tam we incubated leucocytes derived from irradiated ex vivo tumor culture with ph sensitive pe labeled e coli particles in the phagosome the fluorescence of e coli particles increases as demonstrated in a representative example figure 2a phagocytosis by tam was significantly elevated upon irradiation with gy figure 2b demonstrating that the acquired shift towards m1 polarization due to irradiation was also functionally relevant as pd1 was previously shown to inhibit phagocytosis38 we next determined pd1 expression on irradiated ex vivo tumor culture derived tam we found variable pd1 expression on tam in non irradiated rectal cancer and observed a significant decrease of pd1 on tam derived from irradiated cultures figure 2c these stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0cchanges induced by irradiation can be instrumental for phagocytic activity of tamirradiation promotes antigen presentation and cytokines associated with th1 responseone of the hallmarks of m1 polarization of macrophages is the acquisition of antigen presenting features leading to efficient th1 response17 as a first step to determine whether irradiation may result in improved capability of tam to initiate immune responses we analyzed the expression of cd86 on tam derived from irradiated ex vivo tumor cultures irradiation correlated with a significant increase of cd86 on the cell surface of tam as compared with tam derived from non irradiated naïve cultures figure 2d we next investigated hla dr on non irradiated tam versus tam irradiated with gy we observed hla dr low expressing tam and high expressing tam clearly dividing them into two groups figure 2e in non irradiated samples more than of tam expressed high levels of hla dr while irradiation significantly increased the fraction of hla drhigh tam to over figure 2f we next investigated whether irradiation can induce the expression of il12 p70 and il23 p19 as markers for the initiation of adaptive immune responses whereas il23 p19 showed a tendency to be produced by tam on irradiation il12 p70 was significantly induced in irradiated tumor tissue compared with open accesstreatment naïve samples figure 2g together these results suggest that low dose irradiation influences tam polarization in rectal cancers by equipping the cells with an hla drhiil12hi il23hi cytokine profile this observation emphasizes that irradiated tam have a higher probability to participate in antitumor immune responses directly by phagocytosis and cytokine secretion as well as indirectly by exhibiting a broad armamentarium that potentially activates t cells as main players of antitumor adaptive immunityshortcourse irradiation of patients with rectal cancer increases the m1m2 ratio of tamto corroborate our ex vivo data with the in vivo situation on short course irradiation we examined tam polarization and function in rectal cancer patients treated by a routinely applied radiotherapy protocol we made use of a cohort of patients in clinical stage t3 rectal cancer who received neoadjuvant hyperfractionated short course radiotherapy two times gy per day over the course of days figure 3a surgery was performed “ days after radiotherapy in these patients as a control we used surgical specimen from treatment naïve clinical t3 rectal cancer patients importantly the indication for radiotherapy was not correlated with a more severe tumor progression compared with the treatment naïve cohort figure ex vivo γirradiation induces polarization of tumor associated macrophages a tissue samples of naïve rectal cancer were minced and irradiated after incubation a single cell suspension of cancer tissue was prepared b representative example of the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer c percentage of macrophages compared to total of viable cd45 cells in non irradiated and ex vivo irradiated rectal cancer lesions assessed by flow cytometry and presented as mean±sd n10 d mean numbers of macrophages in rectal cancer per gram in non irradiated and ex vivo irradiated rectal cancer presented as mean±sd n10 e expression of intracellular and extracellular markers in macrophages assessed by flow cytometry bars presented as mean percentage of indicated markers±sd of non irradiated and ex vivo irradiated macrophages n10 p005 p0001 p00001 by two tailed paired student™s t test gy gray tam tumor associated macrophages ifnγ interferonγ tnfα tumor necrosis factorα il interleukin inos inducible nitric oxide synthasestary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access irradiation increases phagocytosis and marker associated with antigen presentation and th1 response a figure representative histogram and flow cytometry plots indicating difference in pe fluorescence of non irradiated blue versus irradiated red tam in ex vivo phagocytosis assay total phagocytosis was analyzed by first gating on tam and then gating on pe cells b analysis of tam phagocytosis of phrodo e coli particles data presented as percentage of phagocytosis of non irradiated tam and corresponding irradiated tam gy n5 c percentage of tam positive for pd1 non irradiated versus ex vivo irradiated and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 d percentage of expression of cd86 in non irradiated versus ex vivo irradiated tam and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 e representative plots histogram and f expression of hla dr on tam of non irradiated and ex vivo irradiated rectal cancer data given as mean±sd n10 g expression of il12 p70 and il23 p19 data presented as mean percentage±sd of total tam n10 representative example of il12 p70 and il23 p19 expression on tam p005 p0001 by two tailed paired student™s t test th1 t helper type gy gray tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessfigure short course irradiation in patients modulates the immune infiltrate with induction of macrophage polarization a irradiation protocol of patients with neoadjuvant hyperfractionated × gy per day over the course of five days radiotherapy for clinical t3 rectal cancer surgery was performed the following week b quantitative in situ assessment of infiltrating macrophages cd68 t cells cd3cd56ˆ’ nk cells cd56cd3ˆ’ and nkt cells cd56cd3 in non irradiated n25 and irradiated rectal cancer n45 assessed by multicolor immunofluorescence staining data are given as absolute numbers of positive cells per mm2±sd c analysis of immunohistochemical staining for γh2ax as irradiation response in non irradiated n25 and irradiated tissue sections data are given as mean±sd d representative examples of immunohistochemical staining for γh2ax images below are magnified × e representative immunohistochemical staining of cd68 cells in non irradiated n25 and irradiated tissue sections n45 images below are magnified × f representative example of immunofluorescence multicolor staining of cd68 macrophages pe m1inos fitc m2 cd163 apc g quantitative in situ analysis of immunofluorescence staining of ratio of inos m1 to cd163 m2 tams cd68 macrophages presented as m1m2 in non irradiated n25 compared to irradiated sections n45 data are given as mean±sd h percentage of il10 cells to total cd68 cells data are presented as mean±sd i representative image of multicolor immunofluorescence staining of il10 in macrophages cd68 peil10 fitc of irradiated rectal cancer p0001 p00001 by two tailed unpaired student™s t test gy gray inos inducible nitric oxide synthase tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access since the stage in the tnm classification of malignant tumors and cancer differentiation of the two cohorts were comparable online supplementary table to assess the composition of the leucocytic infiltrate and the impact of radiotherapy we evaluated the distribution of macrophages t cells nk cells and nkt cells via immunofluorescence staining and automated image analysis irradiated tumor sections showed significantly less infiltration of cd68 cells which correlated with a tendency for a decrease of cd3 t cells while the amount of nk cells cd56 cd3ˆ’ or nkt cd56 cd3 cells did not show significant differences figure 3b immunostaining of phosphorylated γh2ax was used to confirm radiotherapy induced dna damage in analyzed tumor specimen irradiated tumors revealed a significantly higher amount of γh2ax positive cells than non irradiated tissue figure 3c γh2ax foci were primarily located in the nuclei of tumor cells rather than in the cells of the tumor infiltrating microenvironment figure 3d in both patient cohorts cd68 cells accumulated in regions surrounding tumor cell clusters figure 3eto evaluate the in vivo effect of irradiation on macrophage polarization and to correlate results with ex vivo irradiated tumor specimens we determined the ratio of m1 like tam to m2 like tam by staining for inos and cd163 in cd68 cells irradiation was associated with higher numbers of inos expressing tam while cd163 tam were drastically reduced resulting in a significant increase in the m1m2 ratio figure 3fg similar to the ex vivo cultures we observed a sixfold decrease of il10 tam in tumors from irradiated patients as compared with treatment naive tumors figure 3hi these data are corroborating hallmarks of our ex vivo irradiation protocols with macrophages becoming active players within the tumor microenvironment upon γirradiationirradiation of anotypic cultures promote macrophage activation towards an m1like phenotypeto be able to further dissect the irradiation induced findings in patients tissue sections and ex vivo cultures we used anotypic cultures which were established with colorectal cancer cell lines hct116 or dld1 primary caf and peripheral blood derived macrophages online supplementary figure a in collagen i gels in contrast to primary cultures ota allowed culture periods of up to days to mimic the clinical protocol of short course irradiation ota containing either hct116 or dld1 were exposed to irradiation with two times gy or two times gy over the course of hours compared with non irradiated controls figure 4ab frozen tissue sections of ota were assessed for macrophage marker using immunostaining and automated image analysis the number of macrophages in ota was comparable and not significantly different among the distinct conditions online supplementary figure b non irradiated gy ota harbored macrophages resembling an m2 like phenotype of cd11bcd68 macrophages as indicated by high expression of cd163 figure 4cd low expression of ccr7 figure 4e and modest expression of il10 figure 4fg upon irradiation of ota cd163 and il10 were reduced whereas the expression of the pro inflammatory marker ccr7 was elevated thus the marker profile of ota correlated with those of primary ex vivo tumor cultures and surgical specimen following neoadjuvant irradiation these observa Answer:

Colon_Cancer

" triple negative breast cancer tnbc remains recalcitrant to most targeted therapy approaches however recent clinical studies suggest that inducing tumor damage can render tnbc responsive to immunotherapy we therefore tested a strategy for immune sensitization of murine tnbc 4t1 tumors through combination of focused ultrasound fus thermal ablation and a chemotherapy gemcitabine gem known to attenuate myeloid derived suppressor cells mdscsmethods we applied a sparse scan thermally ablative fus regimen at the tumor site in combination with systemically administered gem we used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity we also tested this combination in rag1ˆ’ˆ’ mice or t cell depleted wild type mice to determine the essentiality of adaptive immunity further we layered programmed cell death protein pd1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalresults the immune modulatory effect of fus monotherapy was insufficient to promote a robust t cell response against 4t1 consistent with the dominant mdsc driven immunosuppression evident in this model the combination of fusgem significantly constrained primary tnbc tumor outgrowth and extended overall survival of mice tumor control correlated with increased circulating antigen experienced t cells and was entirely dependent on t cell mediated immunity the ability of fusgem to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti pd1 thermally ablative fus in combination with gem restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic tnbc model this treatment strategy promises a novel option for potentiating the role of fus in immunotherapy of metastatic tnbc and is worthy of future clinical evaluationtrial registration numbers nct03237572 and nct04116320 metastatic breast cancer brca particularly the triple negative breast cancer tnbc phenotype is resistant to most chemical and molecularly targeted therapeutic approaches interestingly tnbc is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 early studies in the use of immunotherapies targeting the pd1programmed death ligand pd l1 checkpoint inhibitory axis showed some efficacy2“ in tnbc compared with other brca subtypes which are generally recalcitrant to checkpoint blockade activity in the tnbc subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in tnbc greater immunotherapy efficacy in tnbc has been recently observed with the use of antibodies targeting the pd1pd l1 checkpoint inhibitory axis in combination with nab paclitaxel5 this outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment tme found in tnbcamong the potential networks in tnbc that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets these have the capacity to impair adaptive immunity and promote tumor growth and metastasis among these cell types myeloid derived suppressor cells mdscs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both t cell activation and effector functions6 increased levels of this cell type have been demonstrated in tumor tissues of patients with primary brca while those with metastatic disease bear the highest abundance of circulating mdscs8 studies have sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate mdsc can improve antitumor immunity9“to this end the central premise put forth in this study is that focused ultrasound fus”a safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissues”can synergize with immunotherapy in a murine model of metastatic tnbc fus is capable of rapidly heating tumors to thermally ablative temperatures its extracorporeal application obviates the need for catheterization injection or implantation fus can be targeted with millimeter precision under mri or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues the bioeffects of fus hold distinct implications for tumor antigenicity immune cell activation and trafficking13 thermally active fus regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17“ colon20 kidney21 and brca23 pertaining to the challenge of myeloid cell immunosuppression in tnbc thermally ablative fus has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin il12 interferonÎ ifnÎ and tumor necrosis factorα tnfα from a variety of cancer cell lines and after in vivo treatment of tumors26 whether the ability of fus to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of brca is currently under debate with some studies showing activation of antigen presenting cells and t cell recruitment in patients with brca treated with thermally ablative fus28 while others show that additional innate stimuli are needed to support antitumor immunity23 notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells dcs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation fus regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine tnbc with extensive granulocytic mdsc involvement that is recalcitrant to anti pd1 while some activity is evident with the partial ablation approach significantly greater control was achieved by targeting mdsc inhibition in combination with thermally ablative fus this control was completely dependent on the adaptive immune responsemoreover we demonstrate that layering anti pd1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction these data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative fus once immunosuppressive myeloid cells are accounted for fus treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockademethodscell line maintenance4t1 and e0771 cell lines were maintained in rpmi l glut or dulbecco™s modified eagle™s medium dmem gl d glucose l glutamine respectively supplemented with fetal bovine serum fbs at °c and co2 thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments cells tested negative for mycoplasmaeight week old to week old female balbc or c57bl6 mice were obtained from nci charles river nci crl or the jackson laboratory female balbc rag1ˆ’ˆ’ mice were obtained from the jackson laboratory 4t1 or e0771 cells — were subcutaneously implanted into the right flank of mice mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum tumor outgrowth was monitored via digital caliper measurements tumor volume was calculated as follows volume length—width22 approximately days 4t1 or days e0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsin vivo ultrasoundguided fus partial thermal ablationmice were treated with fus either days 4t1 cohorts or days e0771 postimplantation on treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg zoetis and dexdomitor mgkg pfizer in sterilized saline mouse flanks were shaved and depilated following which ultrasound guided fus thermal ablation was performed using one of the two systems system and treatment details are provided in online supplementary materials and methods mice that did not receive fus treatment consistently underwent anesthesia and depilation of the flank additionally these mice underwent a ˜sham™ treatment consisting of exposure to the °c degassed water bath exposure for min following ˜sham™ or fus treatment all mice were moved to a heating pad and given antisedan for anesthesia reversal and recoverygemcitabine therapygemcitabine gem mgmouse in µl volume mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of fus treatment following which administration was repeated for an additional weeks administration of gem doses was based on existing literature demonstrating the use of gem for inhibition of mdscs in 4t112 the initial dose of gem was administered immediately prior to ˜sham™ or fus treatment sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cmice that did not receive gem received an intraperitoneal injection of ˜vehicle™ treatment µl of sterile saline at the time points specifiedpd1 blockade therapyfor checkpoint inhibitor therapy the rat anti mouse pd1 antibody αpd1 rmp114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse treatment was initiated on day ˜early αpd1™ or day ˜delayed αpd1™t cell depletionst cell depletion antibodies”anti cd8 clone bio x cell and anti cd4 gk15 clone bio x cell”were diluted in sterilized saline and administered intraperitoneally every to days starting at day days post fus for a total of seven doses µg of each antibody for a total µg per mouseimmunohistochemistryon day sham or fus exposed tumors were excised and fixed in neutral buffered formalin sigma fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin digital images of stained slides were acquired using the vectra automated quantitative pathology imaging system akoya biosciences whole slide screening and image capture were subsequently performed using phenochart akoya biosciencesflow cytometrymice were bled at days and via tail vein and samples were rbc lysed hybri max sigma and stained for flow cytometry analysis at days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment in order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse cd45 fitc clone f11 bd biosciences min prior to euthanasia 4t1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor dlns pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis additional details are provided in online supplementary materials and methodssamples were acquired on an attune nxt flow cytometer thermofisher scientific and data were analyzed with flowjo treestar or fcs express de novo software a representative gating strategy for granulocytic myeloid derived suppressor cell g mdsc and cd44 t cells is provided in online supplementary figure statistical analysisall statistical analyses were performed in graphpad prism graphpad software a detailed description of statistical methods for each experiment is provided in the corresponding figure legendopen accessanimal study approvalall animal work was performed under a protocol approved by the animal care and use committee at the university of virginia and conformed to the national institutes of health guidelines for the use of animals in researchresultspartial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsetsto achieve partial thermal ablation of 4t1 tumors we used an ultrasound guided fus system equipped with a single element therapeutic transducer driven at mhz figure 1a online supplementary figure a grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under b mode ultrasound guidance figure 1b“c the exceptionally small focus of this system rendered a low ablation fraction “ of total tumor volume immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1d one week following fus partial thermal ablation tumors and secondary lymphoid organs were excised for immunological characterization by flow cytometry figure 1b fus partial thermal ablation of 4t1 tumors conferred a significant increase fold in the absolute number of cd11c hi dcs within the axillary tumor draining lymph node adln of mice figure 1e while this was accompanied by a nearly threefold elevation in the absolute number of cd86 dcs within the adln figure 1f the percentage of dcs expressing cd86 did not change figure 1g increased numbers of dcs”and cd86 dcs in particular”suggest fus is promoting the maturation or trafficking of these cells in the dlns where they could encounter and activate t cells however this did not translate to tumor growth restriction data not shown we also did not observe significant differences in the absolute number of activated t cells in 4t1 tumors figure 1h or dlns data not shown following fus exposure suggesting limitations in the ability of fus activated dc to further drive an antitumor t cell responseimmune profiling by flow cytometry revealed that irrespective of fus exposure of the intratumoral cd45 immune cell population is comprised of cd11b myeloid cells figure 1i similarly approximately of the circulating immune cell population in 4t1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure notably ly6g granulocytic myeloid derived suppressor cells g mdscs significantly dominated the immune cell repertoire within 4t1 tumors relative to other myeloid including f480 macrophages ly6c cell subsets monocytic myeloid derived suppressor cells m mdscs and cd11c hi dcs figure 1j fus partial thermal ablation did not significantly alter the absolute number per sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure partial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsets a design overview of a custom ultrasound guided fus system consisting of a mhz single element transducer orthogonally co registered to an mhz linear ultrasound imaging array the tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °c ˜sham™ mice were similarly positioned but did not undergo sonications b schematic illustration of fus partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4t1 tumor bearing mice a grid of sonications was applied in a raster pattern onto the b mode ultrasound visible tumor in total two planes of sonication spaced mm apart were applied to each tumor grid points were spaced mm apart within a single plane one week following thermal ablation tumors and secondary lymphoid organs were excised for sham n6 or fus treated n5 mice and processed for flow cytometry c representative b mode ultrasound images of ectopic 4t1 tumors either before top or during bottom fus exposure sonication grid depicting targets red points is superimposed on b mode image during treatment subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed d representative he staining of either sham 4t1 tumors or those resected immediately following fus partial thermal ablation zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively e absolute number of cd11c hi dcs in the axillary tumor draining lymph node adln of 4t1 tumor bearing mice p00136 vs sham f absolute number of cd86 cd11c hi dcs in the adln p00063 vs sham g percentage of cd86 subset out of total cd11c hi dcs within adln h absolute number of intratumoral cd44 cd8 and cd44 cd4 t cells and regulatory t cells tregs per gram tumor i percentage of cd11b myeloid cells out of total cd45 immune cells across tumor spleen adln inguinal dln idln and nontumor draining axillary and inguinal lns ndlns p005 vs all other groups irrespective of fus exposure specifically tumor vs spleen p00226 tumor spleen vs all other organs p00001 j absolute number of intratumoral myeloid cells cd11c hi dcs f480 macrophages ly6c monocytic myeloid derived suppressor cells m mdscs ly6g granulocytic myeloid derived suppressor cells g mdscs per gram 4t1 tumor p00001 vs all other cell types irrespective of fus exposure all data represented as mean±sem significance assessed by unpaired t test f“h or two way analysis of variance followed by tukey multiple comparison correction i“k ˜ns™not significant dcs dendritic cells fus focused ultrasound hifu high intensityfocused ultrasoundsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets these observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4t1 tme must be addressed in order to facilitate the t cell response to fusfus partial thermal ablation in combination with gem constrains primary tnbc tumor outgrowth and extends overall survivalour observation of the overwhelming mdsc burden following 4t1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier to this end we tested a combinatorial paradigm incorporating gem a myelosuppressive chemotherapy demonstrated to inhibit mdscs transiently in the 4t1 model without consequence to t cell phenotype or function12to evaluate the efficacy of fus and gem in combination we used a preclinical ultrasound guided fus system to achieve partial thermal ablation of established 4t1 tumors 14d after tumor implantation average tumor volume of mm3 in combination with the single session of fus thermal ablation we initiated gem therapy mgmouse which was then readministered weekly for a total of three gem doses figure 2a combinatorial therapy synergized to produce significant constraint of 4t1 tumor outgrowth compared with sham and monotherapy groups figure 2b“cby termination of treatments at day 4t1 tumors exposed to fusgem combination saw nearly — and — reductions in average volume compared with sham or gem exposed tumors respectively figure 2b two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2d“e in a fraction of mice treated with fusgem we observed complete regression of 4t1 tumors although transient figure 2c tumor outgrowth eventually rebounded after termination of treatments 4t1 tumor bearing mice receiving fusgem treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and gem groups respectively hrs and for fusgem relative to sham and gem groups respectively figure 2f we additionally observed that fusgem significantly constrained outgrowth in a separate c57bl6 metastatic mammary carcinoma model e0771 online supplementary figure to further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided fus system theraclion echopulse that is already ce marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging fus thermal ablation in combination with cancer immunotherapy we observed that partial thermal ablation using the theraclion visualization and treatment unit mhz in combination open accesswith gem controlled 4t1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure these findings lend credence to the notion that the impact of combining gem with fus may be conserved across partial thermal ablation regimens moreover they demonstrate that the efficacy of fus partial thermal ablation in combination with gem can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallycombination of fus partial thermal ablation with gem increases the levels of circulating t cellslymphocytes”in particular cd8 and cd4 t cells”play an important role in responding to tumor antigen and generating a durable antitumor response based on the extended protective effect observed in mice treated with fusgem flow cytometry analysis was performed to evaluate the contribution of t cells in generating systemic and local tumor control we sampled the circulating immune cell repertoire in 4t1 tumor bearing mice via serial tail bleeds days and prior to readministration of gem and a terminal cardiac bleed at the time of spleen harvest day figure 3a combinatorial therapy significantly elevated absolute number of cd8 and cd4 t cells in the circulation at days and figure 3b“c and e“f moreover a trend threefold to fivefold increase in circulating t cells was noted in the fus group relative to sham figure 3b“c and e“f from days to systemic cd44 expressing antigen experienced t cell populations both cd8 and cd4 saw a steady significant increase after combinatorial therapy figure 3d and g a similar modest trend was noted for the fus monotherapy group relative to sham and gem figure 3d and g these changes were concordant with a decrease in circulating myeloid cd11b cells in gem recipient groups demonstrating the ability of gem to partially alleviate circulating myeloid burden figure 3hsplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4t1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression we observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6a“b consistent with this observation immunological characterization of spleens revealed a significant decrease in cd11b myeloid cells”a “ reduction in fusgem spleens relative to sham or monotherapy figure 3i while there appeared to be a trend toward more cd11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute cd11b cell numbers within the spleen data not shown the decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following fusgem treatment relative to these sham and gem groups combination therapy elevated splenic cd8 t lymphocytes by fold and sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem constrains primary triple negative breast cancer outgrowth and extends overall survival a overview of experimental design for evaluation combination of fus with serial gem treatment in murine mammary carcinoma b average 4t1 tumor outgrowth in sham n7 fus monotherapy n5 gem monotherapy n10 and combinatorial fusgem therapy groups n10 data are represented up to select time points corresponding with mouse dropout due to humane endpoints all data represented as mean±sem significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by tukey multiple comparison correction p005 vs all other groups specifically sham vs fusgem p00001 fus vs fusgem p00001 shamgem vs fusgem p00026 c 4t1 tumor outgrowth from individual mice in sham fus shamgem or fusgem groups data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint d representative images of 4t1 tumors excised at day scale bar1 cm e quantification of 2d tumor areas from images in previous panel f kaplan meier curve depicting overall survival of sham treatment n9 fus monotherapy n6 gem monotherapy n10 and combinatorial fusgem therapy n10 recipient mice significance assessed by log rank mantel cox test p005 vs all other groups specifically sham vs fus p02154 sham vs fusgem p00001 sham vs shamgem p00050 fus vs fusgem p00021 fus vs shamgem p00312 fusgem vs shamgem p00041sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen accessfigure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem increases the levels of circulating t cells a overview of experimental design to understand the impact of fus andor gem treatment on circulating immune cells b“c absolute number of circulating cd8 t cells at day b and day c d percentage of circulating cd8 t cells expressing cd44 from days to e“f absolute number of circulating cd4 t cells at day e and day f g percentage of circulating cd4 t cells expressing cd44 from days to h percentage of cd11b myeloid cells out of total cd45 immune cell in circulation from days to i“k percentage of myeloid cells i cd8 t cells j and cd4 t cells k out of total cd452 immune cells all data represented as mean±sem all data representative of sham n6“ fus monotherapy n4“ gem monotherapy n9 and combinatorial fusgem therapy n6“ groups significance assessed by analysis of variance followed by tukey multiple comparison correction for b c e f or fisher™s least significant difference lsd without multiple comparisons correction for i“k significance for d g and h assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by fisher™s lsd without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access fold figure 3j and cd4 t lymphocytes by fold and fold figure 3k these elevations were accompanied by a modest increase in percentage of foxp3 regulatory t cells tregs online supplementary figure 6e additionally increases in percentage of nk and b cells were noted twofold to fivefold online supplementary figure 6c“d these findings indicate that combinatorial therapy with fusgem promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasescombinatorial fusgem therapy does not promote robust local antitumor t cell responsesgiven the robust systemic immune signatures within the blood and spleen following fusgem we assayed 4t1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral t cell response figure 4a approximately hours prior to euthanasia mice received intravenous brefeldin a injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry immune characterization of tumors at days postimplantation”that is days subsequent to final gem administration”revealed no significant changes in absolute number of antigen experienced cd44 cd8 or cd4 t lymphocytes figure 4b“c moreover the polyfunctionality of these t cells as denoted by ifnÎ and granzyme b expression was not significantly altered figure 4d“e however intratumoral functional changes were noted in the myeloid compartment gem monotherapy modestly increased il 12p40 production by dcs fold but this was not conserved in the combinatorial therapy group figure 4f moreover while fus monotherapy generated a trend in elevated tnfα production by intratumoral g mdscs gem recipient groups saw a significant increase threefold relative to sham figure 4g these findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely interestingly intratumoral t cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionprotection conferred by combination of fus and gem is dependent on adaptive immunitysince our findings revealed no obvious advantage or function of adaptive immunity in the local tme we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with fusgem to this end we utilized an rag1ˆ’ˆ’ model that is deficient in t and b cells to address the hypothesis that mature t andor b cells play a role in the observed response wild type wt or rag1ˆ’ˆ’ mice bearing 4t1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes mice were subsequently treated with either gem monotherapy or the combination of fusgem the tumor growth inhibition offered by fusgem was entirely lost in rag1ˆ’ˆ’ mice relative to their wt counterparts with average 4t1 tumor volume in rag1ˆ’ˆ’ mice being over fivefold higher than that of wt mice on termination of treatments figure 5a of note despite a trend toward loss of protection in rag1ˆ’ˆ’ mice tumor outgrowth in response to gem monotherapy did not significantly stratify between wt and rag1ˆ’ˆ’ settings figure 5a we also observed a complete loss of fusgem mediated survival benefit over gem monotherapy in the rag1ˆ’ˆ’ setting figure 5b while these results demonstrate that an intact adaptive immune response is required for both the overall survival benefit and restriction of primary tumor offered by fusgem therapy they do not delineate the relative roles of t andor b cellsthus to address the hypothesis that the protective effect of fusgem is specifically dependent on cd4 and cd8 t cells we depleted these populations via serial coinjections of cd8 depleting and cd4 depleting antibodies in 4t1 tumor bearing wt mice on a fusgem figure 5c depletions were maintained between day and day and flow cytometry analysis of circulating immune cells at day confirmed that the target t cell populations were effectively depleted in all mice online supplementary figure consistent with the tumor escape observ

cancer7511

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " triple negative breast cancer tnbc remains recalcitrant to most targeted therapy approaches however recent clinical studies suggest that inducing tumor damage can render tnbc responsive to immunotherapy we therefore tested a strategy for immune sensitization of murine tnbc 4t1 tumors through combination of focused ultrasound fus thermal ablation and a chemotherapy gemcitabine gem known to attenuate myeloid derived suppressor cells mdscsmethods we applied a sparse scan thermally ablative fus regimen at the tumor site in combination with systemically administered gem we used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity we also tested this combination in rag1ˆ’ˆ’ mice or t cell depleted wild type mice to determine the essentiality of adaptive immunity further we layered programmed cell death protein pd1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalresults the immune modulatory effect of fus monotherapy was insufficient to promote a robust t cell response against 4t1 consistent with the dominant mdsc driven immunosuppression evident in this model the combination of fusgem significantly constrained primary tnbc tumor outgrowth and extended overall survival of mice tumor control correlated with increased circulating antigen experienced t cells and was entirely dependent on t cell mediated immunity the ability of fusgem to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti pd1 thermally ablative fus in combination with gem restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic tnbc model this treatment strategy promises a novel option for potentiating the role of fus in immunotherapy of metastatic tnbc and is worthy of future clinical evaluationtrial registration numbers nct03237572 and nct04116320 metastatic breast cancer brca particularly the triple negative breast cancer tnbc phenotype is resistant to most chemical and molecularly targeted therapeutic approaches interestingly tnbc is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 early studies in the use of immunotherapies targeting the pd1programmed death ligand pd l1 checkpoint inhibitory axis showed some efficacy2“ in tnbc compared with other brca subtypes which are generally recalcitrant to checkpoint blockade activity in the tnbc subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in tnbc greater immunotherapy efficacy in tnbc has been recently observed with the use of antibodies targeting the pd1pd l1 checkpoint inhibitory axis in combination with nab paclitaxel5 this outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment tme found in tnbcamong the potential networks in tnbc that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets these have the capacity to impair adaptive immunity and promote tumor growth and metastasis among these cell types myeloid derived suppressor cells mdscs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both t cell activation and effector functions6 increased levels of this cell type have been demonstrated in tumor tissues of patients with primary brca while those with metastatic disease bear the highest abundance of circulating mdscs8 studies have sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate mdsc can improve antitumor immunity9“to this end the central premise put forth in this study is that focused ultrasound fus”a safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissues”can synergize with immunotherapy in a murine model of metastatic tnbc fus is capable of rapidly heating tumors to thermally ablative temperatures its extracorporeal application obviates the need for catheterization injection or implantation fus can be targeted with millimeter precision under mri or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues the bioeffects of fus hold distinct implications for tumor antigenicity immune cell activation and trafficking13 thermally active fus regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17“ colon20 kidney21 and brca23 pertaining to the challenge of myeloid cell immunosuppression in tnbc thermally ablative fus has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin il12 interferonÎ ifnÎ and tumor necrosis factorα tnfα from a variety of cancer cell lines and after in vivo treatment of tumors26 whether the ability of fus to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of brca is currently under debate with some studies showing activation of antigen presenting cells and t cell recruitment in patients with brca treated with thermally ablative fus28 while others show that additional innate stimuli are needed to support antitumor immunity23 notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells dcs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation fus regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine tnbc with extensive granulocytic mdsc involvement that is recalcitrant to anti pd1 while some activity is evident with the partial ablation approach significantly greater control was achieved by targeting mdsc inhibition in combination with thermally ablative fus this control was completely dependent on the adaptive immune responsemoreover we demonstrate that layering anti pd1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction these data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative fus once immunosuppressive myeloid cells are accounted for fus treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockademethodscell line maintenance4t1 and e0771 cell lines were maintained in rpmi l glut or dulbecco™s modified eagle™s medium dmem gl d glucose l glutamine respectively supplemented with fetal bovine serum fbs at °c and co2 thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments cells tested negative for mycoplasmaeight week old to week old female balbc or c57bl6 mice were obtained from nci charles river nci crl or the jackson laboratory female balbc rag1ˆ’ˆ’ mice were obtained from the jackson laboratory 4t1 or e0771 cells — were subcutaneously implanted into the right flank of mice mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum tumor outgrowth was monitored via digital caliper measurements tumor volume was calculated as follows volume length—width22 approximately days 4t1 or days e0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsin vivo ultrasoundguided fus partial thermal ablationmice were treated with fus either days 4t1 cohorts or days e0771 postimplantation on treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg zoetis and dexdomitor mgkg pfizer in sterilized saline mouse flanks were shaved and depilated following which ultrasound guided fus thermal ablation was performed using one of the two systems system and treatment details are provided in online supplementary materials and methods mice that did not receive fus treatment consistently underwent anesthesia and depilation of the flank additionally these mice underwent a ˜sham™ treatment consisting of exposure to the °c degassed water bath exposure for min following ˜sham™ or fus treatment all mice were moved to a heating pad and given antisedan for anesthesia reversal and recoverygemcitabine therapygemcitabine gem mgmouse in µl volume mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of fus treatment following which administration was repeated for an additional weeks administration of gem doses was based on existing literature demonstrating the use of gem for inhibition of mdscs in 4t112 the initial dose of gem was administered immediately prior to ˜sham™ or fus treatment sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cmice that did not receive gem received an intraperitoneal injection of ˜vehicle™ treatment µl of sterile saline at the time points specifiedpd1 blockade therapyfor checkpoint inhibitor therapy the rat anti mouse pd1 antibody αpd1 rmp114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse treatment was initiated on day ˜early αpd1™ or day ˜delayed αpd1™t cell depletionst cell depletion antibodies”anti cd8 clone bio x cell and anti cd4 gk15 clone bio x cell”were diluted in sterilized saline and administered intraperitoneally every to days starting at day days post fus for a total of seven doses µg of each antibody for a total µg per mouseimmunohistochemistryon day sham or fus exposed tumors were excised and fixed in neutral buffered formalin sigma fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin digital images of stained slides were acquired using the vectra automated quantitative pathology imaging system akoya biosciences whole slide screening and image capture were subsequently performed using phenochart akoya biosciencesflow cytometrymice were bled at days and via tail vein and samples were rbc lysed hybri max sigma and stained for flow cytometry analysis at days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment in order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse cd45 fitc clone f11 bd biosciences min prior to euthanasia 4t1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor dlns pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis additional details are provided in online supplementary materials and methodssamples were acquired on an attune nxt flow cytometer thermofisher scientific and data were analyzed with flowjo treestar or fcs express de novo software a representative gating strategy for granulocytic myeloid derived suppressor cell g mdsc and cd44 t cells is provided in online supplementary figure statistical analysisall statistical analyses were performed in graphpad prism graphpad software a detailed description of statistical methods for each experiment is provided in the corresponding figure legendopen accessanimal study approvalall animal work was performed under a protocol approved by the animal care and use committee at the university of virginia and conformed to the national institutes of health guidelines for the use of animals in researchresultspartial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsetsto achieve partial thermal ablation of 4t1 tumors we used an ultrasound guided fus system equipped with a single element therapeutic transducer driven at mhz figure 1a online supplementary figure a grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under b mode ultrasound guidance figure 1b“c the exceptionally small focus of this system rendered a low ablation fraction “ of total tumor volume immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1d one week following fus partial thermal ablation tumors and secondary lymphoid organs were excised for immunological characterization by flow cytometry figure 1b fus partial thermal ablation of 4t1 tumors conferred a significant increase fold in the absolute number of cd11c hi dcs within the axillary tumor draining lymph node adln of mice figure 1e while this was accompanied by a nearly threefold elevation in the absolute number of cd86 dcs within the adln figure 1f the percentage of dcs expressing cd86 did not change figure 1g increased numbers of dcs”and cd86 dcs in particular”suggest fus is promoting the maturation or trafficking of these cells in the dlns where they could encounter and activate t cells however this did not translate to tumor growth restriction data not shown we also did not observe significant differences in the absolute number of activated t cells in 4t1 tumors figure 1h or dlns data not shown following fus exposure suggesting limitations in the ability of fus activated dc to further drive an antitumor t cell responseimmune profiling by flow cytometry revealed that irrespective of fus exposure of the intratumoral cd45 immune cell population is comprised of cd11b myeloid cells figure 1i similarly approximately of the circulating immune cell population in 4t1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure notably ly6g granulocytic myeloid derived suppressor cells g mdscs significantly dominated the immune cell repertoire within 4t1 tumors relative to other myeloid including f480 macrophages ly6c cell subsets monocytic myeloid derived suppressor cells m mdscs and cd11c hi dcs figure 1j fus partial thermal ablation did not significantly alter the absolute number per sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure partial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsets a design overview of a custom ultrasound guided fus system consisting of a mhz single element transducer orthogonally co registered to an mhz linear ultrasound imaging array the tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °c ˜sham™ mice were similarly positioned but did not undergo sonications b schematic illustration of fus partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4t1 tumor bearing mice a grid of sonications was applied in a raster pattern onto the b mode ultrasound visible tumor in total two planes of sonication spaced mm apart were applied to each tumor grid points were spaced mm apart within a single plane one week following thermal ablation tumors and secondary lymphoid organs were excised for sham n6 or fus treated n5 mice and processed for flow cytometry c representative b mode ultrasound images of ectopic 4t1 tumors either before top or during bottom fus exposure sonication grid depicting targets red points is superimposed on b mode image during treatment subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed d representative he staining of either sham 4t1 tumors or those resected immediately following fus partial thermal ablation zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively e absolute number of cd11c hi dcs in the axillary tumor draining lymph node adln of 4t1 tumor bearing mice p00136 vs sham f absolute number of cd86 cd11c hi dcs in the adln p00063 vs sham g percentage of cd86 subset out of total cd11c hi dcs within adln h absolute number of intratumoral cd44 cd8 and cd44 cd4 t cells and regulatory t cells tregs per gram tumor i percentage of cd11b myeloid cells out of total cd45 immune cells across tumor spleen adln inguinal dln idln and nontumor draining axillary and inguinal lns ndlns p005 vs all other groups irrespective of fus exposure specifically tumor vs spleen p00226 tumor spleen vs all other organs p00001 j absolute number of intratumoral myeloid cells cd11c hi dcs f480 macrophages ly6c monocytic myeloid derived suppressor cells m mdscs ly6g granulocytic myeloid derived suppressor cells g mdscs per gram 4t1 tumor p00001 vs all other cell types irrespective of fus exposure all data represented as mean±sem significance assessed by unpaired t test f“h or two way analysis of variance followed by tukey multiple comparison correction i“k ˜ns™not significant dcs dendritic cells fus focused ultrasound hifu high intensityfocused ultrasoundsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets these observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4t1 tme must be addressed in order to facilitate the t cell response to fusfus partial thermal ablation in combination with gem constrains primary tnbc tumor outgrowth and extends overall survivalour observation of the overwhelming mdsc burden following 4t1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier to this end we tested a combinatorial paradigm incorporating gem a myelosuppressive chemotherapy demonstrated to inhibit mdscs transiently in the 4t1 model without consequence to t cell phenotype or function12to evaluate the efficacy of fus and gem in combination we used a preclinical ultrasound guided fus system to achieve partial thermal ablation of established 4t1 tumors 14d after tumor implantation average tumor volume of mm3 in combination with the single session of fus thermal ablation we initiated gem therapy mgmouse which was then readministered weekly for a total of three gem doses figure 2a combinatorial therapy synergized to produce significant constraint of 4t1 tumor outgrowth compared with sham and monotherapy groups figure 2b“cby termination of treatments at day 4t1 tumors exposed to fusgem combination saw nearly — and — reductions in average volume compared with sham or gem exposed tumors respectively figure 2b two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2d“e in a fraction of mice treated with fusgem we observed complete regression of 4t1 tumors although transient figure 2c tumor outgrowth eventually rebounded after termination of treatments 4t1 tumor bearing mice receiving fusgem treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and gem groups respectively hrs and for fusgem relative to sham and gem groups respectively figure 2f we additionally observed that fusgem significantly constrained outgrowth in a separate c57bl6 metastatic mammary carcinoma model e0771 online supplementary figure to further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided fus system theraclion echopulse that is already ce marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging fus thermal ablation in combination with cancer immunotherapy we observed that partial thermal ablation using the theraclion visualization and treatment unit mhz in combination open accesswith gem controlled 4t1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure these findings lend credence to the notion that the impact of combining gem with fus may be conserved across partial thermal ablation regimens moreover they demonstrate that the efficacy of fus partial thermal ablation in combination with gem can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallycombination of fus partial thermal ablation with gem increases the levels of circulating t cellslymphocytes”in particular cd8 and cd4 t cells”play an important role in responding to tumor antigen and generating a durable antitumor response based on the extended protective effect observed in mice treated with fusgem flow cytometry analysis was performed to evaluate the contribution of t cells in generating systemic and local tumor control we sampled the circulating immune cell repertoire in 4t1 tumor bearing mice via serial tail bleeds days and prior to readministration of gem and a terminal cardiac bleed at the time of spleen harvest day figure 3a combinatorial therapy significantly elevated absolute number of cd8 and cd4 t cells in the circulation at days and figure 3b“c and e“f moreover a trend threefold to fivefold increase in circulating t cells was noted in the fus group relative to sham figure 3b“c and e“f from days to systemic cd44 expressing antigen experienced t cell populations both cd8 and cd4 saw a steady significant increase after combinatorial therapy figure 3d and g a similar modest trend was noted for the fus monotherapy group relative to sham and gem figure 3d and g these changes were concordant with a decrease in circulating myeloid cd11b cells in gem recipient groups demonstrating the ability of gem to partially alleviate circulating myeloid burden figure 3hsplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4t1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression we observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6a“b consistent with this observation immunological characterization of spleens revealed a significant decrease in cd11b myeloid cells”a “ reduction in fusgem spleens relative to sham or monotherapy figure 3i while there appeared to be a trend toward more cd11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute cd11b cell numbers within the spleen data not shown the decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following fusgem treatment relative to these sham and gem groups combination therapy elevated splenic cd8 t lymphocytes by fold and sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem constrains primary triple negative breast cancer outgrowth and extends overall survival a overview of experimental design for evaluation combination of fus with serial gem treatment in murine mammary carcinoma b average 4t1 tumor outgrowth in sham n7 fus monotherapy n5 gem monotherapy n10 and combinatorial fusgem therapy groups n10 data are represented up to select time points corresponding with mouse dropout due to humane endpoints all data represented as mean±sem significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by tukey multiple comparison correction p005 vs all other groups specifically sham vs fusgem p00001 fus vs fusgem p00001 shamgem vs fusgem p00026 c 4t1 tumor outgrowth from individual mice in sham fus shamgem or fusgem groups data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint d representative images of 4t1 tumors excised at day scale bar1 cm e quantification of 2d tumor areas from images in previous panel f kaplan meier curve depicting overall survival of sham treatment n9 fus monotherapy n6 gem monotherapy n10 and combinatorial fusgem therapy n10 recipient mice significance assessed by log rank mantel cox test p005 vs all other groups specifically sham vs fus p02154 sham vs fusgem p00001 sham vs shamgem p00050 fus vs fusgem p00021 fus vs shamgem p00312 fusgem vs shamgem p00041sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen accessfigure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem increases the levels of circulating t cells a overview of experimental design to understand the impact of fus andor gem treatment on circulating immune cells b“c absolute number of circulating cd8 t cells at day b and day c d percentage of circulating cd8 t cells expressing cd44 from days to e“f absolute number of circulating cd4 t cells at day e and day f g percentage of circulating cd4 t cells expressing cd44 from days to h percentage of cd11b myeloid cells out of total cd45 immune cell in circulation from days to i“k percentage of myeloid cells i cd8 t cells j and cd4 t cells k out of total cd452 immune cells all data represented as mean±sem all data representative of sham n6“ fus monotherapy n4“ gem monotherapy n9 and combinatorial fusgem therapy n6“ groups significance assessed by analysis of variance followed by tukey multiple comparison correction for b c e f or fisher™s least significant difference lsd without multiple comparisons correction for i“k significance for d g and h assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by fisher™s lsd without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access fold figure 3j and cd4 t lymphocytes by fold and fold figure 3k these elevations were accompanied by a modest increase in percentage of foxp3 regulatory t cells tregs online supplementary figure 6e additionally increases in percentage of nk and b cells were noted twofold to fivefold online supplementary figure 6c“d these findings indicate that combinatorial therapy with fusgem promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasescombinatorial fusgem therapy does not promote robust local antitumor t cell responsesgiven the robust systemic immune signatures within the blood and spleen following fusgem we assayed 4t1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral t cell response figure 4a approximately hours prior to euthanasia mice received intravenous brefeldin a injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry immune characterization of tumors at days postimplantation”that is days subsequent to final gem administration”revealed no significant changes in absolute number of antigen experienced cd44 cd8 or cd4 t lymphocytes figure 4b“c moreover the polyfunctionality of these t cells as denoted by ifnÎ and granzyme b expression was not significantly altered figure 4d“e however intratumoral functional changes were noted in the myeloid compartment gem monotherapy modestly increased il 12p40 production by dcs fold but this was not conserved in the combinatorial therapy group figure 4f moreover while fus monotherapy generated a trend in elevated tnfα production by intratumoral g mdscs gem recipient groups saw a significant increase threefold relative to sham figure 4g these findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely interestingly intratumoral t cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionprotection conferred by combination of fus and gem is dependent on adaptive immunitysince our findings revealed no obvious advantage or function of adaptive immunity in the local tme we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with fusgem to this end we utilized an rag1ˆ’ˆ’ model that is deficient in t and b cells to address the hypothesis that mature t andor b cells play a role in the observed response wild type wt or rag1ˆ’ˆ’ mice bearing 4t1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes mice were subsequently treated with either gem monotherapy or the combination of fusgem the tumor growth inhibition offered by fusgem was entirely lost in rag1ˆ’ˆ’ mice relative to their wt counterparts with average 4t1 tumor volume in rag1ˆ’ˆ’ mice being over fivefold higher than that of wt mice on termination of treatments figure 5a of note despite a trend toward loss of protection in rag1ˆ’ˆ’ mice tumor outgrowth in response to gem monotherapy did not significantly stratify between wt and rag1ˆ’ˆ’ settings figure 5a we also observed a complete loss of fusgem mediated survival benefit over gem monotherapy in the rag1ˆ’ˆ’ setting figure 5b while these results demonstrate that an intact adaptive immune response is required for both the overall survival benefit and restriction of primary tumor offered by fusgem therapy they do not delineate the relative roles of t andor b cellsthus to address the hypothesis that the protective effect of fusgem is specifically dependent on cd4 and cd8 t cells we depleted these populations via serial coinjections of cd8 depleting and cd4 depleting antibodies in 4t1 tumor bearing wt mice on a fusgem figure 5c depletions were maintained between day and day and flow cytometry analysis of circulating immune cells at day confirmed that the target t cell populations were effectively depleted in all mice online supplementary figure consistent with the tumor escape observ Answer:

Colon_Cancer

" adenovirus serotype ad5 is a commonly used viral vector for transient delivery of transgenes primarily for vaccination against pathogen and tumor antigens however endemic infections with ad5 produce virus specific neutralizing antibodies nabs that limit transgene delivery and constrain target directed immunity following exposure to ad5 based vaccines indeed clinical trials have revealed the limitations that virus specific nabs impose on the efficacy of ad5 based vaccines in that context the emerging focus on immunological approaches targeting cancer self antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectorsmethods here we evaluated the ability of a chimeric adenoviral vector ad5f35 derived from the capsid of ad5 and fiber of the rare adenovirus serotype ad35 to induce immune responses to the tumor associated antigen guanylyl cyclase c gucy2cresults in the absence of pre existing immunity to ad5 gucy2c specific t cell responses and antitumor efficacy induced by ad5f35 were comparable to ad5 in a mouse model of metastatic colorectal cancer furthermore like ad5 ad5f35 vector expressing gucy2c was safe and produced no toxicity in tissues with or without gucy2c expression importantly this chimeric vector resisted neutralization in ad5 immunized mice and by sera collected from patients with colorectal cancer naturally exposed to ad5s these data suggest that ad5f35 based vaccines targeting gucy2c or other tumor or pathogen antigens may produce clinically relevant immune responses in more ‰¥ patients compared with ad5 based vaccines inhibitor introductiontherapies immune checkpoint have revolutionized cancer treatment and cancer drug development by engaging the immune system to target various cancers1 despite this success many tumors are immunologically œcold characterized by a dearth of immunogenic neoepitopes3 and lack of tumor infiltrating lymphocytes4 and remain refractory to checkpoint inhibitors6 one emerging strategy to modify a cold tumor into one responsive to immunotherapy is through combination with cancer vaccines8 the goal of this strategy is to use cancer vaccines to create a pool of tumor reactive t cells with antitumor activity alone andor in combination with checkpoint therapies however this approach is significantly limited by the paucity of effective vaccine platforms to safely deliver tumor specificassociated antigens to elicit beneficial antitumor immunitythe ability of adenovirus serotype ad5 to mediate gene transfer and induce potent immune responses has made it a popular vector for experimental vaccines infectious diseases10 against cancer and indeed there have been more than clinical trials using the ad5 vector with most trials focused on developing cancer treatments10 however on natural infection the host immune system develops neutralizing antibodies nabs to the ad5 capsid limiting viral spread and blocking reinfection because ad5 infections are endemic in many human populations pre existing nabs present in of the worldwide population limit ad5 based vaccine strategies12“ these considerations highlight the need for improved vectors for use in vaccines targeting cancer and pathogen associated antigens that can create therapeutic immune responses in the greatest number of patients importantly while the adenovirus capsid is composed of hexon penton and fiber proteins nabs elicited by natural ad5 infection in humans are directed primarily to the ad5 fiber15 suggesting that strategies to flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access circumvent pre existing immunity to this element may improve ad5 based vaccineshere we sought to overcome pre existing ad5 nabs by replacing the ad5 fiber with that of a rare adenovirus serotype ad35 international seroprevalence “ to improve antitumor immunity in mouse models expressing the gastrointestinal gi cancer antigen guanylyl cyclase c gucy2c preclinical models demonstrated that an ad5 based gucy2c directed vaccine ad5 gucy2c s1 elicited cd8 t cell and antibody responses without autoimmunity17 further ad5 gucy2c s1 vaccination of mice induced long term t cell mediated protection against metastatic colorectal cancer in lung and liver19 moreover those results were recapitulated in a recent first in human phase i clinical trial nct01972737 demonstrating that a humanized version of the vaccine ad5 gucy2c padre safely induced gucy2c specific cd8 t cell responses in patients with colorectal cancer following conventional therapies21 however patients possessing high pre existing titers of nabs against ad5 failed to generate gucy2c specific immunity following ad5 gucy2c padre vaccination21 to overcome ad5 nabs we generated a chimeric ad5 vector possessing the fiber of ad35 ad5f35 with equivalent safety and antitumor activity to ad5 and resistance to ad5 nabs in mice and humans this chimeric vaccine can be translated to patients with gi cancer to safely induce gucy2c specific immunity not only in those patients with low ad5 immunity but also in those with high pre existing ad5 nabsmaterials and methodsadenovirus vectorsadenovirus containing mouse extracellular domain gucy2c1429 with the influenza ha107119 cd4 t cell epitope known as site s1 was described previously ad5 gucy2c s120 here gucy2c s1 was cloned into pshuttle and subcloned into the e1 region of previously generated replication deficient chimeric adenovirus ad5f35 in which the ad5 fiber was replaced by the ad35 fiber22 to generate ad5f35 gucy2c s1 all adenovirus vaccines used in this study were produced in hek293 cells and purified by cesium chloride ultracentrifugation under good laboratory practices by the baylor college of medicine in the cell and gene therapy vector development lab and certified to be negative for replication competent adenovirus mycoplasma and host cell dna contamination in vitro gucy2c expression experiments dose“response and time“course were carried out in a549 american type culture collection atcc cells virus was added to the cultures at the indicated doses and culture supernatants were collected at the indicated time points relative gucy2c levels were quantified in supernatants by western blot using μgml ms7 mouse anti gucy2c monoclonal antibody23“ and μgml horseradish peroxidase conjugated goat antimouse secondary antibody jackson immunomice and immunizationseight week old male and female balbcj mice were purchased from the jackson laboratory for experiments animal protocols were approved by the thomas jefferson university institutional animal care and use committee protocol for immunizations mice received or vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or ad5f35 gfp control administered as two μl intramuscular injections one in each hind limb using a ml insulin syringequantifying tcell responses by elispotelispot assays were performed using a mouse interferonÎ ifnÎ single color elispot kit cellular technology according to the manufacturer™s protocol26 briefly well plates were coated with ifnÎ capture antibody overnight at °c the next day plates were washed with phosphate buffered saline pbs and splenocytes from immunized mice were plated at cellswell with no peptide or μgml gucy2c254262 peptide in dimethyl sulfoxide dmso in ctl test medium cellular technology for hours at °c for t cell avidity studies splenocytes were plated at “ cellswell with decreasing concentrations of gucy2c254262 peptide μgml to pgml normalized to cellswell26 after incubation cells were removed and development reagents were added to detect ifnÎproducing spot forming cells the number of spot forming cells per well was determined using the smartcount and autogate functions of an immunospot s6 universal analyzer cellular technology gucy2c specific responses were calculated by subtracting mean spot counts of dmso wells from peptide stimulated wells26 tumor studiesgucy2c expressing mouse balbc ct26 colorectal cancer cells were used for in vivo tumor studies17 luciferase expressing cells were generated by transduction with lentiviral supernatants produced by 293ft cells invitrogen with plenti4 v5 gw luciferase28 for tumor experiments balbcj mice were immunized with vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or pbs control days before delivering × ct26 cells into tail veins tumor burden was quantified weekly by subcutaneous injection of mg of d luciferin potassium salt gold biotechnologies in pbs followed by an min incubation and imaging with a s exposure using a caliper ivis lumina xr imaging station perkinelmer total radiance photonssecond was measured using living image in vivo imaging software perkinelmerantibody neutralization assayserum samples were obtained previously from patients before ad5 gucy2c padre nct01972737 approved by the thomas jefferson university institutional review board21 neutralizing antibody titers against ad5 and ad5f35 vectors were quantified as immunization with flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0cdescribed21 briefly dilutions of heat inactivated serum samples were added to well tissue culture plates containing a549 cells atcc and infected with vp of gfp expressing ad5 or ad5f35 virus ad5 cmv egfp or ad5f35 cmv egfp respectively baylor vector development lab following a hour incubation at °c egfp fluorescence nm excitation nm emission was quantified using a polarstar optimate plate reader bmg labtech sample fluorescence was normalized to control wells containing cells and virus neutralization or wells containing cells alone neutralization titers were quantified using non linear regression as the serum dilution producing neutralization prism v8 graphpad softwaread5 neutralizing immunity studiesto induce anti ad5 immunity mice were exposed intranasally to ad5 gfp once or twice at a week interval thirty days after the last exposure ad5 nabs were quantified in sera as described above and mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1biodistribution and toxicology studybalbcj mice were immunized intramuscularly with a single dose of vp of ad5f35 gucy2c s1 three doses of vp of ad5f35 gucy2c s1 at day intervals or pbs control animals were monitored for adverse events once daily with additional evaluations on the day of dosing min hour and hours after dosing on days and designated animals were sacrificed and brain salivary glands stomach small intestine colon heart lungs kidneys liver and injection site were harvested and weighed for histopathological analysis by a blinded pathologist pathology evaluation was performed by idexx bioanalytics and detection of viral dna by quantitative pcr qpcr using the previously described assay for the gucy2c transgene19 also spleens were collected for histopathological analysis and detection of viral dna as described above as well as quantification of gucy2c specific t cell responses by ifnÎ elispot as described abovestatistical analysisstatistical analyses were conducted using graphpad prism software v8 statistical significance was considered as follows nsp p p p and p cohort sizes were powered based on prior studies with β02 and α005 for multiple comparisons of survival outcomes significance thresholds were corrected using the bonferroni method to identify vaccine induced t cell responders and non responders a previously described21 modified distribution free resampling approach was employed and positive t cell responses were defined as × compared with dmso and specific spots106 cells to determine the impact of gender and number of vaccinations on responses log transformed vaccine response magnitude was compared in mice of different genders cohorts and treatment regimens for up to three way interactions with stepwise backward variable selection by akaike information criterion using r29 package mass30open accessresultsad5gucy2cs1 and ad5f35gucy2cs1 vectorswhile ad5 seroprevalence worldwide exceeds in some regions ad35 is and associated with lower titers figure 1a12 thus we constructed a chimeric adenovirus ad5f35 composed of ad5 in which the fiber was replaced by the ad35 fiber and evaluated its ability to induce gucy2c specific immunity and resist ad5 specific immunity in humans and mice ad5 gucy2c s1 is a replication deficient human ad5 expressing the mouse gucy2c extracellular domain fused to the i ed restricted cd4 epitope known as site at its c terminus20 to generate ad5f35 gucy2c s1 the ad5 fiber l5 was replaced with the ad35 fiber figure 1b replication deficient ad5 gucy2c s1 and ad5f35 gucy2c s1 generated in hek293 cells produced dose dependent figure 1c and time dependent figure 1d expression of gucy2c s1 protein in a549 human alveolar basal epithelial cells in vitroad5f35gucy2cs1 induces gucy2cspecific antitumor immunityfollowing in vitro validation of gucy2c expression by ad5f35 gucy2c s1 we confirmed its ability to induce gucy2c specific immune responses after vaccination in vivo balbc mice immunized intramuscularly with vp of ad5f35 gucy2c s1 produced lower gucy2c specific cd8 t cell responses figure 2a and no gucy2c specific antibody responses figure 2b compared with ad5 gucy2c s1 importantly ad5 and ad5f35 vaccines produced gucy2c specific cd8 t cells of comparable avidity figure 2c a critical determinant of the antitumor efficacy of gucy2c targeted vaccines26 in contrast gucy2c specific antibody responses have no detectable antitumor activity20 similarly ad5 and ad5f35 vaccines produced comparable s1 specific cd4 t cell responses figure 2dluciferase this model previous studies revealed that ad5 gucy2c vaccines induced protective antitumor cd8 t cell responses in murine models of metastatic colorectal cancer17“ thus balbc mice were immunized with ad5 or ad5f35 expressing gucy2c s1 and challenged days later with ct26 colorectal cancer cells expressing gucy2c and firefly specifically emulates secondary prevention of metastatic disease the clinical setting for which the gucy2c vaccine is being developed21 as previously demonstrated ad5 vaccination nearly eliminated metastatic tumor burden figure 3ab delayed disease progression figure 3c and improved survival figure 3d similarly ad5f35 also reduced tumor burden figure 3ab disease progression figure 3c and prolonged survival figure 3d importantly the efficacy of ad5 based and ad5f35 based gucy2c vaccines in flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure construction of ad5f35 gucy2c s1 and antigen expression a reported international seroprevalence of ad5 and ad3512 b the l5 gene encoding the fiber protein from ad5 was replaced with the l5 gene from ad35 producing the chimeric adenoviral vector ad5f35 recombinant ad5f35 gucy2c s1 was produced by inserting mouse gucy2c s1 into the e1 region of e1e3 deleted ad5f35 c and d the human alveolar basal epithelial cell line a549 was transduced in duplicate with ad5f35 gucy2c s1 at a multiplicity of infection moi from to for hours c or at an moi of for and hours d supernatants from infected cells were analyzed for gucy2c s1 protein expression by immunoblot protein expression was quantified by densitometry and plotted relative to uninfected cells error bars indicate mean±sem ad5 adenovirus serotype reducing tumor burden opposing disease progression and promoting survival was identical figure 3a“dad5f35 resists ad5directed immunity in mice and humansnabs against ad5 correlated with poor gucy2c specific immune responses in patients receiving ad5 gucy2c padre vaccination and prior exposure of mice to ad5 similarly blunted vaccine induced immunity21 ad5f35 based vaccine resistance to pre existing ad5 immunity was quantified in a model of respiratory pre exposure to ad5 the natural route of infection in patients33 followed by vaccination and quantification of gucy2c specific t cell responses control mice not pre exposed to ad5 naive and those that were pre exposed once × or twice × to intranasal ad5 were vaccinated after weeks with intramuscular ad5 or ad5f35 expressing gucy2c s1 and immune responses were quantified weeks later immunogenicity of ad5 gucy2c s1 and ad5f35 gucy2c s1 a“d balbc mice n4“ micegroup were figure immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and serum and splenocytes were collected days later gucy2c specific cd8 t cell responses were quantified by interferon gamma ifnÎ elispot a and antibodies were quantified by elisa b c gucy2c specific t cell avidity measurements were analyzed by elispot using non linear regression logagonist versus normalized response with comparisons made using the extra sum of squares f test avidity plots depict the regression line solid with cis dashed d s1 specific cd4 t cell responses were measured by ifnÎ elispot t cell responses in a and d were analyzed by one way analysis of variance values in a b and d indicate individual animals and bars in a and d indicate means tcr t cell receptor ad5 adenovirus serotype flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure antitumor efficacy of ad5 gucy2c s1 and ad5f35 gucy2c s1 a“d balbc mice n10 micegroup were immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and challenged days later with a mouse colorectal cancer cell line ct26 expressing gucy2c and luciferase on days and following challenge mice were injected with d luciferin and imaged a to quantify tumor burden day b mice were weighed twice weekly c and monitored for survival d tumor burden b was analyzed by one way analysis of variance and survival comparisons d were analyzed by the mantel cox log rank test in b and d asterisks indicate comparisons of gucy2c vaccines to the control and brackets ] indicate comparisons between ad5 and ad5f35 vaccines ns not significant ad5 adenovirus serotype figure 4a as expected one ad5 pre exposure induced moderate ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses while two pre exposures induced high ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses following ad5 vaccination figure 4b in contrast gucy2c specific t cell responses were reduced only × pre exposure and × pre exposure following ad5f35 vaccination figure 4b importantly ad5f35 produced t cell responses in a substantially greater fraction of the population cohort responses compared with ad5 cohort responses following serial pre exposures to ad5 figure 4cthese observations in mice were recapitulated using sera from patients with colorectal cancer in the ad5 gucy2c padre phase i trial nct0197273721 here nab titers against ad5 and ad5f35 were quantified using an established ad5ad5f35 reporter virus inhibition bioassay in serum samples collected prior to vaccination with ad5 gucy2c padre21 in these patients ad5f35 specific nab titers were substantially lower than ad5 specific titers figure 4d most importantly of patients possessed low ad5 nabs titers figure 4de which closely correlated with a gucy2c specific response rate21 in striking contrast had low ad5f35 nab titers suggesting that the vast majority of patients immunized with ad5f35 based vaccines could produce gucy2c specific responses figure 4e collectively these observations suggest that pre existing viral immunity induced by repeated environmental exposures which neutralizes ad5 delivery platforms may be overcome by the chimeric ad5f35 vector to enhance fractional population vaccine responsessafety biodistribution and toxicity of ad5f35gucy2cs1food and drug administration ind investigational new drug enabling studies quantified the toxicity biodistribution and immunogenicity of ad5f35 gucy2c s1 in balbc mice employing three schemes to examine acute and chronic effects figure 5a cohorts balanced for sex received ad5f35 gucy2c s1 either as a single intramuscular injection or as three intramuscular injections spaced weeks apart monitored daily and sacrificed on day or for analysis as indicated figure 5a there were no signs of acute or chronic toxicity in the in life phase by observation weight changes or survival figure 5b“d similarly there were no clinically significant differences in organ weights online supplementary figure s2 or histopathology not shown at necropsy small statistical differences in organ weights were considered clinically insignificant and were unrelated to vaccine exposure dose time online supplementary figure s2 biodistribution quantified by qpcr detected ad5f35 gucy2c s1 at the injection site and in the spleen but not appreciably in other organs after acute and chronic exposures online supplementary figure s3 moreover robust cd8 t cell responses were quantified at day that persisted through day in of mice after a single administration figure 5e“g as expected cd8 t cell responses were greater and persisted in more mice at days after three vaccinations figure 5e“gdiscussionthrough decades of gene therapy trials ad5 has remained a popular vector while high ad5 seroprevalence remains a barrier to universal vaccination33 natural respiratory infection can generate long lived antibodies that neutralize ad5 based vaccines eliminating transgene delivery and potential therapeutic benefit in that context ad5 seroprevalence is across multiple countries12 highlighting an unmet need for alternative vectors here we demonstrate that the chimeric ad5f35 resists pre existing ad5 immunity and induces transgene specific antitumor immunity indeed ad5f35 is less susceptible to neutralization associated with ad5 exposure in mice and humans and generates a substantially higher proportion of vaccine responders in mice pre exposed to ad5 these observations support the suggestion that ad5f35 flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure ad5f35 resists neutralization associated with pre existing anti ad5 immunity in mice and humans a“c to generate pre existing immunity to ad5 balbc mice n10 micegroup were exposed intranasally once or twice to vp of ad5 gfp at week intervals four weeks after the final ad5 gfp exposure ad5 exposed and naive mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 b two weeks after immunization gucy2c specific cd8 t cell responses in each group were quantified by interferon gamma ifnÎ elispot and calculated as the of mean responses in naive mice values indicate individual animals and bars indicate means ad5 and ad5f35 were compared by two way analysis of variance c the fraction of animals producing a detectable gucy2c specific cd8 t cell response filled regions in naive × and × ad5 exposed mice was determined from b d and e sera from patients with colorectal cancer collected prior to ad5gucy2c padre vaccination were tested for the ability to neutralize ad5 and ad5f35 vectors and titers were quantified d analyzed by paired t test the dotted line indicates a titer of the threshold for high neutralizing antibody nab titers21 e while subjects had high nab titers against ad5 only had high titers to ad5f35 vector filled regions binomial test ad5 adenovirus serotype will produce a higher proportion of vaccine responders in patient populationsthe extent to which nabs to the ad5 fiber limit reinfection is controversial in some studies replacing the ad5 fiber with that of another serotype circumvents pre existing ad5 immunity34 in contrast other studies suggest that these chimeric adenoviruses do not evade pre existing ad5 nabs suggesting the hexon as the major target of antibody neutralization35 in contrast to those previous studies which generated pre existing ad5 immunity by intramuscular35 or intravenous administration36 here ad5 immunity was induced by intranasal exposure in mice recapitulating natural infection33 moreover natural human respiratory pre existing ad5 nabs in patients with colorectal cancer uniformly produced by repeated respiratory infections33 similarly were overcome by the ad5f35 vector importantly the quality of antibody responses following adenovirus infection is dependent on the route of exposure indeed respiratory infections elicit fiber specific nabs while intramuscular exposure induce capsid specific nabs15 these qualitative differences in nab responses reflecting varying routes of immunization may contribute to observational discrepancies between laboratories the present studies using relevant animal models confirmed and validated with patient samples support the suggestion that ad5f35 based vaccines should produce clinically relevant immune responses in a substantial proportion of patientsflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure safety and immunogenicity of multiple ad5f35 gucy2c s1 administrations a“g balbc mice n10 micegroup were immunized intramuscularly with one or three administrations of vp ad5f35 gucy2c s1 or control at week intervals following immunization body weights b female and c male were recorded weekly and mice were monitored for survival d at days and following first immunization mice were euthanized to quantify organ pathology by weight online supplementary figure s2 biodistribution by quantitative pcr online supplementary figure s3 and gucy2c specific cd8 t cell responses by interferon gamma ifnÎ elispot e“g g pie charts indicate proportion of responding animals ad5 adenovirus serotype recognizing the pervasive limitations imposed by endemic ad5 immunity in global populations12 there is an emerging interest in alternative serotypes and chimeric constructs as a tractable strategy in vaccine development ad26 ad35 and ad48 vectors have been advanced into phase i clinical trials37 in that regard a comparison of ad5 ad26 ad35 and ad48 immunity among healthy patients revealed that endemic ad35 seropositivity was lowest across global populations12 reinforcing chimeric strategies employed herein similarly the first hexon chimeric adenovirus comprising ad5 and ad48 components was safe and immunogenic in patients39 interestingly ad5 ad35 chimeric vectors more efficiently transduce a variety of human cell types in vitro compared with either parental vector40 these observations underscore the future potential of intelligently designed chimeric adenoviruses strategically constructed to deliver transgenes for replacement therapy or vaccination and targeted precisely to the cellular or disease context40while antitumor efficacy was equivalent cd8 t cell responses were lower and antibody responses were absent for ad5f35 gucy2c s1 compared with ad5 gucy2c s1 however the antitumor efficacy of gucy2c directed immunotherapy is driven primarily by t cell avidity rather than effector t cell quantity26 in that context the functional avidity of gucy2c specific cd8 t cells following ad5 and ad5f35 immunizations were equivalent consistent with their comparable antitumor efficacy quantitative differences in transgene specific immunity between vectors may reflect a variety of factors thus the quantity and persistence of gucy2c s1 transgene following ad5f35 immunization is lower compared with ad519 consistent with prior observations that ad5 transduction efficiency in vivo may be several fold higher than ad5f3541 moreover the ad5 fiber binds to cxadr coxsackievirus and adenovirus receptor42 while the ad35 fiber binds to cd4643 suggesting the two viruses may infect distinct cell typesflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access while checkpoint inhibitors have generated practice shifting results in the clinic and defined immunotherapy as an effective strategy for the treatment of several malignancies they have not been universally successful in that context the dearth of neoepitopes in many cancer types including microsatellite stable colorectal and pancreatic second and third leading causes of cancer mortality respectively makes them insensitive to checkpoint blockade7 indeed examination of neoepitopes presented on the surface of five colorectal cancer specimens revealed a total of three neoepitopes3 thus vaccines targeting cancer associated self antigens have re emerged alone and in combination with checkpoint inhibitors as a strategy to prevent and treat metastases from these cold tumors44 checkpoint inhibitors have become first line therapy in the metastatic setting for some cancers46 while chimeric antigen receptor expressing t cells car t cells are being deployed in patients with metastatic and refractory disease47 in contrast few cancer immunotherapies have been developed for early stage cancer patients with œno evidence of disease ned following conventional surgicalradiochemotherapies who are at significant risk of disease recurrence indeed25 of stage ii and of stage iii patients with colorectal cancer recur following surgery and chemotherapy49 while of patients with resectable pancreatic cancer experience recurrence50 vaccines targeting tumor associated antigens such as ad5f35 gucy2c padre may provide safe and effective immunotherapies for the secondary prevention of metastatic disease in patients with ned who are otherwise ineligible to receive checkpoint inhibitors or car t cellsthe present studies suggest that the chimeric adenoviral vector ad5f35 may be preferable to the widely used ad5 vector and warrants further investigation indeed they suggest that ongoing clinical investigations of gucy2c directed immunotherapy in patients with gucy2c expressing cancers including colorectal pancreatic gastric and esophageal could benefit from using the ad5f35 rather than the ad5 vector in that context an upcoming clinical trial will examine the safety immunogenicity and resistance to pre existing immunity of ad5f35 gucy2c padre in patients with gi cancer nct04111172 safe generation of gucy2c targeted immunity in a high proportion of patients will lead to efficacy trials to establish the ability of ad5f35 gucy2c padre to prevent recurrence following standard therapy in patients with gi cancer who represent of all cancer deaths51 and for whom established immunotherapies are ineffectivetwitter adam e snook adamsnookphdacknowledgements the authors thank adrian p gee phd zhuyong mei md deborah lyon and malcolm brenner md phd center for cell and gene therapy baylor college of medicine for assistance in vaccine manufacturingcontributors jcf js bb saw and aes designed the studies jcf js rc el trb jb ec ap jar and jr carried out the studies tz carried out data analysis and statistical analysis in discussion with aes jcf and aes wrote the manuscript and all authors critically reviewed and approved the final version of the manuscriptfunding this work was supported by the national institutes of health nih r01 ca204881 r01 ca206026 and p30 ca56036 the defense congressionally directed medical research program w81xwh17 prcrp ttsa and targeted diagnostic therapeutics to saw aes received a research starter grant in translational medicine and therapeutics from the phrma foundation a degregorio family foundation award and was supported by the defense congressionally directed medical research programs nos w81xwh1710299 w81xw

cancer7512

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " adenovirus serotype ad5 is a commonly used viral vector for transient delivery of transgenes primarily for vaccination against pathogen and tumor antigens however endemic infections with ad5 produce virus specific neutralizing antibodies nabs that limit transgene delivery and constrain target directed immunity following exposure to ad5 based vaccines indeed clinical trials have revealed the limitations that virus specific nabs impose on the efficacy of ad5 based vaccines in that context the emerging focus on immunological approaches targeting cancer self antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectorsmethods here we evaluated the ability of a chimeric adenoviral vector ad5f35 derived from the capsid of ad5 and fiber of the rare adenovirus serotype ad35 to induce immune responses to the tumor associated antigen guanylyl cyclase c gucy2cresults in the absence of pre existing immunity to ad5 gucy2c specific t cell responses and antitumor efficacy induced by ad5f35 were comparable to ad5 in a mouse model of metastatic colorectal cancer furthermore like ad5 ad5f35 vector expressing gucy2c was safe and produced no toxicity in tissues with or without gucy2c expression importantly this chimeric vector resisted neutralization in ad5 immunized mice and by sera collected from patients with colorectal cancer naturally exposed to ad5s these data suggest that ad5f35 based vaccines targeting gucy2c or other tumor or pathogen antigens may produce clinically relevant immune responses in more ‰¥ patients compared with ad5 based vaccines inhibitor introductiontherapies immune checkpoint have revolutionized cancer treatment and cancer drug development by engaging the immune system to target various cancers1 despite this success many tumors are immunologically œcold characterized by a dearth of immunogenic neoepitopes3 and lack of tumor infiltrating lymphocytes4 and remain refractory to checkpoint inhibitors6 one emerging strategy to modify a cold tumor into one responsive to immunotherapy is through combination with cancer vaccines8 the goal of this strategy is to use cancer vaccines to create a pool of tumor reactive t cells with antitumor activity alone andor in combination with checkpoint therapies however this approach is significantly limited by the paucity of effective vaccine platforms to safely deliver tumor specificassociated antigens to elicit beneficial antitumor immunitythe ability of adenovirus serotype ad5 to mediate gene transfer and induce potent immune responses has made it a popular vector for experimental vaccines infectious diseases10 against cancer and indeed there have been more than clinical trials using the ad5 vector with most trials focused on developing cancer treatments10 however on natural infection the host immune system develops neutralizing antibodies nabs to the ad5 capsid limiting viral spread and blocking reinfection because ad5 infections are endemic in many human populations pre existing nabs present in of the worldwide population limit ad5 based vaccine strategies12“ these considerations highlight the need for improved vectors for use in vaccines targeting cancer and pathogen associated antigens that can create therapeutic immune responses in the greatest number of patients importantly while the adenovirus capsid is composed of hexon penton and fiber proteins nabs elicited by natural ad5 infection in humans are directed primarily to the ad5 fiber15 suggesting that strategies to flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access circumvent pre existing immunity to this element may improve ad5 based vaccineshere we sought to overcome pre existing ad5 nabs by replacing the ad5 fiber with that of a rare adenovirus serotype ad35 international seroprevalence “ to improve antitumor immunity in mouse models expressing the gastrointestinal gi cancer antigen guanylyl cyclase c gucy2c preclinical models demonstrated that an ad5 based gucy2c directed vaccine ad5 gucy2c s1 elicited cd8 t cell and antibody responses without autoimmunity17 further ad5 gucy2c s1 vaccination of mice induced long term t cell mediated protection against metastatic colorectal cancer in lung and liver19 moreover those results were recapitulated in a recent first in human phase i clinical trial nct01972737 demonstrating that a humanized version of the vaccine ad5 gucy2c padre safely induced gucy2c specific cd8 t cell responses in patients with colorectal cancer following conventional therapies21 however patients possessing high pre existing titers of nabs against ad5 failed to generate gucy2c specific immunity following ad5 gucy2c padre vaccination21 to overcome ad5 nabs we generated a chimeric ad5 vector possessing the fiber of ad35 ad5f35 with equivalent safety and antitumor activity to ad5 and resistance to ad5 nabs in mice and humans this chimeric vaccine can be translated to patients with gi cancer to safely induce gucy2c specific immunity not only in those patients with low ad5 immunity but also in those with high pre existing ad5 nabsmaterials and methodsadenovirus vectorsadenovirus containing mouse extracellular domain gucy2c1429 with the influenza ha107119 cd4 t cell epitope known as site s1 was described previously ad5 gucy2c s120 here gucy2c s1 was cloned into pshuttle and subcloned into the e1 region of previously generated replication deficient chimeric adenovirus ad5f35 in which the ad5 fiber was replaced by the ad35 fiber22 to generate ad5f35 gucy2c s1 all adenovirus vaccines used in this study were produced in hek293 cells and purified by cesium chloride ultracentrifugation under good laboratory practices by the baylor college of medicine in the cell and gene therapy vector development lab and certified to be negative for replication competent adenovirus mycoplasma and host cell dna contamination in vitro gucy2c expression experiments dose“response and time“course were carried out in a549 american type culture collection atcc cells virus was added to the cultures at the indicated doses and culture supernatants were collected at the indicated time points relative gucy2c levels were quantified in supernatants by western blot using μgml ms7 mouse anti gucy2c monoclonal antibody23“ and μgml horseradish peroxidase conjugated goat antimouse secondary antibody jackson immunomice and immunizationseight week old male and female balbcj mice were purchased from the jackson laboratory for experiments animal protocols were approved by the thomas jefferson university institutional animal care and use committee protocol for immunizations mice received or vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or ad5f35 gfp control administered as two μl intramuscular injections one in each hind limb using a ml insulin syringequantifying tcell responses by elispotelispot assays were performed using a mouse interferonÎ ifnÎ single color elispot kit cellular technology according to the manufacturer™s protocol26 briefly well plates were coated with ifnÎ capture antibody overnight at °c the next day plates were washed with phosphate buffered saline pbs and splenocytes from immunized mice were plated at cellswell with no peptide or μgml gucy2c254262 peptide in dimethyl sulfoxide dmso in ctl test medium cellular technology for hours at °c for t cell avidity studies splenocytes were plated at “ cellswell with decreasing concentrations of gucy2c254262 peptide μgml to pgml normalized to cellswell26 after incubation cells were removed and development reagents were added to detect ifnÎproducing spot forming cells the number of spot forming cells per well was determined using the smartcount and autogate functions of an immunospot s6 universal analyzer cellular technology gucy2c specific responses were calculated by subtracting mean spot counts of dmso wells from peptide stimulated wells26 tumor studiesgucy2c expressing mouse balbc ct26 colorectal cancer cells were used for in vivo tumor studies17 luciferase expressing cells were generated by transduction with lentiviral supernatants produced by 293ft cells invitrogen with plenti4 v5 gw luciferase28 for tumor experiments balbcj mice were immunized with vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or pbs control days before delivering × ct26 cells into tail veins tumor burden was quantified weekly by subcutaneous injection of mg of d luciferin potassium salt gold biotechnologies in pbs followed by an min incubation and imaging with a s exposure using a caliper ivis lumina xr imaging station perkinelmer total radiance photonssecond was measured using living image in vivo imaging software perkinelmerantibody neutralization assayserum samples were obtained previously from patients before ad5 gucy2c padre nct01972737 approved by the thomas jefferson university institutional review board21 neutralizing antibody titers against ad5 and ad5f35 vectors were quantified as immunization with flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0cdescribed21 briefly dilutions of heat inactivated serum samples were added to well tissue culture plates containing a549 cells atcc and infected with vp of gfp expressing ad5 or ad5f35 virus ad5 cmv egfp or ad5f35 cmv egfp respectively baylor vector development lab following a hour incubation at °c egfp fluorescence nm excitation nm emission was quantified using a polarstar optimate plate reader bmg labtech sample fluorescence was normalized to control wells containing cells and virus neutralization or wells containing cells alone neutralization titers were quantified using non linear regression as the serum dilution producing neutralization prism v8 graphpad softwaread5 neutralizing immunity studiesto induce anti ad5 immunity mice were exposed intranasally to ad5 gfp once or twice at a week interval thirty days after the last exposure ad5 nabs were quantified in sera as described above and mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1biodistribution and toxicology studybalbcj mice were immunized intramuscularly with a single dose of vp of ad5f35 gucy2c s1 three doses of vp of ad5f35 gucy2c s1 at day intervals or pbs control animals were monitored for adverse events once daily with additional evaluations on the day of dosing min hour and hours after dosing on days and designated animals were sacrificed and brain salivary glands stomach small intestine colon heart lungs kidneys liver and injection site were harvested and weighed for histopathological analysis by a blinded pathologist pathology evaluation was performed by idexx bioanalytics and detection of viral dna by quantitative pcr qpcr using the previously described assay for the gucy2c transgene19 also spleens were collected for histopathological analysis and detection of viral dna as described above as well as quantification of gucy2c specific t cell responses by ifnÎ elispot as described abovestatistical analysisstatistical analyses were conducted using graphpad prism software v8 statistical significance was considered as follows nsp p p p and p cohort sizes were powered based on prior studies with β02 and α005 for multiple comparisons of survival outcomes significance thresholds were corrected using the bonferroni method to identify vaccine induced t cell responders and non responders a previously described21 modified distribution free resampling approach was employed and positive t cell responses were defined as × compared with dmso and specific spots106 cells to determine the impact of gender and number of vaccinations on responses log transformed vaccine response magnitude was compared in mice of different genders cohorts and treatment regimens for up to three way interactions with stepwise backward variable selection by akaike information criterion using r29 package mass30open accessresultsad5gucy2cs1 and ad5f35gucy2cs1 vectorswhile ad5 seroprevalence worldwide exceeds in some regions ad35 is and associated with lower titers figure 1a12 thus we constructed a chimeric adenovirus ad5f35 composed of ad5 in which the fiber was replaced by the ad35 fiber and evaluated its ability to induce gucy2c specific immunity and resist ad5 specific immunity in humans and mice ad5 gucy2c s1 is a replication deficient human ad5 expressing the mouse gucy2c extracellular domain fused to the i ed restricted cd4 epitope known as site at its c terminus20 to generate ad5f35 gucy2c s1 the ad5 fiber l5 was replaced with the ad35 fiber figure 1b replication deficient ad5 gucy2c s1 and ad5f35 gucy2c s1 generated in hek293 cells produced dose dependent figure 1c and time dependent figure 1d expression of gucy2c s1 protein in a549 human alveolar basal epithelial cells in vitroad5f35gucy2cs1 induces gucy2cspecific antitumor immunityfollowing in vitro validation of gucy2c expression by ad5f35 gucy2c s1 we confirmed its ability to induce gucy2c specific immune responses after vaccination in vivo balbc mice immunized intramuscularly with vp of ad5f35 gucy2c s1 produced lower gucy2c specific cd8 t cell responses figure 2a and no gucy2c specific antibody responses figure 2b compared with ad5 gucy2c s1 importantly ad5 and ad5f35 vaccines produced gucy2c specific cd8 t cells of comparable avidity figure 2c a critical determinant of the antitumor efficacy of gucy2c targeted vaccines26 in contrast gucy2c specific antibody responses have no detectable antitumor activity20 similarly ad5 and ad5f35 vaccines produced comparable s1 specific cd4 t cell responses figure 2dluciferase this model previous studies revealed that ad5 gucy2c vaccines induced protective antitumor cd8 t cell responses in murine models of metastatic colorectal cancer17“ thus balbc mice were immunized with ad5 or ad5f35 expressing gucy2c s1 and challenged days later with ct26 colorectal cancer cells expressing gucy2c and firefly specifically emulates secondary prevention of metastatic disease the clinical setting for which the gucy2c vaccine is being developed21 as previously demonstrated ad5 vaccination nearly eliminated metastatic tumor burden figure 3ab delayed disease progression figure 3c and improved survival figure 3d similarly ad5f35 also reduced tumor burden figure 3ab disease progression figure 3c and prolonged survival figure 3d importantly the efficacy of ad5 based and ad5f35 based gucy2c vaccines in flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure construction of ad5f35 gucy2c s1 and antigen expression a reported international seroprevalence of ad5 and ad3512 b the l5 gene encoding the fiber protein from ad5 was replaced with the l5 gene from ad35 producing the chimeric adenoviral vector ad5f35 recombinant ad5f35 gucy2c s1 was produced by inserting mouse gucy2c s1 into the e1 region of e1e3 deleted ad5f35 c and d the human alveolar basal epithelial cell line a549 was transduced in duplicate with ad5f35 gucy2c s1 at a multiplicity of infection moi from to for hours c or at an moi of for and hours d supernatants from infected cells were analyzed for gucy2c s1 protein expression by immunoblot protein expression was quantified by densitometry and plotted relative to uninfected cells error bars indicate mean±sem ad5 adenovirus serotype reducing tumor burden opposing disease progression and promoting survival was identical figure 3a“dad5f35 resists ad5directed immunity in mice and humansnabs against ad5 correlated with poor gucy2c specific immune responses in patients receiving ad5 gucy2c padre vaccination and prior exposure of mice to ad5 similarly blunted vaccine induced immunity21 ad5f35 based vaccine resistance to pre existing ad5 immunity was quantified in a model of respiratory pre exposure to ad5 the natural route of infection in patients33 followed by vaccination and quantification of gucy2c specific t cell responses control mice not pre exposed to ad5 naive and those that were pre exposed once × or twice × to intranasal ad5 were vaccinated after weeks with intramuscular ad5 or ad5f35 expressing gucy2c s1 and immune responses were quantified weeks later immunogenicity of ad5 gucy2c s1 and ad5f35 gucy2c s1 a“d balbc mice n4“ micegroup were figure immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and serum and splenocytes were collected days later gucy2c specific cd8 t cell responses were quantified by interferon gamma ifnÎ elispot a and antibodies were quantified by elisa b c gucy2c specific t cell avidity measurements were analyzed by elispot using non linear regression logagonist versus normalized response with comparisons made using the extra sum of squares f test avidity plots depict the regression line solid with cis dashed d s1 specific cd4 t cell responses were measured by ifnÎ elispot t cell responses in a and d were analyzed by one way analysis of variance values in a b and d indicate individual animals and bars in a and d indicate means tcr t cell receptor ad5 adenovirus serotype flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure antitumor efficacy of ad5 gucy2c s1 and ad5f35 gucy2c s1 a“d balbc mice n10 micegroup were immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and challenged days later with a mouse colorectal cancer cell line ct26 expressing gucy2c and luciferase on days and following challenge mice were injected with d luciferin and imaged a to quantify tumor burden day b mice were weighed twice weekly c and monitored for survival d tumor burden b was analyzed by one way analysis of variance and survival comparisons d were analyzed by the mantel cox log rank test in b and d asterisks indicate comparisons of gucy2c vaccines to the control and brackets ] indicate comparisons between ad5 and ad5f35 vaccines ns not significant ad5 adenovirus serotype figure 4a as expected one ad5 pre exposure induced moderate ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses while two pre exposures induced high ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses following ad5 vaccination figure 4b in contrast gucy2c specific t cell responses were reduced only × pre exposure and × pre exposure following ad5f35 vaccination figure 4b importantly ad5f35 produced t cell responses in a substantially greater fraction of the population cohort responses compared with ad5 cohort responses following serial pre exposures to ad5 figure 4cthese observations in mice were recapitulated using sera from patients with colorectal cancer in the ad5 gucy2c padre phase i trial nct0197273721 here nab titers against ad5 and ad5f35 were quantified using an established ad5ad5f35 reporter virus inhibition bioassay in serum samples collected prior to vaccination with ad5 gucy2c padre21 in these patients ad5f35 specific nab titers were substantially lower than ad5 specific titers figure 4d most importantly of patients possessed low ad5 nabs titers figure 4de which closely correlated with a gucy2c specific response rate21 in striking contrast had low ad5f35 nab titers suggesting that the vast majority of patients immunized with ad5f35 based vaccines could produce gucy2c specific responses figure 4e collectively these observations suggest that pre existing viral immunity induced by repeated environmental exposures which neutralizes ad5 delivery platforms may be overcome by the chimeric ad5f35 vector to enhance fractional population vaccine responsessafety biodistribution and toxicity of ad5f35gucy2cs1food and drug administration ind investigational new drug enabling studies quantified the toxicity biodistribution and immunogenicity of ad5f35 gucy2c s1 in balbc mice employing three schemes to examine acute and chronic effects figure 5a cohorts balanced for sex received ad5f35 gucy2c s1 either as a single intramuscular injection or as three intramuscular injections spaced weeks apart monitored daily and sacrificed on day or for analysis as indicated figure 5a there were no signs of acute or chronic toxicity in the in life phase by observation weight changes or survival figure 5b“d similarly there were no clinically significant differences in organ weights online supplementary figure s2 or histopathology not shown at necropsy small statistical differences in organ weights were considered clinically insignificant and were unrelated to vaccine exposure dose time online supplementary figure s2 biodistribution quantified by qpcr detected ad5f35 gucy2c s1 at the injection site and in the spleen but not appreciably in other organs after acute and chronic exposures online supplementary figure s3 moreover robust cd8 t cell responses were quantified at day that persisted through day in of mice after a single administration figure 5e“g as expected cd8 t cell responses were greater and persisted in more mice at days after three vaccinations figure 5e“gdiscussionthrough decades of gene therapy trials ad5 has remained a popular vector while high ad5 seroprevalence remains a barrier to universal vaccination33 natural respiratory infection can generate long lived antibodies that neutralize ad5 based vaccines eliminating transgene delivery and potential therapeutic benefit in that context ad5 seroprevalence is across multiple countries12 highlighting an unmet need for alternative vectors here we demonstrate that the chimeric ad5f35 resists pre existing ad5 immunity and induces transgene specific antitumor immunity indeed ad5f35 is less susceptible to neutralization associated with ad5 exposure in mice and humans and generates a substantially higher proportion of vaccine responders in mice pre exposed to ad5 these observations support the suggestion that ad5f35 flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure ad5f35 resists neutralization associated with pre existing anti ad5 immunity in mice and humans a“c to generate pre existing immunity to ad5 balbc mice n10 micegroup were exposed intranasally once or twice to vp of ad5 gfp at week intervals four weeks after the final ad5 gfp exposure ad5 exposed and naive mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 b two weeks after immunization gucy2c specific cd8 t cell responses in each group were quantified by interferon gamma ifnÎ elispot and calculated as the of mean responses in naive mice values indicate individual animals and bars indicate means ad5 and ad5f35 were compared by two way analysis of variance c the fraction of animals producing a detectable gucy2c specific cd8 t cell response filled regions in naive × and × ad5 exposed mice was determined from b d and e sera from patients with colorectal cancer collected prior to ad5gucy2c padre vaccination were tested for the ability to neutralize ad5 and ad5f35 vectors and titers were quantified d analyzed by paired t test the dotted line indicates a titer of the threshold for high neutralizing antibody nab titers21 e while subjects had high nab titers against ad5 only had high titers to ad5f35 vector filled regions binomial test ad5 adenovirus serotype will produce a higher proportion of vaccine responders in patient populationsthe extent to which nabs to the ad5 fiber limit reinfection is controversial in some studies replacing the ad5 fiber with that of another serotype circumvents pre existing ad5 immunity34 in contrast other studies suggest that these chimeric adenoviruses do not evade pre existing ad5 nabs suggesting the hexon as the major target of antibody neutralization35 in contrast to those previous studies which generated pre existing ad5 immunity by intramuscular35 or intravenous administration36 here ad5 immunity was induced by intranasal exposure in mice recapitulating natural infection33 moreover natural human respiratory pre existing ad5 nabs in patients with colorectal cancer uniformly produced by repeated respiratory infections33 similarly were overcome by the ad5f35 vector importantly the quality of antibody responses following adenovirus infection is dependent on the route of exposure indeed respiratory infections elicit fiber specific nabs while intramuscular exposure induce capsid specific nabs15 these qualitative differences in nab responses reflecting varying routes of immunization may contribute to observational discrepancies between laboratories the present studies using relevant animal models confirmed and validated with patient samples support the suggestion that ad5f35 based vaccines should produce clinically relevant immune responses in a substantial proportion of patientsflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure safety and immunogenicity of multiple ad5f35 gucy2c s1 administrations a“g balbc mice n10 micegroup were immunized intramuscularly with one or three administrations of vp ad5f35 gucy2c s1 or control at week intervals following immunization body weights b female and c male were recorded weekly and mice were monitored for survival d at days and following first immunization mice were euthanized to quantify organ pathology by weight online supplementary figure s2 biodistribution by quantitative pcr online supplementary figure s3 and gucy2c specific cd8 t cell responses by interferon gamma ifnÎ elispot e“g g pie charts indicate proportion of responding animals ad5 adenovirus serotype recognizing the pervasive limitations imposed by endemic ad5 immunity in global populations12 there is an emerging interest in alternative serotypes and chimeric constructs as a tractable strategy in vaccine development ad26 ad35 and ad48 vectors have been advanced into phase i clinical trials37 in that regard a comparison of ad5 ad26 ad35 and ad48 immunity among healthy patients revealed that endemic ad35 seropositivity was lowest across global populations12 reinforcing chimeric strategies employed herein similarly the first hexon chimeric adenovirus comprising ad5 and ad48 components was safe and immunogenic in patients39 interestingly ad5 ad35 chimeric vectors more efficiently transduce a variety of human cell types in vitro compared with either parental vector40 these observations underscore the future potential of intelligently designed chimeric adenoviruses strategically constructed to deliver transgenes for replacement therapy or vaccination and targeted precisely to the cellular or disease context40while antitumor efficacy was equivalent cd8 t cell responses were lower and antibody responses were absent for ad5f35 gucy2c s1 compared with ad5 gucy2c s1 however the antitumor efficacy of gucy2c directed immunotherapy is driven primarily by t cell avidity rather than effector t cell quantity26 in that context the functional avidity of gucy2c specific cd8 t cells following ad5 and ad5f35 immunizations were equivalent consistent with their comparable antitumor efficacy quantitative differences in transgene specific immunity between vectors may reflect a variety of factors thus the quantity and persistence of gucy2c s1 transgene following ad5f35 immunization is lower compared with ad519 consistent with prior observations that ad5 transduction efficiency in vivo may be several fold higher than ad5f3541 moreover the ad5 fiber binds to cxadr coxsackievirus and adenovirus receptor42 while the ad35 fiber binds to cd4643 suggesting the two viruses may infect distinct cell typesflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access while checkpoint inhibitors have generated practice shifting results in the clinic and defined immunotherapy as an effective strategy for the treatment of several malignancies they have not been universally successful in that context the dearth of neoepitopes in many cancer types including microsatellite stable colorectal and pancreatic second and third leading causes of cancer mortality respectively makes them insensitive to checkpoint blockade7 indeed examination of neoepitopes presented on the surface of five colorectal cancer specimens revealed a total of three neoepitopes3 thus vaccines targeting cancer associated self antigens have re emerged alone and in combination with checkpoint inhibitors as a strategy to prevent and treat metastases from these cold tumors44 checkpoint inhibitors have become first line therapy in the metastatic setting for some cancers46 while chimeric antigen receptor expressing t cells car t cells are being deployed in patients with metastatic and refractory disease47 in contrast few cancer immunotherapies have been developed for early stage cancer patients with œno evidence of disease ned following conventional surgicalradiochemotherapies who are at significant risk of disease recurrence indeed25 of stage ii and of stage iii patients with colorectal cancer recur following surgery and chemotherapy49 while of patients with resectable pancreatic cancer experience recurrence50 vaccines targeting tumor associated antigens such as ad5f35 gucy2c padre may provide safe and effective immunotherapies for the secondary prevention of metastatic disease in patients with ned who are otherwise ineligible to receive checkpoint inhibitors or car t cellsthe present studies suggest that the chimeric adenoviral vector ad5f35 may be preferable to the widely used ad5 vector and warrants further investigation indeed they suggest that ongoing clinical investigations of gucy2c directed immunotherapy in patients with gucy2c expressing cancers including colorectal pancreatic gastric and esophageal could benefit from using the ad5f35 rather than the ad5 vector in that context an upcoming clinical trial will examine the safety immunogenicity and resistance to pre existing immunity of ad5f35 gucy2c padre in patients with gi cancer nct04111172 safe generation of gucy2c targeted immunity in a high proportion of patients will lead to efficacy trials to establish the ability of ad5f35 gucy2c padre to prevent recurrence following standard therapy in patients with gi cancer who represent of all cancer deaths51 and for whom established immunotherapies are ineffectivetwitter adam e snook adamsnookphdacknowledgements the authors thank adrian p gee phd zhuyong mei md deborah lyon and malcolm brenner md phd center for cell and gene therapy baylor college of medicine for assistance in vaccine manufacturingcontributors jcf js bb saw and aes designed the studies jcf js rc el trb jb ec ap jar and jr carried out the studies tz carried out data analysis and statistical analysis in discussion with aes jcf and aes wrote the manuscript and all authors critically reviewed and approved the final version of the manuscriptfunding this work was supported by the national institutes of health nih r01 ca204881 r01 ca206026 and p30 ca56036 the defense congressionally directed medical research program w81xwh17 prcrp ttsa and targeted diagnostic therapeutics to saw aes received a research starter grant in translational medicine and therapeutics from the phrma foundation a degregorio family foundation award and was supported by the defense congressionally directed medical research programs nos w81xwh1710299 w81xw Answer:

Colon_Cancer

clinical manifestations of sarscov2 infection include more frequently fever and cough failure can occur in persons with additional comorbidities liver dysfunction is one of the most striking affections among patients suggesting that sarscov2 may represent a new king of liver aggressor however the molecular process underlying this phenomenon is 0cstill unclear in this work we overview the most recent findings between the molecular biology of the virus pathogenic mechanisms and its relationship to liver disease observed in patients abbreviations aado2 ace2 aih alt ast covid19 ggt gi gtex alveolararterial oxygen gradient angiotensinconverting enzyme autoimmune hepatitis alanine transaminase aspartate aminotransferase coronavirus infectious disease gamma glutamyl transpeptidase gastrointestinal genotypetissue expression metabolicassociated fatty liver disease nonstructural proteins open reading frame preproofcomplex disease in many severely ill patients in other infected subjects an infection is keywords sarscov2 liver liver impairment covid19 ace2 mafld nsp orf introduction sarscov2 is the etiological agent of the disease known as covid19 which causes a disease characterized by pneumonia cough fever occasional diarrhea headache cardiac injury and in some patients™ liver alterations covid19 has been found to be an extremely reported to be so severe that it can lead to a disproportionate and mortal reaction of the immune system known as a cytokine storm all of these factors make covid19 highly unpredictable it is what specialists call a multisystem disease 0caround the world cases of liver dysfunction denoted by elevated hepatic enzymes in serum such as ast aspartate aminotransferase and alt alanine transaminase have been documented among patients infected with sarscov2 there is still no certainty whether the covid19related liver damagedysfunction is due mainly to the viral replication per se drugs toxicity or other coexisting comorbidities whether sexrelated difference could help to explain why infected men are more healthy or harmful relationship between the liver and its viral aggressor in this paper we describe a brief overview of the implications for researchers in the field of it is important to understand how liver function can be altered by direct infection with this predisposed to develop covid19associated liver dysfunction than infected women to analyze if there is any genetic predisposition related to impaired liver function during the œrespiratory virus which mechanisms of viral pathogenesis are involved to evaluate disease and of course the crosstalk between viral and cellular proteins that mediate this preproofliver disease of the most recent findings between the molecular biology of the virus this emerging viral illness is typically characterized by fever dry cough myalgia headache sore throat diarrhea and may be aggravated with shortness of breath and respiratory failure the angiotensinconverting enzyme ace2 the functional receptor of the spike glycoprotein of sarscov2 is widely distributed in the anism historically hamming and colleagues reported ace2 expression in the surface of lung alveolar epithelial cells enterocytes of the small intestine arterial and venous endothelial pathogenic mechanisms and its relationship to liver disease observed in patients clinical characteristics and liver injury in patients with covid19 0ccells and arterial smooth muscle cells posterior transcriptomic and proteomic analyses confirmed their findings and added high ace2 expression in adipose tissue bone marrow duodenum endometrium heart kidney small intestine smooth muscle testis and thyroid ace2 is also expressed in liver but in lesser extent one of the most worrisome severe cases of covid19 regarding the gastrointestinal gi tract and liver over covid19associated liver injury is defined as any liver damage that occurs during disease progression andor covid19 treatment in patients with or without a history of previous complications is the unusual formation of blood clots in many patients with covid19 even those who were receiving anticoagulants researchers at mount sinai in new york published studies suggesting that clots in the lungs play an important role in the most of covid19 patients develop gi symptoms such as anorexia diarrhea nausea and vomiting and a significant proportion present with altered liver function tests preproofdecreased albumin levels are associated with severe infection and poor prognosis still ast elevation is the most common abnormality in patients presenting with covid19 observed more frequently in men and is mainly documented in more severe cases liver disease in general the incidence of increased liver biochemical markers in hospitalized patients with covid19 mainly ast and alt and slightly elevated bilirubin varies between to of cases the increase in liver enzymes is there are no reports of acute or subacute liver failure in patients with covid19 the largest cohort study that included cases of covid19 from china showed that had preexisting chronic liver disease lei and colleagues reported that impaired liver function was related to mortality in covid19 patients elevated ast was more frequent and significant than the increase of alt in severe 0chospitalized patients moreover elevated ast was shown to be associated with highest mortality risk in the study reported by yijin wang they found that of covid patients had elevated ast activity the median levels of alt were ul vs ul respectively ast were ul vs ul respectively in abnormal and normal aminotransferase groups liver enzymes abnormality were associated with disease severity protein levels in addition they found by ultrastructural examination of coronavirus ps in hepatocytes in covid19 cases sarscov2 infected hepatocytes displayed as well as a series of laboratory tests including higher alveolararterial oxygen gradient aado2 higher gamma glutamyl transpeptidase ggt lower albumin and lower total conspicuous mitochondrial swelling endoplasmic reticulum dilatation and glycogen granule decreased histological findings showed apoptosis and binuclear hepatocytes preproofshowed a disease course similar to that reported in non immunosuppressed population coronavirus the interaction of its proteins with cellular proteins and consequently the immunosuppressive therapy for autoimmune hepatitis aih developing covid19 taken together both ultrastructural and histological evidence indicated a typical lesion of viral infection all these findings by different reports demonstrates that sarscov2 infection in liver is a crucial cause of hepatic impairment in covid19 patients however alteration of cellular metabolism that give rise to systematic alterations and metabolic report from alessio gerussi demonstrated that patients under today the cellular and molecular mechanisms that are altered by infection with this changes are still unknown 0cmolecular biology of sarscov2 coronaviruses are enveloped viruses that contain a positively polarized unsegmented rna genome belonging to the coronaviridae family and the order of nidovirales they are distributed in humans and other mammals the size of the sarscov2 virions is approximately to nm in diameter [“] sarscov2 has a genome that consists polymerase rdrp which is nsp12 and is responsible of the replication and transcription of the virus which are encoded by the various genetic loci on the genome at the center of the virion lies a nucleocapsid composed of the genomic rna and the nucleocapsid protein the virus glycoprotein s consists of two subunits s1 which is at the amino terminus and that encodes for structural proteins and a larger region that encodes two open reading frames orf 1a and 1b which together encode for the nonstructural virus proteins from nucleocapsid protein which is within the phospholipid bilayer and nonstructural proteins nsp1 to nsp16 the virions have a structural sspike protein outer spiky glycoprotein mmembrane protein a type iii transmembrane glycoprotein nof nucleotides encoding amino acids and it is composed of a region preproofvirus as well as protein m which is a type iii transmembrane glycoprotein and participates in the cellular membrane rearrangements for the replication and transcription complexes among the encoded proteins is an rnadependent rna provides the receptor binding site and s2 which is at the carboxyl terminus responsible for membrane fusion the envelope protein e has a role in the assembly and release of the nonstructural proteins have several functions during de viral cycle for example nsp 0cthe virus enters the cell by endocytosis through the interaction between envelope glycoprotein s with the cell receptor ace2 and with the participation of the type ii transmembrane serine protease tmprss2 once it enters the cell the n protein with viral genome are released within the cytoplasm then cellular proteases degrade the capsid and the virus genome is left free next the polyprotein containing the viral proteins that are how does the virus select which cells to infect viruses can infect only certain species of hosts and only permissive cells within that host permissive cells make all the necessary proteins and viral factors to allow virus to replicate once a virus gets inside a cell it hijacks the cellular processes to produce virally encoded proteins that will replicate the virus™s genetic material viral replication may cause exocytosis will translate into viral proteins this entire process will occur in the cell cytoplasm the processed to form the replication complex is translated and then the complementary strand of negative sense pregenomic rna is synthesized to be used as a template to replicate the structural proteins that will be synthesized in the endoplasmic reticulum membrane to assembly the viral p and finish the cycle through the release of the viruses by positive sense viral genome furthermore the replication and transcription complex will lead to a series of smaller positive sense subgenomic rnas these are the ones that preproofboth sarscov and the new sarscov2 are very similar in structure and pathogenicity but the major structural protein s protein is slightly different between them compared to other beta coronaviruses the presence of a furinlike cleavage site in sarscov2 enables the s protein priming and facilitates an increase on the efficiency of the spread of sarscov2 as is reported wide world 0cbiochemical changes producing cell damage called cytopathic effects like other coronaviruses sarscov2 requires cellular receptors to initiate its internalization to the cells that carry these factors li sarscov2 uses the angiotensinconverting enzyme ace2 as a host cell receptor sarscov2 spike s protein binds ace2 with significantly high affinity in addition the main host protease that suggested to promote the pathogenesis of this coronavirus program httpsportalgdccancergov they compared ace2 expression levels across human tissues between males and females and between younger and older persons in these individual tissues furthermore other reports have analyzed the correlations between ace2 in order to provide insights into the mechanism of sarscov2 infection li analyzed the expression of ace2 in various normal human tissues using the datasets from the genotypetissue expression gtex project and the cancer genome atlas tcga transmembrane serine protease other host proteases such as furin have also been mediates sprotein activation on primary target cells and initial viral entry is the type ii preproofreported by li ace2 expression levels showed no significant difference between have no significant association with sex age or race is the liver a direct target for sarscov2 males and females between younger and older persons or between asian and nonasian races this finding suggests that the infection risk of sarscov2 and sarscov may expression levels and immune signature enrichment levels in individual tissues as as we expected because the systemic manifestations of covid19 it has been reported that sarscov2 has an anotropism beyond the respiratory tract including the kidneys 0cliver heart and brain and possibly that anotropism influences the course of covid19 disease and aggravates preexisting conditions the ace2 protein is found at high levels in the gi tract as the colon biliary system and liver on the other hand it is well documented a sarscov2 rna shedding in the gi tract supporting its tropism for architecture express ace tmprss1 receptors the presence of these two host factors in the liver suggests that a direct viral cytopathic effect occurs also in sars infection the presence of viral rna in liver tissue was documented but not as extensively as the new coronavirus data published by gordon suggest that mitochondrial proteins interact directly with the virus which helps to understand the potential mechanism by which elevated ast the gi tract and liver cells and these may be sites of active viral replication and either direct or indirect tissue injury indeed a large part of the cells distributed in the liver preproofeffect the exacerbated inflammatory response in covid19 may play a central role in profiles are detected in these patients furthermore in addition to this intracellular more recently identified the clinical and laboratory characteristics of covid19 patients with abnormal liver transaminases and they reported that sarscov2 is able to which high levels of il6 have been reported which are involved in both infect liver cells and cause liver impairment by direct cytopathic effect inflammatory and repair responses in liver disease mechanisms of liver pathogenicity 0cif sarscov2 replication has direct adverse effects on liver function it is still unknown findings in liver biopsy of patients killed by covid19 showed moderate micro vesicular steatosis and mild portal and necroinflammatory activity this seems to indicate that a direct injury occurred while the infection that could have been directly caused by sarscov2 another possibility is that a druginduced liver injury occurred to date there are the following possible mechanisms figure infection the massive release of cytokines by the immune system in response to the viral infection can result in a cytokine storm and symptoms of sepsis that are the cause of death in of fatal covid19 cases in these cases uncontrolled inflammation induces multian damage leading including liver failure biomarkers of inflammation such as creactive protein pcr serum ferritin ldh ddimer il6 il2 are have been found to be significantly elevated in immune damage from exacerbated inflammation in response to sarscov2 preproofpathogenesis of sars cov “related liver disease more studies should be liver is a potential target for direct infection with this virus to understand the performed for evidence of viral mechanisms of replication in different cell anelles as cytoplasm endoplasmic reticulum golgi apparatus and lipidrafts cov2 enters cells through the ace2 molecule which is abundantly expressed in the liver and in particular in bile epithelial cells based on this expression the direct cytopathic effect due to active viral replication in various liver cells sarscritically ill patients with covid19 into hepatocytes and liver histology characterization it is also important to know in cells their capacity to efficiently produce both infectious and defective non 0cinfective whole virions there are not yet enough data to know the viral dynamics in the different tissues and the associated pathogenesis anoxia respiratory failure is one of the main characteristics of covid19 anoxic hypoxic hepatitis is common in patients with severe symptoms reactivation of preexisting liver disease patients with preexisting chronic liver medications such as tocilizumab and baricitinib used to combat the adverse immune cholestatic liver disease various studies such as lopinavir ritonavir remdesivir chloroquine tocilizumab uminefovir traditional chinese medicine so it is important to consider that they could be potentially hepatotoxic in some patients druginduced liver damage dili initial clinical guidelines recommended antiviral agents for covid19 so a variety of drugs have been administered in disease may be more susceptible to liver damage from sarscov2 biological preproof genetic factors genetics may well be one of the determining factors in some reaction may also cause hbv reactivation and induce eventual impairment of liver function in those patients with hbv on the other hand it is still unknown whether sarscov2 infection exacerbates cholestasis in people with underlying patients who become seriously ill with covid19 but until now we cannot be sure it is possible for example that the genetic variation that makes an individual more susceptible to high blood pressure or diabetes also makes him more vulnerable to the virus it will be important to find out what role genetic factors predisposing to liver steatosis have and their sensitivity to severe symptoms of covid19 ji and 0ccolleagues showed that subjects with metabolicassociated fatty liver disease mafld have a higher risk of covid19 severity disease and abnormal liver blood tests than patients without mafld in contrast louise biquard demonstrated that mafld is not associated with changes in liver expression blood test abnormalities reported by ji and colleagues is thus likely not explained by concluding remarks the scenario is not yet complete which does not allow us to establish or understand the natural history of the disease and the participation or commitment of the liver in this disease certainly the application of new technological platforms such as singlecell increased hepatic sarscov2 uptake still several contradictory reports will help of genes implicated in sarscov2 infection the observed persistence of liver to find the real role of genetic factors in the evolution of this disease preprooftranscriptomic assays will allow us to quickly know the commitment of each cell type in affected ans and the meaning of viral dynamics in the various affected systems including the liver however as we have already learned from the old hepatotropic viruses history still is ongoing and we have much to learn and understand about the virologic characteristics of this emerging rna virus that allow us to develop specific antivirals such as the case of hcv and the vaccine to decrease the impact of this œacute infection declarations of interest none ethical approval not required 0c references chen n zhou m dong x qu j gong f han y epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan china httpsdoi101002path1570 wang d eraslan b wieland t hallström b hopf t zolg dp a deep proteome and transcriptome abundance atlas of healthy human tissues mol syst hamming i timens w bulthuis mlc lely at navis gj van goor h tissue distribution of ace2 protein the 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0cimmunosuppressed patients hepatol commun 2020na httpsdoi101002hep41557 richman d whitley r hayden f clinical virology 4th ed asm press ksiazek tg erdman d goldsmith cs zaki sr peret t emery s a novel “ httpsdoi101056nejmoa030781 kuiken t fouchier ram schutten m rimmelzwaan gf van amerongen g van respiratory syndrome lancet “ httpsdoi101016s0140 drosten c günther s preiser w van der werf s brodt hr becker s identification of a novel coronavirus in patients with severe acute respiratory riel d newly discovered coronavirus as the primary cause of severe acute coronavirus associated with severe acute respiratory syndrome n engl j med preproofsyndrome n engl j med “ httpsdoi101056nejmoa030747 outbreak associated with a new coronavirus of probable bat origin nature “ httpsdoi101038s4158602020127 mortola e roy p efficient assembly and release of sars coronaviruslike ps by a heterologous expression system febs lett “ httpsdoi101016jfebslet200409009 fehr a perlman s coronaviruses methods and protocols methods in molecular biology chapter coronaviruses an overview of their replication and zhou p yang xl lou wang xgg hu b zhang l zhang w a pneumonia 0cpathogenesis springer berlin heidelberg belouzard s millet jk licitra bn whittaker gr mechanisms of coronavirus cell entry mediated by the viral spike protein viruses “ httpsdoi103390v4061011 vennema h godeke gj rossen jw voorhout wf horzinek mc opstelten dj et snijder ej decroly e ziebuhr j the nonstructural proteins directing coronavirus neuman bw buchmeier mj supramolecular architecture of the coronavirus p adv virus res “ httpsdoi101016bsaivir201608005 van der hoeven b oudshoorn d koster aj snijder ej kikkert m barcena m neuman bw kiss g kunding ah bhella d baksh mf connelly s a structural analysis of m protein in coronavirus assembly and morphology j struct biol “ httpsdoi101016jjsb201011021 expression of viral envelope protein genes embo j “ al nucleocapsidindependent assembly of coronaviruslike ps by co preproofbiogenesis and architecture of arterivirus replication anelles virus res “ httpsdoi101016jvirusres201604001 rna synthesis and processing vol 1st ed elsevier inc httpsdoi101016bsaivir201608008 rabi f al zoubi m kasasbeh g salameh d alnasser a sarscov2 and coronavirus disease what we know so far pathogens masters p the molecular biology of coronaviruses adv virus res “ 0c shang j ye g shi k wan y luo c aihara h structural basis of receptor recognition by sarscov2 nature “ httpsdoi101038s415860202179y rabaan aa alahmed sh haque s sah r tiwari r malik ys sarscov“ li w moore mj vasilieva n sui j wong sk berne ma angiotensin“ httpsdoi101038nature02145 hoffmann m kleineweber h pöhlmann s a multibasic cleavage site in the cohen fs how viruses invade cells biophys j “ httpsdoi101016jbpj201602006 sarscov and merscov a comparative overview le infez med converting enzyme is a functional receptor for the sars coronavirus nature preproofem structure of the 2019ncov spike in the prefusion conformation science “ httpsdoi101126scienceabb2507 spike protein of sarscov2 is essential for infection of human lung cells mol cell 202078779784e5 httpsdoi101016jmolcel202004022 ziegler cgk allon sj nyquist sk mbano im miao vn tzouanas cn sarscov2 receptor ace2 is an interferonstimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues cell “ httpsdoi101016jcell202004035 wrapp d wang n corbett ks goldsmith ja hsieh cl abiona o cryo 0c follis k york j nunberg j furin cleavage of the sars coronavirus spike glycoprotein enhances cell“cell fusion but does not affect virion entry virology “ httpsdoi101016jvirol200602003 millet jk whittaker gr host cell proteases critical determinants of coronavirus li myy li l zhang y wang xss expression of the sarscov2 cell receptor httpsdoi101186s4024902000662x xu h zhong l deng j peng j dan h zeng x high expression of ace2 receptor of 2019ncov on the epithelial cells of oral mucosa int j oral sci httpsdoi101038s413680200074x tropism and pathogenesis virus res “ httpsdoi101016jvirusres201411021 gene ace2 in a wide variety of human tissues infect dis poverty 20209na preproofcov2 protein interaction map reveals targets for drug repurposing nature infection of sarscov2 gastroenterology 202015818311833e3 httpsdoi101053jgastro202002055 httpsdoi101038s4158602022869 xu l liu j lu m yang d zheng x liver injury during highly pathogenic human coronavirus infections liver int off j int assoc study liver “ httpsdoi101111liv14435 coomes ea haghbayan h interleukin6 in covid19 a systematic review and xiao f tang m zheng x liu y li x shan h evidence for gastrointestinal gordon de jang gm bouhaddou m xu j obernier k white km a sars 0cmetaanalysis medrxiv httpsdoi1011012020033020048058 xu z shi l wang y zhang j huang l zhang c pathological findings of covid19 associated with acute respiratory distress syndrome lancet respir med “ httpsdoi101016s221326002030076x zhang b zhou x qiu y feng f feng j jia y clinical characteristics of chen g wu d guo w cao y huang d wang h clinical and chai x hu l zhang y han w lu z ke a specific ace2 expression in invest “ httpsdoi101172jci137244 death cases with covid19 medrxiv httpsdoi1011012020022620028191 immunological features of severe and moderate coronavirus disease j clin preproofcholangiocytes may cause liver damage after 2019ncov infection biorxiv patients medrxiv httpsdoi1011012020040120047381 httpsdoi10110120200203931766 herold t jurinovic v arnreich c hellmuth jc von bergweltbaildon m klein m level of il6 predicts respiratory failure in hospitalized symptomatic covid grein j ohmagari n shin d diaz g asperges e castagna a compassionate use of remdesivir for patients with severe covid19 n engl j med “ httpsdoi101056nejmoa2007016 u s food and drug administration fact sheet for health care providers emergency 0cuse authorization eua of hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of covid19 in certain hospitalized patients varona pérez j rodriguez chinesta jm riesgo de reactivación de la hepatitis b asociado al tratamiento con corticoides frente a sarscov2 covid19 rev clínica española httpsdoi101016jrce202004012 ji d qin e xu j zhang d cheng g wang y nonalcoholic fatty liver httpsdoi101016jjhep202003044 sarscov2 in metabolicassociated fatty liver disease j hepatol diseases in patients with covid19 a retrospective study j hepatol preproof biquard l valla d rautou pe no evidence for an increased liver uptake of httpsdoi101016jjhep202004035 0cfigure legends preprooffig1 proposed mechanisms of liver pathogenicity of sarscov2 in infected cells sars cov2 infection2 cytokinestorm3 drugeffects4 hypoxia5 previousliverdamagebiochemicallabmarkerswhite bloodcellsgenomereleasereplicationtranslationvirionassemblyviral proteinsmaturevirus release\uf0e9aado2mitochondrialproteinshypoxicisquemicliverinjuryliver damagelopinavirritonavirremdesivirchloroquinetocilizumaboxidativeimbalancesteatosisace2s proteincytopathiceffect\uf0e9gmcsf\uf0e9il6\uf0e9il1β\uf0e9il2\uf0e9il8\uf0e9ccl2\uf0e9ccl3\uf0e9ccl5\uf0e9cxcl \uf0e9alt\uf0e9ast\uf0eaalbumin\uf0e9pcr\uf0e9ldh\uf0e9ddimer\uf0e9ferritin\uf0e9bilirubin 0c'

cancer7513

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: clinical manifestations of sarscov2 infection include more frequently fever and cough failure can occur in persons with additional comorbidities liver dysfunction is one of the most striking affections among patients suggesting that sarscov2 may represent a new king of liver aggressor however the molecular process underlying this phenomenon is 0cstill unclear in this work we overview the most recent findings between the molecular biology of the virus pathogenic mechanisms and its relationship to liver disease observed in patients abbreviations aado2 ace2 aih alt ast covid19 ggt gi gtex alveolararterial oxygen gradient angiotensinconverting enzyme autoimmune hepatitis alanine transaminase aspartate aminotransferase coronavirus infectious disease gamma glutamyl transpeptidase gastrointestinal genotypetissue expression metabolicassociated fatty liver disease nonstructural proteins open reading frame preproofcomplex disease in many severely ill patients in other infected subjects an infection is keywords sarscov2 liver liver impairment covid19 ace2 mafld nsp orf introduction sarscov2 is the etiological agent of the disease known as covid19 which causes a disease characterized by pneumonia cough fever occasional diarrhea headache cardiac injury and in some patients™ liver alterations covid19 has been found to be an extremely reported to be so severe that it can lead to a disproportionate and mortal reaction of the immune system known as a cytokine storm all of these factors make covid19 highly unpredictable it is what specialists call a multisystem disease 0caround the world cases of liver dysfunction denoted by elevated hepatic enzymes in serum such as ast aspartate aminotransferase and alt alanine transaminase have been documented among patients infected with sarscov2 there is still no certainty whether the covid19related liver damagedysfunction is due mainly to the viral replication per se drugs toxicity or other coexisting comorbidities whether sexrelated difference could help to explain why infected men are more healthy or harmful relationship between the liver and its viral aggressor in this paper we describe a brief overview of the implications for researchers in the field of it is important to understand how liver function can be altered by direct infection with this predisposed to develop covid19associated liver dysfunction than infected women to analyze if there is any genetic predisposition related to impaired liver function during the œrespiratory virus which mechanisms of viral pathogenesis are involved to evaluate disease and of course the crosstalk between viral and cellular proteins that mediate this preproofliver disease of the most recent findings between the molecular biology of the virus this emerging viral illness is typically characterized by fever dry cough myalgia headache sore throat diarrhea and may be aggravated with shortness of breath and respiratory failure the angiotensinconverting enzyme ace2 the functional receptor of the spike glycoprotein of sarscov2 is widely distributed in the anism historically hamming and colleagues reported ace2 expression in the surface of lung alveolar epithelial cells enterocytes of the small intestine arterial and venous endothelial pathogenic mechanisms and its relationship to liver disease observed in patients clinical characteristics and liver injury in patients with covid19 0ccells and arterial smooth muscle cells posterior transcriptomic and proteomic analyses confirmed their findings and added high ace2 expression in adipose tissue bone marrow duodenum endometrium heart kidney small intestine smooth muscle testis and thyroid ace2 is also expressed in liver but in lesser extent one of the most worrisome severe cases of covid19 regarding the gastrointestinal gi tract and liver over covid19associated liver injury is defined as any liver damage that occurs during disease progression andor covid19 treatment in patients with or without a history of previous complications is the unusual formation of blood clots in many patients with covid19 even those who were receiving anticoagulants researchers at mount sinai in new york published studies suggesting that clots in the lungs play an important role in the most of covid19 patients develop gi symptoms such as anorexia diarrhea nausea and vomiting and a significant proportion present with altered liver function tests preproofdecreased albumin levels are associated with severe infection and poor prognosis still ast elevation is the most common abnormality in patients presenting with covid19 observed more frequently in men and is mainly documented in more severe cases liver disease in general the incidence of increased liver biochemical markers in hospitalized patients with covid19 mainly ast and alt and slightly elevated bilirubin varies between to of cases the increase in liver enzymes is there are no reports of acute or subacute liver failure in patients with covid19 the largest cohort study that included cases of covid19 from china showed that had preexisting chronic liver disease lei and colleagues reported that impaired liver function was related to mortality in covid19 patients elevated ast was more frequent and significant than the increase of alt in severe 0chospitalized patients moreover elevated ast was shown to be associated with highest mortality risk in the study reported by yijin wang they found that of covid patients had elevated ast activity the median levels of alt were ul vs ul respectively ast were ul vs ul respectively in abnormal and normal aminotransferase groups liver enzymes abnormality were associated with disease severity protein levels in addition they found by ultrastructural examination of coronavirus ps in hepatocytes in covid19 cases sarscov2 infected hepatocytes displayed as well as a series of laboratory tests including higher alveolararterial oxygen gradient aado2 higher gamma glutamyl transpeptidase ggt lower albumin and lower total conspicuous mitochondrial swelling endoplasmic reticulum dilatation and glycogen granule decreased histological findings showed apoptosis and binuclear hepatocytes preproofshowed a disease course similar to that reported in non immunosuppressed population coronavirus the interaction of its proteins with cellular proteins and consequently the immunosuppressive therapy for autoimmune hepatitis aih developing covid19 taken together both ultrastructural and histological evidence indicated a typical lesion of viral infection all these findings by different reports demonstrates that sarscov2 infection in liver is a crucial cause of hepatic impairment in covid19 patients however alteration of cellular metabolism that give rise to systematic alterations and metabolic report from alessio gerussi demonstrated that patients under today the cellular and molecular mechanisms that are altered by infection with this changes are still unknown 0cmolecular biology of sarscov2 coronaviruses are enveloped viruses that contain a positively polarized unsegmented rna genome belonging to the coronaviridae family and the order of nidovirales they are distributed in humans and other mammals the size of the sarscov2 virions is approximately to nm in diameter [“] sarscov2 has a genome that consists polymerase rdrp which is nsp12 and is responsible of the replication and transcription of the virus which are encoded by the various genetic loci on the genome at the center of the virion lies a nucleocapsid composed of the genomic rna and the nucleocapsid protein the virus glycoprotein s consists of two subunits s1 which is at the amino terminus and that encodes for structural proteins and a larger region that encodes two open reading frames orf 1a and 1b which together encode for the nonstructural virus proteins from nucleocapsid protein which is within the phospholipid bilayer and nonstructural proteins nsp1 to nsp16 the virions have a structural sspike protein outer spiky glycoprotein mmembrane protein a type iii transmembrane glycoprotein nof nucleotides encoding amino acids and it is composed of a region preproofvirus as well as protein m which is a type iii transmembrane glycoprotein and participates in the cellular membrane rearrangements for the replication and transcription complexes among the encoded proteins is an rnadependent rna provides the receptor binding site and s2 which is at the carboxyl terminus responsible for membrane fusion the envelope protein e has a role in the assembly and release of the nonstructural proteins have several functions during de viral cycle for example nsp 0cthe virus enters the cell by endocytosis through the interaction between envelope glycoprotein s with the cell receptor ace2 and with the participation of the type ii transmembrane serine protease tmprss2 once it enters the cell the n protein with viral genome are released within the cytoplasm then cellular proteases degrade the capsid and the virus genome is left free next the polyprotein containing the viral proteins that are how does the virus select which cells to infect viruses can infect only certain species of hosts and only permissive cells within that host permissive cells make all the necessary proteins and viral factors to allow virus to replicate once a virus gets inside a cell it hijacks the cellular processes to produce virally encoded proteins that will replicate the virus™s genetic material viral replication may cause exocytosis will translate into viral proteins this entire process will occur in the cell cytoplasm the processed to form the replication complex is translated and then the complementary strand of negative sense pregenomic rna is synthesized to be used as a template to replicate the structural proteins that will be synthesized in the endoplasmic reticulum membrane to assembly the viral p and finish the cycle through the release of the viruses by positive sense viral genome furthermore the replication and transcription complex will lead to a series of smaller positive sense subgenomic rnas these are the ones that preproofboth sarscov and the new sarscov2 are very similar in structure and pathogenicity but the major structural protein s protein is slightly different between them compared to other beta coronaviruses the presence of a furinlike cleavage site in sarscov2 enables the s protein priming and facilitates an increase on the efficiency of the spread of sarscov2 as is reported wide world 0cbiochemical changes producing cell damage called cytopathic effects like other coronaviruses sarscov2 requires cellular receptors to initiate its internalization to the cells that carry these factors li sarscov2 uses the angiotensinconverting enzyme ace2 as a host cell receptor sarscov2 spike s protein binds ace2 with significantly high affinity in addition the main host protease that suggested to promote the pathogenesis of this coronavirus program httpsportalgdccancergov they compared ace2 expression levels across human tissues between males and females and between younger and older persons in these individual tissues furthermore other reports have analyzed the correlations between ace2 in order to provide insights into the mechanism of sarscov2 infection li analyzed the expression of ace2 in various normal human tissues using the datasets from the genotypetissue expression gtex project and the cancer genome atlas tcga transmembrane serine protease other host proteases such as furin have also been mediates sprotein activation on primary target cells and initial viral entry is the type ii preproofreported by li ace2 expression levels showed no significant difference between have no significant association with sex age or race is the liver a direct target for sarscov2 males and females between younger and older persons or between asian and nonasian races this finding suggests that the infection risk of sarscov2 and sarscov may expression levels and immune signature enrichment levels in individual tissues as as we expected because the systemic manifestations of covid19 it has been reported that sarscov2 has an anotropism beyond the respiratory tract including the kidneys 0cliver heart and brain and possibly that anotropism influences the course of covid19 disease and aggravates preexisting conditions the ace2 protein is found at high levels in the gi tract as the colon biliary system and liver on the other hand it is well documented a sarscov2 rna shedding in the gi tract supporting its tropism for architecture express ace tmprss1 receptors the presence of these two host factors in the liver suggests that a direct viral cytopathic effect occurs also in sars infection the presence of viral rna in liver tissue was documented but not as extensively as the new coronavirus data published by gordon suggest that mitochondrial proteins interact directly with the virus which helps to understand the potential mechanism by which elevated ast the gi tract and liver cells and these may be sites of active viral replication and either direct or indirect tissue injury indeed a large part of the cells distributed in the liver preproofeffect the exacerbated inflammatory response in covid19 may play a central role in profiles are detected in these patients furthermore in addition to this intracellular more recently identified the clinical and laboratory characteristics of covid19 patients with abnormal liver transaminases and they reported that sarscov2 is able to which high levels of il6 have been reported which are involved in both infect liver cells and cause liver impairment by direct cytopathic effect inflammatory and repair responses in liver disease mechanisms of liver pathogenicity 0cif sarscov2 replication has direct adverse effects on liver function it is still unknown findings in liver biopsy of patients killed by covid19 showed moderate micro vesicular steatosis and mild portal and necroinflammatory activity this seems to indicate that a direct injury occurred while the infection that could have been directly caused by sarscov2 another possibility is that a druginduced liver injury occurred to date there are the following possible mechanisms figure infection the massive release of cytokines by the immune system in response to the viral infection can result in a cytokine storm and symptoms of sepsis that are the cause of death in of fatal covid19 cases in these cases uncontrolled inflammation induces multian damage leading including liver failure biomarkers of inflammation such as creactive protein pcr serum ferritin ldh ddimer il6 il2 are have been found to be significantly elevated in immune damage from exacerbated inflammation in response to sarscov2 preproofpathogenesis of sars cov “related liver disease more studies should be liver is a potential target for direct infection with this virus to understand the performed for evidence of viral mechanisms of replication in different cell anelles as cytoplasm endoplasmic reticulum golgi apparatus and lipidrafts cov2 enters cells through the ace2 molecule which is abundantly expressed in the liver and in particular in bile epithelial cells based on this expression the direct cytopathic effect due to active viral replication in various liver cells sarscritically ill patients with covid19 into hepatocytes and liver histology characterization it is also important to know in cells their capacity to efficiently produce both infectious and defective non 0cinfective whole virions there are not yet enough data to know the viral dynamics in the different tissues and the associated pathogenesis anoxia respiratory failure is one of the main characteristics of covid19 anoxic hypoxic hepatitis is common in patients with severe symptoms reactivation of preexisting liver disease patients with preexisting chronic liver medications such as tocilizumab and baricitinib used to combat the adverse immune cholestatic liver disease various studies such as lopinavir ritonavir remdesivir chloroquine tocilizumab uminefovir traditional chinese medicine so it is important to consider that they could be potentially hepatotoxic in some patients druginduced liver damage dili initial clinical guidelines recommended antiviral agents for covid19 so a variety of drugs have been administered in disease may be more susceptible to liver damage from sarscov2 biological preproof genetic factors genetics may well be one of the determining factors in some reaction may also cause hbv reactivation and induce eventual impairment of liver function in those patients with hbv on the other hand it is still unknown whether sarscov2 infection exacerbates cholestasis in people with underlying patients who become seriously ill with covid19 but until now we cannot be sure it is possible for example that the genetic variation that makes an individual more susceptible to high blood pressure or diabetes also makes him more vulnerable to the virus it will be important to find out what role genetic factors predisposing to liver steatosis have and their sensitivity to severe symptoms of covid19 ji and 0ccolleagues showed that subjects with metabolicassociated fatty liver disease mafld have a higher risk of covid19 severity disease and abnormal liver blood tests than patients without mafld in contrast louise biquard demonstrated that mafld is not associated with changes in liver expression blood test abnormalities reported by ji and colleagues is thus likely not explained by concluding remarks the scenario is not yet complete which does not allow us to establish or understand the natural history of the disease and the participation or commitment of the liver in this disease certainly the application of new technological platforms such as singlecell increased hepatic sarscov2 uptake still several contradictory reports will help of genes implicated in sarscov2 infection the observed persistence of liver to find the real role of genetic factors in the evolution of this disease preprooftranscriptomic assays will allow us to quickly know the commitment of each cell type in affected ans and the meaning of viral dynamics in the various affected systems including the liver however as we have already learned from the old hepatotropic viruses history still is ongoing and we have much to learn and understand about the virologic characteristics of this emerging rna virus that allow us to develop specific antivirals such as the case of hcv and the vaccine to decrease the impact of this œacute infection declarations of interest none ethical approval not required 0c references chen n zhou m dong x qu j gong f han y epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan china httpsdoi101002path1570 wang d eraslan b wieland t hallström b hopf t zolg dp a deep proteome and transcriptome abundance atlas of healthy human tissues mol syst hamming i timens w bulthuis mlc lely at navis gj van goor h tissue distribution of ace2 protein the functional receptor for sars coronavirus a first step in understanding sars pathogenesis j pathol “ a descriptive study lancet “ httpsdoi101016s0140 preproofa manifestation of covid19 rev gastroenterol méxico english ed biol 201915e8503 httpsdoi1015252msb20188503 patients with covid19 j am coll cardiol httpsdoi101016jjacc202005001 paranjpe i fuster v lala a russak a glicksberg bs levin ma association of treatment dose anticoagulation with inhospital survival among hospitalized schmulson m dávalos mf berumen j beware gastrointestinal symptoms can be httpsdoi101016jrgmxen202004001 cai q huang d yu h zhu z xia z su y covid19 abnormal liver function tests j hepatol httpsdoi101016jjhep202004006 0c siddiqi hk mehra mr covid19 illness in native and immunosuppressed states a clinicaltherapeutic staging proposal j hear lung transplant off publ int soc hear transplant “ httpsdoi101016jhealun202003012 feng g zheng ki yan qq rios rs targher g byrne cd covid19 and between markers of liver injury and mortality in covid19 in china hepatology httpsdoi101002hep31301 de la rica r bes m aranda m del castillo a socias a payeras a low transl hepatol “ httpsdoi1014218jcth202000018 albumin levels are associated with poorer outcomes in a case series of covid19 patients in spain a retrospective cohort study medrxiv liver dysfunction current insights and emergent therapeutic strategies j clin lei f liu ym zhou f qin jj zhang p zhu l longitudinal association preproofmortality of adult inpatients with covid19 in wuhan china a retrospective cohort study lancet “ httpsdoi101016s0140liver directly contributes to hepatic impairment in patients with covid19 j hepatol httpsdoi101016jjhep202005002 httpsdoi1011012020050720094987 zhou f yu t du r fan g liu y liu z clinical course and risk factors for wang y liu s liu h li w lin f jiang l sarscov2 infection of the gerussi a rigamonti c elia c cazzagon n floreani a pozzi r coronavirus disease covid19 in autoimmune hepatitis a lesson from 0cimmunosuppressed patients hepatol commun 2020na httpsdoi101002hep41557 richman d whitley r hayden f clinical virology 4th ed asm press ksiazek tg erdman d goldsmith cs zaki sr peret t emery s a novel “ httpsdoi101056nejmoa030781 kuiken t fouchier ram schutten m rimmelzwaan gf van amerongen g van respiratory syndrome lancet “ httpsdoi101016s0140 drosten c günther s preiser w van der werf s brodt hr becker s identification of a novel coronavirus in patients with severe acute respiratory riel d newly discovered coronavirus as the primary cause of severe acute coronavirus associated with severe acute respiratory syndrome n engl j med preproofsyndrome n engl j med “ httpsdoi101056nejmoa030747 outbreak associated with a new coronavirus of probable bat origin nature “ httpsdoi101038s4158602020127 mortola e roy p efficient assembly and release of sars coronaviruslike ps by a heterologous expression system febs lett “ httpsdoi101016jfebslet200409009 fehr a perlman s coronaviruses methods and protocols methods in molecular biology chapter coronaviruses an overview of their replication and zhou p yang xl lou wang xgg hu b zhang l zhang w a pneumonia 0cpathogenesis springer berlin heidelberg belouzard s millet jk licitra bn whittaker gr mechanisms of coronavirus cell entry mediated by the viral spike protein viruses “ httpsdoi103390v4061011 vennema h godeke gj rossen jw voorhout wf horzinek mc opstelten dj et snijder ej decroly e ziebuhr j the nonstructural proteins directing coronavirus neuman bw buchmeier mj supramolecular architecture of the coronavirus p adv virus res “ httpsdoi101016bsaivir201608005 van der hoeven b oudshoorn d koster aj snijder ej kikkert m barcena m neuman bw kiss g kunding ah bhella d baksh mf connelly s a structural analysis of m protein in coronavirus assembly and morphology j struct biol “ httpsdoi101016jjsb201011021 expression of viral envelope protein genes embo j “ al nucleocapsidindependent assembly of coronaviruslike ps by co preproofbiogenesis and architecture of arterivirus replication anelles virus res “ httpsdoi101016jvirusres201604001 rna synthesis and processing vol 1st ed elsevier inc httpsdoi101016bsaivir201608008 rabi f al zoubi m kasasbeh g salameh d alnasser a sarscov2 and coronavirus disease what we know so far pathogens masters p the molecular biology of coronaviruses adv virus res “ 0c shang j ye g shi k wan y luo c aihara h structural basis of receptor recognition by sarscov2 nature “ httpsdoi101038s415860202179y rabaan aa alahmed sh haque s sah r tiwari r malik ys sarscov“ li w moore mj vasilieva n sui j wong sk berne ma angiotensin“ httpsdoi101038nature02145 hoffmann m kleineweber h pöhlmann s a multibasic cleavage site in the cohen fs how viruses invade cells biophys j “ httpsdoi101016jbpj201602006 sarscov and merscov a comparative overview le infez med converting enzyme is a functional receptor for the sars coronavirus nature preproofem structure of the 2019ncov spike in the prefusion conformation science “ httpsdoi101126scienceabb2507 spike protein of sarscov2 is essential for infection of human lung cells mol cell 202078779784e5 httpsdoi101016jmolcel202004022 ziegler cgk allon sj nyquist sk mbano im miao vn tzouanas cn sarscov2 receptor ace2 is an interferonstimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues cell “ httpsdoi101016jcell202004035 wrapp d wang n corbett ks goldsmith ja hsieh cl abiona o cryo 0c follis k york j nunberg j furin cleavage of the sars coronavirus spike glycoprotein enhances cell“cell fusion but does not affect virion entry virology “ httpsdoi101016jvirol200602003 millet jk whittaker gr host cell proteases critical determinants of coronavirus li myy li l zhang y wang xss expression of the sarscov2 cell receptor httpsdoi101186s4024902000662x xu h zhong l deng j peng j dan h zeng x high expression of ace2 receptor of 2019ncov on the epithelial cells of oral mucosa int j oral sci httpsdoi101038s413680200074x tropism and pathogenesis virus res “ httpsdoi101016jvirusres201411021 gene ace2 in a wide variety of human tissues infect dis poverty 20209na preproofcov2 protein interaction map reveals targets for drug repurposing nature infection of sarscov2 gastroenterology 202015818311833e3 httpsdoi101053jgastro202002055 httpsdoi101038s4158602022869 xu l liu j lu m yang d zheng x liver injury during highly pathogenic human coronavirus infections liver int off j int assoc study liver “ httpsdoi101111liv14435 coomes ea haghbayan h interleukin6 in covid19 a systematic review and xiao f tang m zheng x liu y li x shan h evidence for gastrointestinal gordon de jang gm bouhaddou m xu j obernier k white km a sars 0cmetaanalysis medrxiv httpsdoi1011012020033020048058 xu z shi l wang y zhang j huang l zhang c pathological findings of covid19 associated with acute respiratory distress syndrome lancet respir med “ httpsdoi101016s221326002030076x zhang b zhou x qiu y feng f feng j jia y clinical characteristics of chen g wu d guo w cao y huang d wang h clinical and chai x hu l zhang y han w lu z ke a specific ace2 expression in invest “ httpsdoi101172jci137244 death cases with covid19 medrxiv httpsdoi1011012020022620028191 immunological features of severe and moderate coronavirus disease j clin preproofcholangiocytes may cause liver damage after 2019ncov infection biorxiv patients medrxiv httpsdoi1011012020040120047381 httpsdoi10110120200203931766 herold t jurinovic v arnreich c hellmuth jc von bergweltbaildon m klein m level of il6 predicts respiratory failure in hospitalized symptomatic covid grein j ohmagari n shin d diaz g asperges e castagna a compassionate use of remdesivir for patients with severe covid19 n engl j med “ httpsdoi101056nejmoa2007016 u s food and drug administration fact sheet for health care providers emergency 0cuse authorization eua of hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of covid19 in certain hospitalized patients varona pérez j rodriguez chinesta jm riesgo de reactivación de la hepatitis b asociado al tratamiento con corticoides frente a sarscov2 covid19 rev clínica española httpsdoi101016jrce202004012 ji d qin e xu j zhang d cheng g wang y nonalcoholic fatty liver httpsdoi101016jjhep202003044 sarscov2 in metabolicassociated fatty liver disease j hepatol diseases in patients with covid19 a retrospective study j hepatol preproof biquard l valla d rautou pe no evidence for an increased liver uptake of httpsdoi101016jjhep202004035 0cfigure legends preprooffig1 proposed mechanisms of liver pathogenicity of sarscov2 in infected cells sars cov2 infection2 cytokinestorm3 drugeffects4 hypoxia5 previousliverdamagebiochemicallabmarkerswhite bloodcellsgenomereleasereplicationtranslationvirionassemblyviral proteinsmaturevirus release\uf0e9aado2mitochondrialproteinshypoxicisquemicliverinjuryliver damagelopinavirritonavirremdesivirchloroquinetocilizumaboxidativeimbalancesteatosisace2s proteincytopathiceffect\uf0e9gmcsf\uf0e9il6\uf0e9il1β\uf0e9il2\uf0e9il8\uf0e9ccl2\uf0e9ccl3\uf0e9ccl5\uf0e9cxcl \uf0e9alt\uf0e9ast\uf0eaalbumin\uf0e9pcr\uf0e9ldh\uf0e9ddimer\uf0e9ferritin\uf0e9bilirubin 0c' Answer:

Colon_Cancer

" ethnopharmacological relevance herba patriniae has been used for thousands of years in china as a traditional chinese medicine with heatclearing and detoxicating effects it is applied widly for the treatment of rheumatoid arthritis diarrhea acute hepatitis pelvic inflammatory disease and ulcerative colitis in clinic two species namely patrinia scabiosaefolia fisch ps and patrinia villosa juss pv from the caprifoliaceae family are considered as herba patriniae in the pharmaceutical industry aim of the review this paper aims to comprehensively outline the traditional uses botanical description phytochemistry pharmacology toxicology quality control pharmacokinetics and patents of herba patriniae and elaborate the samedifferent characteristics between ps and pv materials and methods detailed information of herba patriniae was collected from various online databases pubmed web of science google schola china national preproof 0c knowledge infrastructure database national intellectual property administration prc national medical products administration and those published resources msc thesis and books results a total of compounds have been identified in herba patriniae including triterpenoid saponins flavonoids anic acids irids and volatiles a very distinct difference was observed that ps is rich in triterpenoid saponins and volatiles while pv contains more flavonoids two source species of herba patriniae gave similar pharmacological effects on anticancer antiinflammatory antioxidant antimicrobial sedative and hypnotic effects but there were no reports were on antipruritic proangiogenic and antidiarrheal effects for ps and no studies on antidiabetic effects for pv generally herba patriniae showed nontoxic in the clinical dose but mild side effects such as temporary leukopenia dizziness and nausea could be found when large and excessive dosage is used a variety of compounds have been quantified for the quality control of ps and pv the variety growth environment growth time and harvest time not only affected the contents but also the pharmacological activities of the bioactive compounds in the past year patents for compositions containing pv and ps have been filed mainly involving human health hygiene agriculture and animal husbandry unfortunately the research on pharmacokinetics is insufficient only the prototype components and metabolites were repored after intragastric administration of total flavonoids extract from pv in rats herba patriniae has displayed a significant medicinal value in clinic but the differences in phytochemistry pharmacological effects and the content of compounds have been found between two official recorded species about side effects and pharmacokinetic characteristics the differeces between two species have not been well studied for a better clinical use of herba patriniae it is urgent to establish systematic pharmacology quality control pharmacokinetics and clinical researches on the samedifferent characteristics between ps and pv keywords herba patriniae traditional uses phytochemistry pharmacology quality control preproof 0c cells a549 human lung cells cancer aspartate polysaccharide mixture ast list of abbreviations 3t3l1 preadipocytes 5fuhct8 human ileocecal adenocarcinoma cells a2780 human ovarian cancer cells a375s2 human melanoma cells a498 human renal abts carcinoma 'azinobis3ethylbenzothiazoline6sulphonic acid ags human gastric cancer cells akt protein kinase b alt alanine aminotransferase ap acute pancreatitis ap3 aminotransferase bax bcl2associated x protein bcl2 bcell lymphoma2 bclxl b cell lymphoma factor xl bel7402 human hepatoma cells bv2 mouse microglia cells caco2 human colon cancer cells cox2 cyclooxygenase2 crc colorectal cancer dai disease activity index dpph 22diphenyl1picrylhydrazyl ec50 half maximal effective concentration emt epithelialmesenchymal transition fak focal adhesion kinase gcms gas chromatographymass spectrometer glut4 glucose transporter gsh glutathione h2o2 hydrogen peroxide hela human cervical cancer cells hepg2 human hepatoma cells hl60 human promyelocytic leukemia cells ho1 heme oxygenase1 hplc high performance liquid chromatography hsp heat shock proteins hsp heat shock proteins ht1080 human fibrosarcoma cells ht29 human colon carcinoma cells huvecs human umbilical vein endothelial cells ic50 inhibitory concentration icam1 intercellular adhesion molecule icr institute of cancer research il1 interleukin1 beta il6 interleukin il8 interleukin inos inducible nitric oxide synthase irs insulin receptor substrate k562 human malignant myeloid cells ldh lactate dehydrogenase lps lipopolysaccharides mcf7 human breast cancer cells mda malondialdehyde mdamb231 human breast cancer cells mpo myeloperoxidase mrna messenger ribonucleic acid nfκb nuclear factor κb ngf nerve growth factor no nitric oxide nqo1 quinine oxidoreductase nrf2 nuclear factor erythroid 2related factor o2 superoxide anion oh hydroxyl radical pcna proliferating cell nuclear antigen pid pelvic inflammatory disease ps patrinia scabiosaefolia fisch pv patrinia villosa juss raw2647 mouse leukemic monocyte macrophage ros reactive oxygen species rsv respiratory syncytial virus sars severe acute respiratory syndrome sd sprague dawley sgc7901 human gastric cancer cells smmc7721 hepatocellular carcinoma cells stat3 signal transducer and activator of transcription sw480 human colon carcinoma cells tc50 half toxic concentration tcm traditional chinese medicine tgf transforming growth factor beta ti drug treatment index tnfα tumor necrosis factor alpha taoc total antioxidant capacity tsod total superoxide dismutase u14 mice cervical cancer cells u266 human multiple myeloma cancer cells u937 human lymphoma cells uc ulcerative colitis uv ultraviolet preproof 0c table of contents introduction traditional uses botany phytochemistry triterpenoid aglycones and triterpenoid saponins flavonoids anic acids irids volatiles other compounds pharmacology anticancer effect antiinflammatory effect antioxidant effect antimicrobial antiviral and antifungi effects sedative and hypnotic effects others toxicity quality control pharmacokinetics patented formulations and perspectives acknowledgements conflict of interest author contribution references preproof 0c introduction herba patriniae as known as œbai jiang cao in chinese is a traditional chinese medicine tcm originally recorded in œshen nong™s herbal classic as a middle grade medicinal material which has been used for thousands years besides korean ancient pharmacopaea œdonguibogam also record its medical value and it has been used for more than years in korea jeon et al it possesses the tcm properties of pungent and bitter in flavor and slightly cold in nature and has been classified to the stomach large intestine and liver meridians xiao two official species of patrinia scabiosaefolia fisch ps and patrinia villosa juss pv figure were considered as herba patriniae in chinese pharmacopoeia edition and chinese provincial pharmacopoeias these two plants have been widely used for more than years with good biological activities of clearing heat and detoxification eliminating carbuncle and expelling pus dispelling blood stasis and relieving pain through an analysis of ancient and modern literatures herba patriniae was mostly used in intestinal carbuncle lung carbuncle gynecological epigastric pain postpartum blood stasis and eczema in ancient times chen and han modern pharmacological studies have found that it has effects of anticancer antiinflammation antipathogenic microanisms antioxidation sedation and hypnosis wang et al 2019a nowadays herba patriniae is widely used in the respiratory system digestive system genitourinary system gynecology dermatology and other multidisciplinary diseases in clinical practice zhu and jiang and the number of applied patents increases every year httppsssystemcnipagovcn in view of its high content of amino acids vitamins minerals and other nutrients herba patriniae is not only regarded as a potherb with healthy value but also processed into tea products su et al zeng et al zhong et al in the past decades an increasing number of scholars have studied the chemical constituents and pharmacological effects of herba patriniae interestingly based on these studies we found that there are many differentsame characteristics between ps and pv both of them are official species for herba patriniae but differentiated clinical uses of them in different diseases may be better for the clinical outcome unfortunately we cannot found a comprehensive and updated review on the samedifferent characteristics of the two sources of herba patriniae and actually these two species also have not been differentiated in clinical uses therefore this review aims to systematically summarize the similarities and differences from the preproof 0c aspects of the traditional uses botanical description phytochemistry pharmacology and quality control of these two species of herba patriniae as well as being evidences for their clinical application and further research figure two species of herba patriniae a patrinia villosa juss b patrinia scabiosaefolia fisch a httpwwwcvhaccnspmcshcsh0005548 b httpwwwcvhaccnspmsyaufsyauf010108 traditional uses herba patriniae has a wide geographical distribution mainly in east asia and north america he et al some plants such as sonchus arvensis l sonchus asper vill sonchus oleraceus l etc may be confused as herba patriniae lu and hence these adulterants of herba patriniae should be exclude when clinical use traditionally according to records of œshen nong™s herbal classic 神农本草经 œcompendium of materia medica 本 草 纲 目 and œsynopsis of the golden chamber 金匮要略 œtai ping sheng hui fang 太平圣惠方 œpu ji fang 普 济方 œsheng ji zong lu 圣济æ»å½• œqian jin yi fang 千金翼方 and œqian jin fang 千金方 ancient doctors have used the whole herbs and roots of herba patriniae for disease treatment such as the stomach intestine liver gallbladder and gynecological diseases tian and tian zhu and jiang herba patriniae was recorded in chinese pharmacopoeia edition for the treatments of appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain swollen wellingabscess and clove sores pharmacopoeia committee of the ministry of health of p r china in addition herba patriniae is also preproof 0c recorded in the standards of traditional chinese medicine in many provinces of china table in miao nationality herba patriniae is also called œjia jiang le and used to treat rheumatoid arthritis colds and diarrheal qiu wang in dong medicine lu yi medicine drug control institute of yunnan chuxiong health bureau and dai medicine shi ps is called œnyangt ngeec liongc bail jangl œshe wei long and œpa hong respectively its whole herb is used to treat infantile diarrhea schizophrenia and infantile tinea capitis respectively ps is also called ba gai bao in zhuang medicine and its root is used to treat icteric hepatitis furuncles and snakebites shi pv is called œbitter vegetable by she nationality biological products identification institute of the ministry of health and œpao zi tong by tujia nationality peng and guan its whole herb can be used to treat appendicitis intestinal febrile symptoms constipation mammary abscess blister carbuncle and qi stagnation pv is also called œba gai lan and œhong pa in zhuang medicine biological products identification institute of the ministry of health and dai medicine shi respectively and its root is used to treat jaundice hepatitis furuncle local ulceration caused by snake injury and infantile convulsion moreover in korea people usually use the roots or whole plants of ps and pv as a traditional herbal medicine to treat appendicitis inflammation wound healing edema abscesses endometritis and abdominal pain after childbirth kang et al yang et al in recent years herba patriniae has been extensively applied in clinical practice in china especially in gynecology such as postpartum pain mastitis dysmenorrhea and tubal obstructive infertility liu 2019a it is noteworthy that herba patriniae is one of the most important ingredients in many prescriptions of tcm which is effective in diarrhea he acute hepatitis song pelvic inflammatory disease zhang 1997a typhoid fever paratyphoid fever sun ulcerative colitis liu anal cryptitis shi pelvic endometriosis yan and qiu acute pancreatitis he et al 2019b itching wang and wang gastroesophageal reflux disease benign prostatic hyperplasia rhinosinusitis mumps and phlebitis kong and zhao zhu and jiang a powder composed of coicis semen radix aconiti lateralis preparata and herba patriniae is a classic prescription for treating intestinal carbuncle in the œsynopsis of the golden chamber which is clinically used to treat chronic appendicitis chronic pelvic inflammatory disease and chronic prostatitis ji in addition a powder containing herba preproof 0c patriniae in the prescription is also used to treat sinusitis acute purulent tonsillitis and recurrent upper respiratory tract infection qin and diao zhu and jiang moreover it showed significant efficacy in the treatment of psoriasis vulgaris yan et al keshan disease scientific research cooperation group of herba patriniae in yan'an city for the prevention and control of keshan disease and chronic pelvic inflammation jia in the form of tablets in the chinese pharmacopoeia edition there are chinese herbal medicine prescriptions containing herba patriniae among which kangfu xiaoyan shuan and yifei qinghua gao are used to treat gynecological diseases and respiratory diseases respectively while longqing pian nankang pian niaosaitong pian and qianliexin jiaonang are used to treat genitourinary diseases state pharmacopoeia commission of p r china a summary of the traditional and traditional and clinical preparation of herba patriniae in china is given in table the tender stems and leaves of herba patriniae are rich in nutrients fresh in taste and grow in the mountains without environmental pollution it is a highquality vegetable that urban and rural residents like to eat pv tea is also abundant in hubei province and fujian province jiang 2019a xu et al herba patriniae is not only used in human health but also in agriculture fishery and animal husbandry interplanting herba patriniae in the newly reclaimed tea garden can increase the natural vegetation and reduce soil erosion and surface runoff caused by rainstorm erosion in the rainy season chen the combination of herba patriniae and other medicinal plants can be used to treat poisoned wound of cattle by agkistrodon acutus bitting crawling bee disease liver and skin diseases of turtle and fish and postpartum abdominal pain in cattle chen li shi zhao preproof 0c table the information of herba patriniae in national and local standards in china standards standard of traditional chinese medicine in hunan province standard of traditional chinese medicine in shandong province standard of traditional chinese medicine in heilongjiang province standard of traditional chinese medicine in liaoning province standard of traditional chinese medicine in sichuan province standard of traditional chinese medicine in guizhou province chinese pharmacopoeia edition application acute appendicitis diarrhea enteritis hemorrhagic leucorrhea red eye pterygium postpartum abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles acute appendicitis diarrhea hemorrhagic leucorrhea postpartum blood stasis abdominal pain swelling and pain of eye hepatitis boils and carbuncles acute appendicitis diarrhea dysentery postpartum blood stasis abdominal pain conjunctivitis boils and carbuncles acute appendicitis and its abdominal pain postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles dosage g standardsetting department hunan food and drug administration g g g g g g shandong medical products administration heilongjiang medical products administration liaoning food and drug administration sichuan food and drug administration guizhou medical products administration pharmacopoeia committee of the ministry of health of p r china preparation name yiyi fuzi baijiang san 薏苡附子败酱散 machixian heji 马齿苋合剂 aiye san 艾叶散 baijiang san 败酱散 baijiang san 败酱散 baijiang tang 败酱汤 baijiang tang 败酱汤 table traditional and clinical preparation of herba patriniae in china formulation main compositions powder coicis semen aconiti lateralis radix praeparata herba patriniae decoction portulacae herba isatidis folium arnebiae radix herba patriniae persicae semen carthami flos paeoniae radix rubar powder powder artemisiae argyi folium angelicae sinensis radix paeoniae radix alba dipsaci radix achyranthis bidentatae radix herba patriniae herba patriniae angelicae sinensis radix chuanxiong rhizoma paeoniae radix alba cinnamomi cortex powder herba patriniae moutan cortex cinnamomi cortex siphonostegiae herba aucklandiae radix decoction herba patriniae notopterygii rhizoma et radix dianthi herba aurantii fructus cinnamomi cortex persicae semen decoction herba patriniae reference jin gui yao lüe ãŠé‡‘匮要略㋠zhang 1997b surgery of chinese medicine ãŠä¸­åŒ»å¤–科学㋠beijing traditional chinese medicine hospital tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang pu ji fang ãŠæ™®æµŽæ–¹ã‹ volume zhu tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao qian jin yi fang ãŠåƒé‡‘翼方㋠volume sun preproof 0cpreparation name baijiang tang 败酱汤 baijiang tang 败酱汤 baijiang yin 败酱饮 changyong tang 肠痈汤 decoction chenzhou sheyao pian 郴州蛇药片 chure jili wan 除热蒺藜丸 tablets pills danggui xi tang 当归洗汤 danggui yin 当归饮 ganyan chongji 肝炎冲剂 decoction decoction electuary formulation main compositions decoction herba patriniae rhei radix et rhizoma persicae semen decoction herba patriniae cinnamomi cortex siphonostegiae herba moutan cortex aucklandiae radix decoction herba patriniae angelicae sinensis radix bambusae caulis in taenias rehmanniae radix moutan cortex glycyrrhizae radix et rhizoma herba patriniae zingiberis rhizoma recens poria coicis semen platycodonis radix liriopes radix salviae miltiorrhizae radix et rhizoma paeoniae radix alba rehmanniae radix pv tribuli fructus rhei radix et rhizoma herba patriniae cinnamomi cortex ginseng radix et rhizoma aconiti lateralis radix praeparata coicis semen coptidis rhizoma astragali radix abri herba angelicae sinensis radix aurantii fructus immaturus paeoniae radix alba tetrapanacis medulla angelicae sinensis radix angelicae pubescentis radix angelicae dahuricae radix sanguisorbae radix herba patriniae angelicae sinensis radix herba patriniae dipsaci radix paeoniae radix alba rehmanniae radix bambusae caulis in taenias bupleuri radix angelicae sinensis radix paeoniae radix alba paeoniae radix rubra citri reticulatae pericarpium aurantii fructus curcumae radix cyperi rhizoma salviae miltiorrhizae radix et rhizoma scrophulariae radix artemisiae scopariae herba isatidis radix herba patriniae huangdan tang 黄疸汤 decoction jiedu dihuang wan 解毒地黄丸 pills gualou san powder artemisiae scopariae herba gardeniae fructus lonicerae japonicae flos forsythiae fructus herba patriniae isatidis radix paeoniae radix rubra paeoniae radix alba bupleuri radix perillae caulis platycodonis radix sojae semen germinatum rehmanniae radix astragali radix trichosanthis radix scutellariae radix liriopes radix mantidis ootheca rhei radix et rhizoma ginseng radix et rhizoma gardeniae fructus cistanches herba peucedani radix cimicifugae rhizoma paeoniae radix alba anemarrhenae rhizoma vaccariae semen polygalae radix herba patriniae jujubae fructus trichosanthis semen herba patriniae asari radix et rhizoma zingiberis rhizoma magnoliae officinalis cortex platycodonis radix ginseng radix et rhizoma saposhnikoviae radix reference sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao qian jin fang ãŠåƒé‡‘方㋠volume （sun ） gu jin ming fang ãŠå¤ä»Šåæ–¹ã‹ yan and liu qian jin fang ãŠåƒé‡‘方㋠volume （sun ） qian jin fang ãŠåƒé‡‘方㋠volume （sun ） sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao study on the treatment of common diseases with traditional chinese medicine ãŠå¸¸è§ç—…的中 医 æ²» 疗 研 究 ㋠teaching and research group of traditional chinese medicine the first affiliated hospital of xi'an medical college lin zheng yi an yi fang ãŠä¸´è¯åŒ»æ¡ˆåŒ»æ–¹ã‹sun sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume preproof 0c preparation name 栝蒌散 lanwei xiaoyan wan 阑尾消炎丸 lanweiyan heji 阑尾炎合剂 formulation main compositions pills lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba spatholobi caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix decoction lonicerae japonicae flos taraxaci herba herba patriniae forsythiae fructus rhei radix et rhizoma paeoniae radix rubra toosendan fructus aucklandiae radix persicae semen lanweiyan tang 阑尾炎汤 lanwei yihao xiaoyan wan 阑尾ä¸å·æ¶ˆç‚Žç‰‡ lenge xiaoji tang 棱莪消积汤 lishi zhiyang pu yao 理湿止痒扑药 lidan tuihuang tang 利胆é黄汤 neibu wuxiang wan 内补五香丸 qianliexian tang 前列腺汤 qumai wan 瞿麦丸 decoction pills decoction powder decoction pills decoction pills yinqiao hongjiang jiedu tang decoction rhei radix et rhizoma moutan cortex persicae semen paeoniae radix alba salviae miltiorrhizae radix et rhizoma bupleuri radix lonicerae japonicae flos forsythiae fructus herba patriniae coicis semen lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix talci pulvis sargentodoxae caulis sparganii rhizoma curcumae rhizoma salviae miltiorrhizae radix et rhizoma paeoniae radix rubra corydalis rhizoma moutan cortex persicae semen coicis semen sargentodoxae caulis herba patriniae kochiae fructus bombyx batryticatus dictamni cortex angelicae dahuricae radix schizonepetae spica artemisiae scopariae herba herba patriniae alumen glycyrrhizae radix et rhizoma talcum cinnabaris artemisiae scopariae herba herba patriniae isatidis radix curcumae radix gardeniae fructus aquilariae lignum resinatum olibanum aucklandiae radix caryophylli flos dipsaci radix rehmanniae radix praeparata paeoniae radix alba magnoliae officinalis cortex herba patriniae ginseng radix et rhizoma poria cervi cornu salviae miltiorrhizae radix et rhizoma lycopi herba paeoniae radix rubra persicae semen carthami flos olibanum myrrha vaccariae semen citri reticulatae pericarpium toosendan fructus foeniculi fructus angelicae dahuricae radix herba patriniae taraxaci herba dianthi herba realgar vaccariae semen rehmanniae radix ephedrae herba imperatae rhizoma herba patriniae saposhnikoviae radix achyranthis bidentatae radix rhei radix et rhizoma lonicerae japonicae flos forsythiae fructus sargentodoxae caulis herba patriniae moutan cortex gardeniae fructus paeoniae radix rubra persicae semen coicis semen corydalis rhizoma toosendan fructus olibanum myrrha reference zhao beijing chinese traditional patent medicine specification ãŠåŒ—京市中成药规范㋠volume beijing municipal bureau of health selected data of acute abdomen treated by integrated traditional chinese and western medicine ãŠä¸­è¥¿åŒ»ç»“合治疗æ¥è…¹ç—‡èµ„æ–™é‰ç¼–ã‹ affiliated hospital of guangzhou college of traditional chinese medicine lin zheng yi an yi fang ãŠä¸´è¯åŒ»æ¡ˆåŒ»æ–¹ã‹ sun compilation of traditional chinese medicine preparations ãŠä¸­è¯åˆ¶å‰‚汇编㋠cao obstetrics and gynecology ㊠妇 产 科 å­¦ ã‹shanghai college of traditional chinese medicine selection of medical prescriptions of cixi and guangxu ãŠæ…ˆç¦§å…‰ç»ªåŒ»æ–¹é‰è®®ã‹ chen gu jin ming fang ãŠå¤ä»Šåæ–¹ã‹ yan and liu tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang surgery of chinese medicine ãŠä¸­åŒ»å¤–科学㋠beijing traditional chinese medicine hospital qian jin yi fang ãŠåƒé‡‘翼方㋠volume sun obstetrics and gynecology ㊠妇 产 科 å­¦ ã‹shanghai college of traditional chinese preproof 0c formulation main compositions preparation name 银翘红酱解毒汤 danhuang quyu jiaonang 丹黄祛ç˜èƒ¶å›Š qianlieping jiaonang 前列平胶囊 fuping jiaonang 妇平胶囊 fuyan kangfu jujue pian 妇炎康复å’嚼片 fuyan kangfu pian 妇炎康复片 fuyan kangfu jiaonang 妇炎康复胶囊 fuyan kangfu keli 妇炎康复颗粒 fuyanxiao jiaonang 妇炎消胶囊 fuyanqing xiji 妇炎清洗剂 capsules capsules capsules tablets tablets capsules granules capsules lotion xiaoer reke koufuye 小儿热咳口服液 oral liquid kangfu xiaoyan shuan 康复消炎栓 kangfu xiaoyan jiaonang suppository capsules astragali radix salviae miltiorrhizae radix et rhizoma dioscoreae rhizoma smilacis glabrae rhizoma angelicae sinensis radix spatholobi caulis euryales semen houttuyniae herba sparganii rhizoma curcumae rhizoma scorpio herba patriniae cinnamomi cortex atractylodis macrocephalae rhizoma zingiberis rhizoma praepatum eupolyphaga steleophaga corydalis rhizoma toosendan fructus sophorae flavescentis radix herba patriniae salviae miltiorrhizae radix et rhizoma paeoniae radix rubra persicae semen carthami flos lycopi herba pyrrosiae folium olibanum myrrha reference medicine httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa fagopyri dibotryis rhizoma violae herba curcumae rhizoma herba patriniae polygoni perfoliati herba solidaginis herba httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae trichosanthis radix rhei radix et rhizoma moutan cortex atractylodis rhizoma linderae radix httpswwwyaozhcom nmpa taraxaci herba herba patriniae coicis semen paeoniae radix rubra atractylodis rhizoma angelicae sinensis radix chuanxiong rhizoma cyperi rhizoma corydalis rhizoma alismatis rhizoma ephedrae herba armeniacae semen amarum forsythiae fructus rhei radix et rhizoma trichosanthis fructus mori cortex herba patriniae carthami flos glycyrrhizae radix et rhizoma sophorae flavescentis radix violae herba herba patriniae andrographis herba suis fellis pulvis taraxaci herba arnebiae radix aloe httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa state pharmacopoeia commission of p r china httpswwwyaozhcom taraxaci herba herba patriniae paeoniae radix rubra coicis semen angelicae sinensis radix atractylodis rhizoma chuanxiong rhizoma cyperi rhizoma alismatis rhizoma corydalis rhizoma httpswwwyaozhcom nmpa preproof 0c formulation main compositions reference preparation name 康妇炎胶囊 manshenning heji 慢肾宁合剂 nankang pian 男康片 decoction tablets astragali radix cinnamomi ramulus epimedii folium rehmanniae radix asini corii colla poria alismatis rhizoma scutellariae radix herba patriniae moutan cortex leonuri herba httpswwwyaozhcom nmpa paeoniae radix rubra rehmanniae radix praeparata cistanches herba glycyrrhizae radix et rhizoma taraxaci herba pyrolae herba phellodendri chinensis cortex carthami flos houttuyniae herba epimedii folium fubi fructus atractylodis macrocephalae rhizoma astragali radix cuscutae semen violae herba herba patriniae chrysanthemi indici flos angelicae sinensis radix sophorae fructus notoginseng radix et rhizoma sophorae flavescentis radix bletillae rhizoma cnidii fructus artemisiae argyi folium herba patriniae lonicerae japonicae flos portulacae herba saposhnikoviae radix alumen borneolum syntheticum glycyrrhizae radix et rhizoma pv sucrose dextrin astragali radix codonopsis radix glehniae radix liriopes radix agrimoniae herba bistortae rhizoma fritillariae cirrhosae bulbus asteris radix et rhizoma platycodonis radix armeniacae semen amarum herba patriniae glycyrrhizae radix et rhizoma codonopsis radix trionycis carapax paridis rhizoma atractylodis macrocephalae rhizoma astragali radix citri reticulatae pericarpium eupolyphaga steleophaga rhei radix et rhizoma persicae semen scutellariae barbatae herba herba patriniae poria coicis semen curcumae radix sappan lignum ostreae concha artemisiae scopariae herba cyperi rhizoma polygoni cuspidati rhizoma et radix ardisiae japonicae herba sedi herba scutellariae barbatae herba magnoliae officinalis cortex herba patriniae arnebiae radix wenyujin rhizoma concisum lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba spatholobi caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba sargentodoxae caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix lonicerae japonicae flos herba patriniae taraxaci herba moutan cortex toosendan fructus paeoniae radix rubra rhei radix et rhizoma persicae semen aucklandiae radix state pharmacopoeia commission of p r china httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa state pharmacopoeia commission of p r china httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa atractylodis rhizoma poria acori tatarinowii rhizoma plantaginis semen indigo naturalis phellodendri amurensis cortex talcum herba patriniae salviae miltiorrhizae radix et rhizoma ht

cancer7514

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " ethnopharmacological relevance herba patriniae has been used for thousands of years in china as a traditional chinese medicine with heatclearing and detoxicating effects it is applied widly for the treatment of rheumatoid arthritis diarrhea acute hepatitis pelvic inflammatory disease and ulcerative colitis in clinic two species namely patrinia scabiosaefolia fisch ps and patrinia villosa juss pv from the caprifoliaceae family are considered as herba patriniae in the pharmaceutical industry aim of the review this paper aims to comprehensively outline the traditional uses botanical description phytochemistry pharmacology toxicology quality control pharmacokinetics and patents of herba patriniae and elaborate the samedifferent characteristics between ps and pv materials and methods detailed information of herba patriniae was collected from various online databases pubmed web of science google schola china national preproof 0c knowledge infrastructure database national intellectual property administration prc national medical products administration and those published resources msc thesis and books results a total of compounds have been identified in herba patriniae including triterpenoid saponins flavonoids anic acids irids and volatiles a very distinct difference was observed that ps is rich in triterpenoid saponins and volatiles while pv contains more flavonoids two source species of herba patriniae gave similar pharmacological effects on anticancer antiinflammatory antioxidant antimicrobial sedative and hypnotic effects but there were no reports were on antipruritic proangiogenic and antidiarrheal effects for ps and no studies on antidiabetic effects for pv generally herba patriniae showed nontoxic in the clinical dose but mild side effects such as temporary leukopenia dizziness and nausea could be found when large and excessive dosage is used a variety of compounds have been quantified for the quality control of ps and pv the variety growth environment growth time and harvest time not only affected the contents but also the pharmacological activities of the bioactive compounds in the past year patents for compositions containing pv and ps have been filed mainly involving human health hygiene agriculture and animal husbandry unfortunately the research on pharmacokinetics is insufficient only the prototype components and metabolites were repored after intragastric administration of total flavonoids extract from pv in rats herba patriniae has displayed a significant medicinal value in clinic but the differences in phytochemistry pharmacological effects and the content of compounds have been found between two official recorded species about side effects and pharmacokinetic characteristics the differeces between two species have not been well studied for a better clinical use of herba patriniae it is urgent to establish systematic pharmacology quality control pharmacokinetics and clinical researches on the samedifferent characteristics between ps and pv keywords herba patriniae traditional uses phytochemistry pharmacology quality control preproof 0c cells a549 human lung cells cancer aspartate polysaccharide mixture ast list of abbreviations 3t3l1 preadipocytes 5fuhct8 human ileocecal adenocarcinoma cells a2780 human ovarian cancer cells a375s2 human melanoma cells a498 human renal abts carcinoma 'azinobis3ethylbenzothiazoline6sulphonic acid ags human gastric cancer cells akt protein kinase b alt alanine aminotransferase ap acute pancreatitis ap3 aminotransferase bax bcl2associated x protein bcl2 bcell lymphoma2 bclxl b cell lymphoma factor xl bel7402 human hepatoma cells bv2 mouse microglia cells caco2 human colon cancer cells cox2 cyclooxygenase2 crc colorectal cancer dai disease activity index dpph 22diphenyl1picrylhydrazyl ec50 half maximal effective concentration emt epithelialmesenchymal transition fak focal adhesion kinase gcms gas chromatographymass spectrometer glut4 glucose transporter gsh glutathione h2o2 hydrogen peroxide hela human cervical cancer cells hepg2 human hepatoma cells hl60 human promyelocytic leukemia cells ho1 heme oxygenase1 hplc high performance liquid chromatography hsp heat shock proteins hsp heat shock proteins ht1080 human fibrosarcoma cells ht29 human colon carcinoma cells huvecs human umbilical vein endothelial cells ic50 inhibitory concentration icam1 intercellular adhesion molecule icr institute of cancer research il1 interleukin1 beta il6 interleukin il8 interleukin inos inducible nitric oxide synthase irs insulin receptor substrate k562 human malignant myeloid cells ldh lactate dehydrogenase lps lipopolysaccharides mcf7 human breast cancer cells mda malondialdehyde mdamb231 human breast cancer cells mpo myeloperoxidase mrna messenger ribonucleic acid nfκb nuclear factor κb ngf nerve growth factor no nitric oxide nqo1 quinine oxidoreductase nrf2 nuclear factor erythroid 2related factor o2 superoxide anion oh hydroxyl radical pcna proliferating cell nuclear antigen pid pelvic inflammatory disease ps patrinia scabiosaefolia fisch pv patrinia villosa juss raw2647 mouse leukemic monocyte macrophage ros reactive oxygen species rsv respiratory syncytial virus sars severe acute respiratory syndrome sd sprague dawley sgc7901 human gastric cancer cells smmc7721 hepatocellular carcinoma cells stat3 signal transducer and activator of transcription sw480 human colon carcinoma cells tc50 half toxic concentration tcm traditional chinese medicine tgf transforming growth factor beta ti drug treatment index tnfα tumor necrosis factor alpha taoc total antioxidant capacity tsod total superoxide dismutase u14 mice cervical cancer cells u266 human multiple myeloma cancer cells u937 human lymphoma cells uc ulcerative colitis uv ultraviolet preproof 0c table of contents introduction traditional uses botany phytochemistry triterpenoid aglycones and triterpenoid saponins flavonoids anic acids irids volatiles other compounds pharmacology anticancer effect antiinflammatory effect antioxidant effect antimicrobial antiviral and antifungi effects sedative and hypnotic effects others toxicity quality control pharmacokinetics patented formulations and perspectives acknowledgements conflict of interest author contribution references preproof 0c introduction herba patriniae as known as œbai jiang cao in chinese is a traditional chinese medicine tcm originally recorded in œshen nong™s herbal classic as a middle grade medicinal material which has been used for thousands years besides korean ancient pharmacopaea œdonguibogam also record its medical value and it has been used for more than years in korea jeon et al it possesses the tcm properties of pungent and bitter in flavor and slightly cold in nature and has been classified to the stomach large intestine and liver meridians xiao two official species of patrinia scabiosaefolia fisch ps and patrinia villosa juss pv figure were considered as herba patriniae in chinese pharmacopoeia edition and chinese provincial pharmacopoeias these two plants have been widely used for more than years with good biological activities of clearing heat and detoxification eliminating carbuncle and expelling pus dispelling blood stasis and relieving pain through an analysis of ancient and modern literatures herba patriniae was mostly used in intestinal carbuncle lung carbuncle gynecological epigastric pain postpartum blood stasis and eczema in ancient times chen and han modern pharmacological studies have found that it has effects of anticancer antiinflammation antipathogenic microanisms antioxidation sedation and hypnosis wang et al 2019a nowadays herba patriniae is widely used in the respiratory system digestive system genitourinary system gynecology dermatology and other multidisciplinary diseases in clinical practice zhu and jiang and the number of applied patents increases every year httppsssystemcnipagovcn in view of its high content of amino acids vitamins minerals and other nutrients herba patriniae is not only regarded as a potherb with healthy value but also processed into tea products su et al zeng et al zhong et al in the past decades an increasing number of scholars have studied the chemical constituents and pharmacological effects of herba patriniae interestingly based on these studies we found that there are many differentsame characteristics between ps and pv both of them are official species for herba patriniae but differentiated clinical uses of them in different diseases may be better for the clinical outcome unfortunately we cannot found a comprehensive and updated review on the samedifferent characteristics of the two sources of herba patriniae and actually these two species also have not been differentiated in clinical uses therefore this review aims to systematically summarize the similarities and differences from the preproof 0c aspects of the traditional uses botanical description phytochemistry pharmacology and quality control of these two species of herba patriniae as well as being evidences for their clinical application and further research figure two species of herba patriniae a patrinia villosa juss b patrinia scabiosaefolia fisch a httpwwwcvhaccnspmcshcsh0005548 b httpwwwcvhaccnspmsyaufsyauf010108 traditional uses herba patriniae has a wide geographical distribution mainly in east asia and north america he et al some plants such as sonchus arvensis l sonchus asper vill sonchus oleraceus l etc may be confused as herba patriniae lu and hence these adulterants of herba patriniae should be exclude when clinical use traditionally according to records of œshen nong™s herbal classic 神农本草经 œcompendium of materia medica 本 草 纲 目 and œsynopsis of the golden chamber 金匮要略 œtai ping sheng hui fang 太平圣惠方 œpu ji fang 普 济方 œsheng ji zong lu 圣济æ»å½• œqian jin yi fang 千金翼方 and œqian jin fang 千金方 ancient doctors have used the whole herbs and roots of herba patriniae for disease treatment such as the stomach intestine liver gallbladder and gynecological diseases tian and tian zhu and jiang herba patriniae was recorded in chinese pharmacopoeia edition for the treatments of appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain swollen wellingabscess and clove sores pharmacopoeia committee of the ministry of health of p r china in addition herba patriniae is also preproof 0c recorded in the standards of traditional chinese medicine in many provinces of china table in miao nationality herba patriniae is also called œjia jiang le and used to treat rheumatoid arthritis colds and diarrheal qiu wang in dong medicine lu yi medicine drug control institute of yunnan chuxiong health bureau and dai medicine shi ps is called œnyangt ngeec liongc bail jangl œshe wei long and œpa hong respectively its whole herb is used to treat infantile diarrhea schizophrenia and infantile tinea capitis respectively ps is also called ba gai bao in zhuang medicine and its root is used to treat icteric hepatitis furuncles and snakebites shi pv is called œbitter vegetable by she nationality biological products identification institute of the ministry of health and œpao zi tong by tujia nationality peng and guan its whole herb can be used to treat appendicitis intestinal febrile symptoms constipation mammary abscess blister carbuncle and qi stagnation pv is also called œba gai lan and œhong pa in zhuang medicine biological products identification institute of the ministry of health and dai medicine shi respectively and its root is used to treat jaundice hepatitis furuncle local ulceration caused by snake injury and infantile convulsion moreover in korea people usually use the roots or whole plants of ps and pv as a traditional herbal medicine to treat appendicitis inflammation wound healing edema abscesses endometritis and abdominal pain after childbirth kang et al yang et al in recent years herba patriniae has been extensively applied in clinical practice in china especially in gynecology such as postpartum pain mastitis dysmenorrhea and tubal obstructive infertility liu 2019a it is noteworthy that herba patriniae is one of the most important ingredients in many prescriptions of tcm which is effective in diarrhea he acute hepatitis song pelvic inflammatory disease zhang 1997a typhoid fever paratyphoid fever sun ulcerative colitis liu anal cryptitis shi pelvic endometriosis yan and qiu acute pancreatitis he et al 2019b itching wang and wang gastroesophageal reflux disease benign prostatic hyperplasia rhinosinusitis mumps and phlebitis kong and zhao zhu and jiang a powder composed of coicis semen radix aconiti lateralis preparata and herba patriniae is a classic prescription for treating intestinal carbuncle in the œsynopsis of the golden chamber which is clinically used to treat chronic appendicitis chronic pelvic inflammatory disease and chronic prostatitis ji in addition a powder containing herba preproof 0c patriniae in the prescription is also used to treat sinusitis acute purulent tonsillitis and recurrent upper respiratory tract infection qin and diao zhu and jiang moreover it showed significant efficacy in the treatment of psoriasis vulgaris yan et al keshan disease scientific research cooperation group of herba patriniae in yan'an city for the prevention and control of keshan disease and chronic pelvic inflammation jia in the form of tablets in the chinese pharmacopoeia edition there are chinese herbal medicine prescriptions containing herba patriniae among which kangfu xiaoyan shuan and yifei qinghua gao are used to treat gynecological diseases and respiratory diseases respectively while longqing pian nankang pian niaosaitong pian and qianliexin jiaonang are used to treat genitourinary diseases state pharmacopoeia commission of p r china a summary of the traditional and traditional and clinical preparation of herba patriniae in china is given in table the tender stems and leaves of herba patriniae are rich in nutrients fresh in taste and grow in the mountains without environmental pollution it is a highquality vegetable that urban and rural residents like to eat pv tea is also abundant in hubei province and fujian province jiang 2019a xu et al herba patriniae is not only used in human health but also in agriculture fishery and animal husbandry interplanting herba patriniae in the newly reclaimed tea garden can increase the natural vegetation and reduce soil erosion and surface runoff caused by rainstorm erosion in the rainy season chen the combination of herba patriniae and other medicinal plants can be used to treat poisoned wound of cattle by agkistrodon acutus bitting crawling bee disease liver and skin diseases of turtle and fish and postpartum abdominal pain in cattle chen li shi zhao preproof 0c table the information of herba patriniae in national and local standards in china standards standard of traditional chinese medicine in hunan province standard of traditional chinese medicine in shandong province standard of traditional chinese medicine in heilongjiang province standard of traditional chinese medicine in liaoning province standard of traditional chinese medicine in sichuan province standard of traditional chinese medicine in guizhou province chinese pharmacopoeia edition application acute appendicitis diarrhea enteritis hemorrhagic leucorrhea red eye pterygium postpartum abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles acute appendicitis diarrhea hemorrhagic leucorrhea postpartum blood stasis abdominal pain swelling and pain of eye hepatitis boils and carbuncles acute appendicitis diarrhea dysentery postpartum blood stasis abdominal pain conjunctivitis boils and carbuncles acute appendicitis and its abdominal pain postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles dosage g standardsetting department hunan food and drug administration g g g g g g shandong medical products administration heilongjiang medical products administration liaoning food and drug administration sichuan food and drug administration guizhou medical products administration pharmacopoeia committee of the ministry of health of p r china preparation name yiyi fuzi baijiang san 薏苡附子败酱散 machixian heji 马齿苋合剂 aiye san 艾叶散 baijiang san 败酱散 baijiang san 败酱散 baijiang tang 败酱汤 baijiang tang 败酱汤 table traditional and clinical preparation of herba patriniae in china formulation main compositions powder coicis semen aconiti lateralis radix praeparata herba patriniae decoction portulacae herba isatidis folium arnebiae radix herba patriniae persicae semen carthami flos paeoniae radix rubar powder powder artemisiae argyi folium angelicae sinensis radix paeoniae radix alba dipsaci radix achyranthis bidentatae radix herba patriniae herba patriniae angelicae sinensis radix chuanxiong rhizoma paeoniae radix alba cinnamomi cortex powder herba patriniae moutan cortex cinnamomi cortex siphonostegiae herba aucklandiae radix decoction herba patriniae notopterygii rhizoma et radix dianthi herba aurantii fructus cinnamomi cortex persicae semen decoction herba patriniae reference jin gui yao lüe ãŠé‡‘匮要略㋠zhang 1997b surgery of chinese medicine ãŠä¸­åŒ»å¤–科学㋠beijing traditional chinese medicine hospital tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang pu ji fang ãŠæ™®æµŽæ–¹ã‹ volume zhu tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao qian jin yi fang ãŠåƒé‡‘翼方㋠volume sun preproof 0cpreparation name baijiang tang 败酱汤 baijiang tang 败酱汤 baijiang yin 败酱饮 changyong tang 肠痈汤 decoction chenzhou sheyao pian 郴州蛇药片 chure jili wan 除热蒺藜丸 tablets pills danggui xi tang 当归洗汤 danggui yin 当归饮 ganyan chongji 肝炎冲剂 decoction decoction electuary formulation main compositions decoction herba patriniae rhei radix et rhizoma persicae semen decoction herba patriniae cinnamomi cortex siphonostegiae herba moutan cortex aucklandiae radix decoction herba patriniae angelicae sinensis radix bambusae caulis in taenias rehmanniae radix moutan cortex glycyrrhizae radix et rhizoma herba patriniae zingiberis rhizoma recens poria coicis semen platycodonis radix liriopes radix salviae miltiorrhizae radix et rhizoma paeoniae radix alba rehmanniae radix pv tribuli fructus rhei radix et rhizoma herba patriniae cinnamomi cortex ginseng radix et rhizoma aconiti lateralis radix praeparata coicis semen coptidis rhizoma astragali radix abri herba angelicae sinensis radix aurantii fructus immaturus paeoniae radix alba tetrapanacis medulla angelicae sinensis radix angelicae pubescentis radix angelicae dahuricae radix sanguisorbae radix herba patriniae angelicae sinensis radix herba patriniae dipsaci radix paeoniae radix alba rehmanniae radix bambusae caulis in taenias bupleuri radix angelicae sinensis radix paeoniae radix alba paeoniae radix rubra citri reticulatae pericarpium aurantii fructus curcumae radix cyperi rhizoma salviae miltiorrhizae radix et rhizoma scrophulariae radix artemisiae scopariae herba isatidis radix herba patriniae huangdan tang 黄疸汤 decoction jiedu dihuang wan 解毒地黄丸 pills gualou san powder artemisiae scopariae herba gardeniae fructus lonicerae japonicae flos forsythiae fructus herba patriniae isatidis radix paeoniae radix rubra paeoniae radix alba bupleuri radix perillae caulis platycodonis radix sojae semen germinatum rehmanniae radix astragali radix trichosanthis radix scutellariae radix liriopes radix mantidis ootheca rhei radix et rhizoma ginseng radix et rhizoma gardeniae fructus cistanches herba peucedani radix cimicifugae rhizoma paeoniae radix alba anemarrhenae rhizoma vaccariae semen polygalae radix herba patriniae jujubae fructus trichosanthis semen herba patriniae asari radix et rhizoma zingiberis rhizoma magnoliae officinalis cortex platycodonis radix ginseng radix et rhizoma saposhnikoviae radix reference sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao qian jin fang ãŠåƒé‡‘方㋠volume （sun ） gu jin ming fang ãŠå¤ä»Šåæ–¹ã‹ yan and liu qian jin fang ãŠåƒé‡‘方㋠volume （sun ） qian jin fang ãŠåƒé‡‘方㋠volume （sun ） sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao study on the treatment of common diseases with traditional chinese medicine ãŠå¸¸è§ç—…的中 医 æ²» 疗 研 究 ㋠teaching and research group of traditional chinese medicine the first affiliated hospital of xi'an medical college lin zheng yi an yi fang ãŠä¸´è¯åŒ»æ¡ˆåŒ»æ–¹ã‹sun sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume preproof 0c preparation name 栝蒌散 lanwei xiaoyan wan 阑尾消炎丸 lanweiyan heji 阑尾炎合剂 formulation main compositions pills lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba spatholobi caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix decoction lonicerae japonicae flos taraxaci herba herba patriniae forsythiae fructus rhei radix et rhizoma paeoniae radix rubra toosendan fructus aucklandiae radix persicae semen lanweiyan tang 阑尾炎汤 lanwei yihao xiaoyan wan 阑尾ä¸å·æ¶ˆç‚Žç‰‡ lenge xiaoji tang 棱莪消积汤 lishi zhiyang pu yao 理湿止痒扑药 lidan tuihuang tang 利胆é黄汤 neibu wuxiang wan 内补五香丸 qianliexian tang 前列腺汤 qumai wan 瞿麦丸 decoction pills decoction powder decoction pills decoction pills yinqiao hongjiang jiedu tang decoction rhei radix et rhizoma moutan cortex persicae semen paeoniae radix alba salviae miltiorrhizae radix et rhizoma bupleuri radix lonicerae japonicae flos forsythiae fructus herba patriniae coicis semen lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix talci pulvis sargentodoxae caulis sparganii rhizoma curcumae rhizoma salviae miltiorrhizae radix et rhizoma paeoniae radix rubra corydalis rhizoma moutan cortex persicae semen coicis semen sargentodoxae caulis herba patriniae kochiae fructus bombyx batryticatus dictamni cortex angelicae dahuricae radix schizonepetae spica artemisiae scopariae herba herba patriniae alumen glycyrrhizae radix et rhizoma talcum cinnabaris artemisiae scopariae herba herba patriniae isatidis radix curcumae radix gardeniae fructus aquilariae lignum resinatum olibanum aucklandiae radix caryophylli flos dipsaci radix rehmanniae radix praeparata paeoniae radix alba magnoliae officinalis cortex herba patriniae ginseng radix et rhizoma poria cervi cornu salviae miltiorrhizae radix et rhizoma lycopi herba paeoniae radix rubra persicae semen carthami flos olibanum myrrha vaccariae semen citri reticulatae pericarpium toosendan fructus foeniculi fructus angelicae dahuricae radix herba patriniae taraxaci herba dianthi herba realgar vaccariae semen rehmanniae radix ephedrae herba imperatae rhizoma herba patriniae saposhnikoviae radix achyranthis bidentatae radix rhei radix et rhizoma lonicerae japonicae flos forsythiae fructus sargentodoxae caulis herba patriniae moutan cortex gardeniae fructus paeoniae radix rubra persicae semen coicis semen corydalis rhizoma toosendan fructus olibanum myrrha reference zhao beijing chinese traditional patent medicine specification ãŠåŒ—京市中成药规范㋠volume beijing municipal bureau of health selected data of acute abdomen treated by integrated traditional chinese and western medicine ãŠä¸­è¥¿åŒ»ç»“合治疗æ¥è…¹ç—‡èµ„æ–™é‰ç¼–ã‹ affiliated hospital of guangzhou college of traditional chinese medicine lin zheng yi an yi fang ãŠä¸´è¯åŒ»æ¡ˆåŒ»æ–¹ã‹ sun compilation of traditional chinese medicine preparations ãŠä¸­è¯åˆ¶å‰‚汇编㋠cao obstetrics and gynecology ㊠妇 产 科 å­¦ ã‹shanghai college of traditional chinese medicine selection of medical prescriptions of cixi and guangxu ãŠæ…ˆç¦§å…‰ç»ªåŒ»æ–¹é‰è®®ã‹ chen gu jin ming fang ãŠå¤ä»Šåæ–¹ã‹ yan and liu tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang surgery of chinese medicine ãŠä¸­åŒ»å¤–科学㋠beijing traditional chinese medicine hospital qian jin yi fang ãŠåƒé‡‘翼方㋠volume sun obstetrics and gynecology ㊠妇 产 科 å­¦ ã‹shanghai college of traditional chinese preproof 0c formulation main compositions preparation name 银翘红酱解毒汤 danhuang quyu jiaonang 丹黄祛ç˜èƒ¶å›Š qianlieping jiaonang 前列平胶囊 fuping jiaonang 妇平胶囊 fuyan kangfu jujue pian 妇炎康复å’嚼片 fuyan kangfu pian 妇炎康复片 fuyan kangfu jiaonang 妇炎康复胶囊 fuyan kangfu keli 妇炎康复颗粒 fuyanxiao jiaonang 妇炎消胶囊 fuyanqing xiji 妇炎清洗剂 capsules capsules capsules tablets tablets capsules granules capsules lotion xiaoer reke koufuye 小儿热咳口服液 oral liquid kangfu xiaoyan shuan 康复消炎栓 kangfu xiaoyan jiaonang suppository capsules astragali radix salviae miltiorrhizae radix et rhizoma dioscoreae rhizoma smilacis glabrae rhizoma angelicae sinensis radix spatholobi caulis euryales semen houttuyniae herba sparganii rhizoma curcumae rhizoma scorpio herba patriniae cinnamomi cortex atractylodis macrocephalae rhizoma zingiberis rhizoma praepatum eupolyphaga steleophaga corydalis rhizoma toosendan fructus sophorae flavescentis radix herba patriniae salviae miltiorrhizae radix et rhizoma paeoniae radix rubra persicae semen carthami flos lycopi herba pyrrosiae folium olibanum myrrha reference medicine httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa fagopyri dibotryis rhizoma violae herba curcumae rhizoma herba patriniae polygoni perfoliati herba solidaginis herba httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae trichosanthis radix rhei radix et rhizoma moutan cortex atractylodis rhizoma linderae radix httpswwwyaozhcom nmpa taraxaci herba herba patriniae coicis semen paeoniae radix rubra atractylodis rhizoma angelicae sinensis radix chuanxiong rhizoma cyperi rhizoma corydalis rhizoma alismatis rhizoma ephedrae herba armeniacae semen amarum forsythiae fructus rhei radix et rhizoma trichosanthis fructus mori cortex herba patriniae carthami flos glycyrrhizae radix et rhizoma sophorae flavescentis radix violae herba herba patriniae andrographis herba suis fellis pulvis taraxaci herba arnebiae radix aloe httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa state pharmacopoeia commission of p r china httpswwwyaozhcom taraxaci herba herba patriniae paeoniae radix rubra coicis semen angelicae sinensis radix atractylodis rhizoma chuanxiong rhizoma cyperi rhizoma alismatis rhizoma corydalis rhizoma httpswwwyaozhcom nmpa preproof 0c formulation main compositions reference preparation name 康妇炎胶囊 manshenning heji 慢肾宁合剂 nankang pian 男康片 decoction tablets astragali radix cinnamomi ramulus epimedii folium rehmanniae radix asini corii colla poria alismatis rhizoma scutellariae radix herba patriniae moutan cortex leonuri herba httpswwwyaozhcom nmpa paeoniae radix rubra rehmanniae radix praeparata cistanches herba glycyrrhizae radix et rhizoma taraxaci herba pyrolae herba phellodendri chinensis cortex carthami flos houttuyniae herba epimedii folium fubi fructus atractylodis macrocephalae rhizoma astragali radix cuscutae semen violae herba herba patriniae chrysanthemi indici flos angelicae sinensis radix sophorae fructus notoginseng radix et rhizoma sophorae flavescentis radix bletillae rhizoma cnidii fructus artemisiae argyi folium herba patriniae lonicerae japonicae flos portulacae herba saposhnikoviae radix alumen borneolum syntheticum glycyrrhizae radix et rhizoma pv sucrose dextrin astragali radix codonopsis radix glehniae radix liriopes radix agrimoniae herba bistortae rhizoma fritillariae cirrhosae bulbus asteris radix et rhizoma platycodonis radix armeniacae semen amarum herba patriniae glycyrrhizae radix et rhizoma codonopsis radix trionycis carapax paridis rhizoma atractylodis macrocephalae rhizoma astragali radix citri reticulatae pericarpium eupolyphaga steleophaga rhei radix et rhizoma persicae semen scutellariae barbatae herba herba patriniae poria coicis semen curcumae radix sappan lignum ostreae concha artemisiae scopariae herba cyperi rhizoma polygoni cuspidati rhizoma et radix ardisiae japonicae herba sedi herba scutellariae barbatae herba magnoliae officinalis cortex herba patriniae arnebiae radix wenyujin rhizoma concisum lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba spatholobi caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba sargentodoxae caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix lonicerae japonicae flos herba patriniae taraxaci herba moutan cortex toosendan fructus paeoniae radix rubra rhei radix et rhizoma persicae semen aucklandiae radix state pharmacopoeia commission of p r china httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa state pharmacopoeia commission of p r china httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa atractylodis rhizoma poria acori tatarinowii rhizoma plantaginis semen indigo naturalis phellodendri amurensis cortex talcum herba patriniae salviae miltiorrhizae radix et rhizoma ht Answer:

Colon_Cancer

"melatonin is a sleepregulating hormone created by the pineal glandand is released at night it has been found to have biological activityin almost all living anisms including plants animals and microbes itcan quickly enter cells through the bilipid bilayer and exhibit scavenging activity towards oxygen free radicals as well as antioxidant properties due to its low molecular weight and amphiphilic nature peripheral tissues have been found to show a high affinity towardsthis hormone melatonin and hence act on receptors and bindingsites studies reported by tan show that melatonin can alsobe produced in the mitochondria and hence tissue melatonin levelsare more than that of serum levels hence can be used as a moleculethat targets mitochondria the main physicochemical and biologicalproperties of melatonin are sleepinducing effects antioxidant behavior [“] anti‚ammatory activity [“] antiapoptotic effects and neuroprotective effects it also regulates variousŽ corresponding authoremail address renjithsbcollegeacin r thomas101016jmolliq2020114082 elsevier bv all rights reservedphysiological functions of the brain as melatonin can diffuse quicklythrough the bloodbrain barrier it is effectively used in the treatmentof brain injuries rapid eyemovement sleepbehavior disorder patients are managed by a combination treatment of melatoninand clonazepam [“] the sensing of bacteria through tolllikereceptor4 and regulation of bacteria through altered goblet cells andantimicrobial peptides are all involved in the anticolitic effects of melatonin in ‚ammatory bowel disease melatonin is involved in theaging process growth towards puberty and modulation of blood pressure this versatile compound blocks proangiogenic andantiangiogenic effects caused by docetaxel and vinorelbine which areantitumor drugs and it enhances their tumorfighting behavior this molecule can modify the redox state of the rat pancreaticstellate cellmelatonin is an endogenous hormone that is involved in circadianrhythm control it is inexpensive and safe as it has a significant effectas an antioxidant and anti‚ammatory melatonin chemically nacetyl5methoxytryptamine is a tryptophan derivative has multiplephysiological effects and can be used to treat many diseases related tovirus infections especially respiratory diseases in covid19 patientswith digestive complications melatonin has positive effects 0cn alzaqri of molecular liquids melatonin can thus be used as an adjunctive or even as a regular therapyas no antiviral treatment is currently available electrochemical measurements of melatonin overflow demonstrate that melatonin secretiondecreases with age melatonin treatments result in the enhancement of essential oil production in salvia species in the context of the recent covid pandemic melatonin can beresearched as a potential molecule to control the dangerous effect ofthis disease rising patients' tolerance and decreasing the mortality infatal virus infections would control the innate immune response and reduce ‚ammation during this period melatonin is a molecule with respective properties as it decreases the overreaction of the innateimmune response and overshoots ‚ammation but also facilitatesadaptive immune function even though melatonin is a critical biomolecule few works havebeen reported on the electronic structure and reactivity of this moleculeexcept for a preliminary work reported by turjanski and coworkers in using semiempirical methods in this manuscript we describe a detailed investigation into the quantum mechanical propertiesof melatonin its spectral features reactivity preferences and the resultsof docking studies with three known structural protein receptors of thenovel coronavirus2 we found that melatonin docks strongly with thethree proteins we hypothesise that this compound can be used as anadjuvant medicine for the treatment of covid19 also significant restby a person peacefully sleeping in dark surroundings will enhance theproduction of this hormone which could help in the management ofcurrent patients or as a preventive measure in the vulnerablepopulation material and methodsthe melatonin molecule was optimised using the gaussian09 software package with the dftb3lyp functional and the 6311g 2dp basis set b3lyp is a commonly used functional and 63112dpbasis set is mediumsized basis set with diffused functions over heavyatoms and polarization functions to bring accuracy we performed frequency calculations to ensure that no imaginary frequency exists suchthat the geometry determined would correspond to a global minimumfor reaching the optimised geometry we used the same geometry for calculating frontier molecular analysis natural bonding orbitals and nonlinear optical studies for uv“visible spectrum simulation we usedtimedependent density functional theory tddft with longrangecorrected camb3lyp functionals with 6311g 2dp as the basis setbecause electronic transitions are timedependent phenomena tddftcalculations are done using the optimised geometry obtained fromb3lyp6311g2dp simulations the frontier molecular orbitals wereviewed from the checkpoint file generated during the optimisation calculations a wavefunction file was generated during a single point groundstate calculation job using which the subsequent analysis performedthe melatonin molecule has more than two reaction sites for examplemethoxy carbonyl“amide and purine ring reaction sites of melatonincalculated using the multiwavefunction software for calculatingtotal electrostatic potential average localised ionization energies and noncovalent interactions as it is reported that melatonincan be used as an adjuvant therapeutic material to fight covid19 we decided to dock the molecule with three ncov2019 protein's rcsb site melatonin is effective in critical care patients by decreasing vessel permeability anxiety use of sedation and increasing the quality ofsleep which may also be beneficial to covid19 patients for improvedclinical outcome melatonin especially has a high health profile significant data indicate that melatonin reduces virusrelated diseases and willpossibly also be effective in patients with covid19 the target proteinswere downloaded cleaned removed alien atoms and molecules andthen used for docking the energy received from the swissdock software and the score values received from patchdock as well as thedocked results collected from biodiscovery studio software arepresented results and discussion geometry of melatoninthe geometry of the molecule explains its rigid structure thestructure of melatonin can be explained based on its physical parametersof bond lengths and bond angles between important atoms or groups asshown in fig the bond lengths of and arebonds of 1c14n 7c14n and 12h14n respectively and the corresponding bond angles are1090343° ° and ° for 1c14n7c 1c14n12h and 7c14n12h respectively the bond angle of° for 4c25o26c having bond lengths of and for 4c\\\\25o and 25o\\\\26c respectively the bond lengths of 18c21n21n22h 21n23c 23c\\\\24o and 23c\\\\30c are and respectively with the corresponding bondangles of ° ° ° ° ° and° for 18c21c22h 18c21c23c 22h22n23c 21n23c24o21n23c30c and 24o23c30c respectively frontier molecular orbital fmo properties of melatoninthe frontier molecular orbitals are highly reactive orbitals of othermolecules and some chemical descriptors are shown in table the higher occupied molecular orbital homo lower unoccupied molecular orbital lumo and energy gap of melatonin are ˆ’ˆ’ and kcalmol respectively the energygap is significant indicating that the molecule is inherently stable theionization energy electron affinity hardness softness chemical potential electronegativity electrophilicity index and nucleophilicity indexof melatonin are ˆ’ and kcalmol respectively interactionof the melatonin with the biological target can be explained by the softness value the softness value is high kcalmol indicatingthat the molecule can positively interact with biological systems andshow the desired effect electron transition study and excitedstate properties of melatonin insolutionthe electron transition study explains electrontransfer excitedstates we used the td“dft formalism using camb3lyp functionalsand 6311g 2dp basis sets in an implicit solvation atmosphere ofmethanol using the iefpcm model as transmission occurs some energyis also emitted melatonin electron transitions to homo having a pyrrole ring and oxygen in methoxy homo1 over the pyrrole ring andethyl carbons and homo2 over acetamide oxygen nitrogen andethyl carbons with energies of ˆ’ ˆ’ and ˆ’ ev respectively melatonin electron transitions to lumo which is over the pyrrolering lumo which is over the pyrrole ring oxygen in methoxyethyl carbons and acetamide nitrogen and carbon and lumo2 having acetamide carbons acetamide nitrogen and carbons with energiesof and ev respectively the electronic spectral datausing td“dft simulations indicate a significant λmax of nm in amethanol solvent the transitions are due to the movement of electronsfrom homo1 to lumo and homo to lumo with an oscillator strength of the electronic transitions are due to chargetransfer transitions from one region of the molecule to another whichindicates its inherent stability due to electronic excitations nonlinear optical behavior of melatoninscientists and technologists working in the molecular electronics fieldare continuously searching for compounds with substantial nonlinearoptical nlo activity such compounds find immense application in electronic displays surveillance equipment and consumer electronic gadgetscomputationally the ability of a molecule to act as an nlo material can be 0cn alzaqri of molecular liquids fig geometry of melatonindetermined from the polarizability and hyperpolarizability data [“]the nlo properties of melatonin are shown in table this is an essentialbehavior of melatonin that has a light absorption nature movement ofelectrons or protons as compared with a standard nlo material such asurea the dipole moment of melatonin is d which is times greater than urea hyperpolarizability mean polarizabilityand anisotropy of the polarizability of melatonin are and esu and which are and times greater than urea respectively the compound is not centrosymmetric hence generates secondorder spherical harmonics and betahyperpolarizability functions this compound can hence be used as an anic nonlinear optically active substance in anic electronicappliances nature of nbo study of melatonina molecule especially one with profound biological activity may havemany intramolecular electron delocalisation and hyperconjugativestabilisation regions natural bond orbital analysis which is a quantummechanical method is useful for this type of study the molecular orbitaltable frontier molecular orbital properties for melatoninchemical descriptorshomolumoionization energy i ɛhomo ˆ’homoelectron affinity a ɛlumo ˆ’lumoenergy gap homo ˆ’ lumoglobal hardness η i ˆ’ a global softness s ηchemical potential μ i a electronegativity χ ˆ’μelectrophilicity index ω μ2 2ηnucleophilicity index n ωenergy in kcalmolˆ’ˆ’ˆ’properties of melatonin for the occupancy of the natural orbitals wereperformed by the nbo suite embedded in the gaussian softwarefrom donorbonding orbital σ c1c2 with occupancy is toacceptor antibonding orbitals σ c3c4 σ c5c6 and σ c7c8exhibiting the transition the energies are and kcalmol respectively from σ c3c4 with occupancy is to σ c5c6 and the rydberg orbital r c30 with the energies are and kcalmol respectively from σ c5c6 having an occupancy is to σ c3c4 r and r h33 with the energies are and kcalmol respectively from σ n21c23 has occupancy to rydberg orbital r c30 and r h33 with the energies are and kcalmol respectively from σ c23o24 having the occupancy is to σ c1c2 σ c2c3 r c23 and r h33 withthe energies are and kcalmol respectivelyfrom σ c23c30 has occupancy is to r h17 r c18 rh19 r c23 r o24 r c30 r h33 σ c1c2 σ c1c6σ c2c3 σ c26h27 σ c30h31 σ c30h32 and σ c30h33 having the energies are and kcalmol respectively from σ o25c26 having occupancy is to r c30 andσ c1c2 with the energies are and kcalmol respectivelyfrom σ c26h27 to σ c1c2 having the energy kcalmol withthe occupancy is from σ c30h31 with the occupancy is to r h17 r c18 r h19 r c23 r o24 r c30 rh33 σ c1c2 σ c2c3 σ c23o24 σ c30h31 and σc30h32 having the energies are and kcalmol respectively from σ c30h32 with occupancy is to r h17 rc18 r h19 r c23 r o24 r c30 r h33 σ c1c2 σc2c3 σ c30h32 and σ c30h33 having the energies are and kcalmol respectively and from σ c30h33 with the occupancy is to r h17 r c18 r c19 r c23 r c24 ro25 r c26 r h28 r c30 r h33 σ c1c2 σ c1c6σ c2c3 σ c2c4 σ c18h19 σ c23o24 σ c26h27 σ 0cn alzaqri of molecular liquids table nonlinear optics property for melatoninnonlinear propertydipole moment μhyperpolarizability βmean polarizability α0anisotropy of the polarizability δαmelatonin d esu esu esuurea d esu esu esucomparison of melatonin with urea times greater than urea times greater than urea times greater than urea times greater than ureac30c31 σ c30h32 and σ c30h33 having the energies are and kcalmol respectivelyfrom core bonding orbital c c23 which has the occupancy electrons move to antibonding r c23 r c30 and r h33 withthe transition energies are and kcalmol respectively from c c23 with the occupancy is to r h33 σ c2c8 and σ c26h28 having the energies are and kcalmol respectively from c o24 to σ c26h29 has the energy is kcalmol with occupancy is and from c c30 having the occupancy is to r h17 r c23 r c30 r h33 σc2c8 σ c26h28 σ c30h31 and σ c30h32 with the energies are and kcalmol respectively from lone pair orbital n n14 with the occupancy is to σ c7c8 with the energy kcalmol from nn21 has the occupancy is to σ c23o24 having the energy kcalmol and from n o24 having the occupancy is to rc23 r c30 r h33 σ c1c2 and σ c2c3 with the energiesare and kcalmol respectively fromantibonding orbital σ c1c2 having the occupancy to rc30 and σ c2c3 with the energies are and kcalmolrespectively from σ c5c6 has occupancy is to r c30 withthe energy is kcalmol and from σ c23o24 has occupancy is to r c30 with the energy is kcalmol the inherentstabilisation offrom the series ofhyperconjugative interactions presented above these interactions canalso be between the melatonin and the surrounding solvent moleculeswhich reveals its stabilisation in biological medium and also betweenthe molecule and the target proteins used in the dockingthe molecule is evident total electrostatic potentials esp and average localised ionization energy alie of melatoninthe electrostatic potential explains how reactive sites canundergo nucleophilic or electrophilic addition or substitution reactionsof melatonin shown in fig within bohr and a color changefrom blue to red indicates charges on elements from ˆ’ to the blue color appears in both methoxyoxygen and acetamideoxygen these are electronrich sites and electrophiles can quickly attack them the red color appears on all of the hydrogens these areelectronpoor sites and nucleophiles can quickly attack themthe alie clarifies the stability of any molecule based on saturatedand unsaturated bond electron movements which are localised ordelocalised the number of the resonance structure is proportionalto the stability of the molecule the alie of melatonin shown in fig iswithin the range of ± bohr color is from indigo to red and the numerical value is from to the blue color of protons in themethoxy group three protons in the sixmembered ring in the indolegroup methyl protons and both adjacent carbons in the acetamidegroup are all sites that act as electrophiles the red color ofacetamide‘carbon conjugated carbon with oxygen atoms and bothacetamideamide and methoxy groups are all sites that act as nucleophiles these blue and red regions represent saturated bonds thebluishgreen regions are on indole rings to methoxy carbons via oxygenand acetamide chains this indicates that delocalised electrons and unsaturated bonds lead to several resonance structures and explains thestability of melatonin the electrophilic and nucleophilic reactive centres identified above interact with the covid virus proteins and providevarious electrostatic and noncovalent interactions and increases drugaffinity noncovalent interaction nci properties of melatoninnoncovalent interactions are a valuable biological property of molecules and are nonbonded directly but are bound by some forces suchas hydrogen bonding van der waals bonding andor steric constraintsnoncovalent interactions of melatonin are shown in fig plotted as agraph with energy plotted versus a reduced density gradient hydrogen bonds appear from ˆ’ to ˆ’ au between oxygenand protons from the acetamide group the van der waals force rangesfig electrostatic potentials of melatoninfig average localised ionization energy for melatonin 0cn alzaqri of molecular liquids fig noncovalent interactions of melatoninfrom ˆ’ to au between acetamideoxygen and its adjacentprotons in both methyl and methoxy groups the steric force rangesfrom to au between the indole ring the methyl groupand the carbonylamide group noncovalent interactions are a groupof interactions like hydrogen bond pistacking hydrophobic interactions van der waal's forces iondipole interactions and dipoledipoleinteractions responsible for the stabilisation of the molecule and thedocking between melatonin and the covid proteins molecular dockingmolecular docking is one of the essential functions of biologically active molecules this is the theoretical evidence to design the structureand reactivity relationship of a molecule at present the covid19 pandemic caused by a new strain of the coronavirus is creating havocthroughout the world we made efforts to dock the melatonin withthe three proteins isolated from the virus represented through thepdb id 6lu7 6m03 and 6w63 were deposited in the database as mentioned in the methodology sectionwith the rapid spread of the novel coronavirus globally the designof vaccines is of great importance sarscov2 is an enveloped nonsegmented and single stranded positive sense rna virus the bestdrug target among coronaviruses is the main protease mpro also called3cl protease this is a key coronavirus enzyme and plays a vitalrole in mediating viral replication and transcription it is identified ashaving a mechanismbased inhibitor the main targeting protease protein pdb 6lu7 is widely studied a series of frontier molecular orbital based interaction analyseswere performed on the complex between the main protease ofcovid19 and the peptidelike inhibitor whose fundamental structure was obtained from the protein pdb 6lu7 anothertargeted protease protein in an apo form pdb 6m03 shows themost stable form after binding with the selected drug threonine residue with the help of several covalent bond interactionwith a ˆ’ kcalmol docking affinity lasinavir brecanavirtelinavir rotigaptide 13bis2ethoxycarbonylchromon5yloxy2lysyloxypropane and pimelautide can be consideredas the main protease inhibitors of covid19 by docking them tothe binding cavities of apo pdb 6m03 and holo pdb 6lu7 another protease protein pdb 6w63 is a reversible inhibitorthe flavonoid narcissoside is reported to have a high affinity towards the protease protein pdb 6w63 according to moleculardocking studies thus these three protease proteins pdb 6lu7pdb 6m03 and pdb 6w63 can be included in the category ofnonstructural proteins in the structure of sarscov2 from table the result from swissdock explains the biological activity of melatonin with coronavirus proteins pdb id 6lu7 6m03and 6w63 in general the total δg is more than ˆ’ kcalmol isright active luckily melatonin has a total δg of ˆ’ ˆ’ andˆ’ kcalmol with coronavirus2 proteins pdb id 6lu7 6m03 and6w63 respectively and the total δg is directly proportional to the fullfitness energy values which are ˆ’ ˆ’ and kcalmol respectively it can also be seen from this table theinterfull fitness intrafull fitness full solvent fitness full surface fitnessδg complex polar solvent δg complex nonpolar solvent δg proteinpolar solvent δg protein nonpolar solvent δg ligand polar solventδg ligand nonpolar solvent δg van der waals force and δg electricforce relationships between melatonin and coronavirus2 proteins asreferredthe result from patchdock are as follows the score values are and total surface interacting area and and the minimum atomic contact energies are ˆ’ˆ’ and ˆ’ kcalmol for melatonin with coronavirus2 proteins pdb id 6lu7 6m03 and 6w63 respectively figs and s1show the skeletal structure and protein residue interactions betweenmelatonin and coronavirus2 protein pdb id 6lu7 6m03 and 6w63table explains what protein residues are interacting with melatoninand details the residue names labels hydrophobic values pka valuesaverage isotropic displacements secondary structures residue solventaccessibility sidechain solvent accessibility percent solvent accessibility and percent sidechain solvent accessibility values of coronavirus2proteinstable and fig s2 show the residue structure of the favorable nonbond interactions between melatonin and coronavirus2 proteinstable lists favorable nonbond interactions of 6lu7 having conventional hydrogen bonds carbonhydrogen bonds pidonor hydrogenbonds pisulfur and pialkyl with melatonin 6m03 has pisigmapipi tshaped and pialkyl with melatonin while 6w63 has pipi tshaped pialkyl and pialkyl with melatonin along with the bond distance from chemistry fig s2 and table show the residue structure 0cn alzaqri of molecular liquids table swissdock result for melatonin with coronovirus2 proteins pdb id 6lu7 6m03 and 6w63energysimple fitnessfull fitnessinter full fitnessintra full fitnesssolvent full fitnesssurface full fitnessextra full fitnessδg complex solvent polarδg complex solvent nonpolarδg protein solvent polarδg protein solvent nonpolarδg ligand solvent polarδg ligand solvent nonpolarδg van der waals forceδg electric forcetotal δg6lu7ˆ’ kcalmolˆ’ kcalmolˆ’ kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol 6m03ˆ’ kcalmolˆ’ kcalmolˆ’ kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol6w63ˆ’ kcalmolˆ’ kcalmolˆ’ kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmolof the unfavorable nonbond interactions between melatonin and coronavirus2 proteins protein 6lu7 does not have any unfavorablestericinteractions protein 6m03 having three unfavorable nonbond interactions and protein 6w63 having unfavorable bumpnonbond interactions fig s2 and table show unsatisfied bonds within melatonininteracting with coronavirus proteins when interacting protein 6lu7has one hydrogen donor and one oxygen acceptor protein 6m03 hastwo hydrogen donors and two oxygen acceptors and protein 6w63has one hydrogen donor and two oxygen acceptors with melatonin table shows noncovalent interactions between melatonin and coronavirus2 proteins hydrophobic groups of protein 6lu7 residues areahis41 aleu141 acys145 ahis164 amet165 and aglu166those of protein 6m03 residues are ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu167 and those ofprotein residues 6w63 are ahis41 acys44 amet49 aleu50 amet165 aglu166 aleu167 and agln189 with melatonin as shownin fig s2 and table the hydrophilic groups of protein 6lu7 residuesare ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189 those of protein 6m03 residues are ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189 andthose of protein 6w63 residues are ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192 with melatonin as shownin fig s3 and table neutral groups of protein 6lu7 residues are atyr54 agly143 and aser144 those of protein 6m03 residues are agly143 aser144 and apro168 and those of protein 6w63 residuesare apro52 atyr54 aarg188 and athr190 with melatonin asshown in fig s4 and table acidic groups of protein 6lu7 residuesare aglu166 and aasp187 that of protein 6m03 residue is aglu166 and protein 6w63 residues are aglu166 and aasp187 withmelatonin as shown in table and fig s5 basic group interactions ofprotein 6lu7 residues are ahis41 ahis164 ahis172 and aarg188those of protein 6m03 residues are ahis41 ahis163 ahis164 andahis172 and those of protein 6w63 residues are ahis41 ahis164and aarg188 as shown in table and fig s6 tan etal has shownthat melatonin and derivatives has excellent biological responses likeacting against oxidative stress and free radical scavenging [“]our studies show that melatonin molecule can interact with differentproteins present in the ncov19 virus and inhibit their proliferationthese results need further clinical follow up and could assist in the management of covid pneumonia conclusionswe conducted a detailed quantummechanical investigation of thehormone melatonin and regulation of the sleepwake cycle naturalbonding orbital studies revealed the intensity of several intramolecularinteractions the various frontier molecular orbital data explain the nature and physical parameters of melatonin and the nonlinear opticalproperties are compared with urea which is a standard materialfig skeletal structure of interactions between melatonin and 6lu7 a 6m03 b and 6w63 c coronavirus2 protein residues 0cn alzaqri of molecular liquids table interactions between melatonin and coronavirus2 protein residuesnamelabelhydrophobicitypkapdbidsavg isotropicdisplacementsecondarystructureresidue solventaccessibilitysidechain solventaccessibilitypercent solventaccessibilitypercent sidechainsolvent accessibility6m03 histidine6lu7 histidineahis41amet49methionineatyr54tyrosinealeu141leucineaasn142asparagineagly143glycineaser144serineacys145cysteineahis164histidinemethionineamet165glutamic acid aglu166ahis172histidineaasp187aspartic acidaarg188arginineglutamineagln189ahis41methionineamet49phenylalanine aphe140aleu141leucineaasn142asparagineglycineagly143aser144serineacys145cysteineahis163histidineahis164histidinemethionineamet165glutamic acid aglu166aleu167leucineapro168prolineahis172histidineagln189glutamine6w63 histidineahis41cysteineacys44methionineamet49leucinealeu50prolineapro52tyrosineatyr54histidineahis164methionineamet165glutamic acid aglu166aleu167leucineapro168prolineaasp187aspartic acidarginineaarg188agln189glutamineathr190threonineglutamineagln192ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’““““““““““““““““““““““““““helixturnhelixcoilturnturncoilturnsheetsheetsheetsheetcoilcoilcoilhelixhelixcoilcoilturnturncoilturnsheetsheetsheetsheetcoilturnsheetcoilhelixcoilcoilcoilcoilhelixsheetsheetsheetcoilturncoilcoilcoilcoilcoilwavefunction studies gave information about electrostatic potentialsaverage localised ionization and noncovalent interactions these datahelped to predict the reactivity and identify the active site of the reactivity of the molecule melatonin docks with novel coronavirus proteinsand shows a variety of interactions with an excellent docking scorewhich leads to inhibition of the virus proteins leading to its destructionhence clinicians can consider incorporating melatonin also in thecovid19 treatment regime after further studiestable favorable nonbond interactions between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63hydrogen bondhydrogen bondhydrogen bondhydrogen bond otherhydrophobichydrophobichydrophobichydrophobichydrogen bondhydrophobichydrophobichydrophobicconventional hydrogen bondconventional hydrogen bondcarbon hydrogen bondpidonor hydrogen bond pisulfurpialkylpisigmapipi tshapedpialkylconventional hydrogen bondpipi tshapedpialkylpialkylaglu166nunk0hunk0hacys145sgunk0amet165caahis41unk0unk0hahis41unk0unk0hdonorhdonorhdonorhdonor sulfurpiorbitalschpiorbitalspiorbitalshdonorpiorbitalspiorbitalspiorbitalsunk0oahis164oaglu166ounk0acys145unk0unk0acys145aglu166ounk0amet165amet165hacceptorhacceptorhacceptorpiorbitals piorbitalsalkylpiorbitalspiorbitalsalkylhacceptorpiorbitalsalkylalkyl 0cn alzaqri of molecular liquids table unfavorable nonbond between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63nilunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorable bumpunfavorable bumpunfavorable bump carbon hydrogen bondunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpacys145sgaglu166ounk0hagln189caagln189caagln189cbagln189cgagln189cgagln189cgagln189cgagln189cdagln189cdagln189cdagln189cdagln189oe1agln189oe1agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189haagln189haagln189haagln189hg1agln189hg2agln189hg2agln189he21agln189he21agln189he21agln189he21agln189he22agln189he22agln189he22stericstericsteric hdonorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericunk0cunk0caglu166ounk0cunk0hunk0cunk0cunk0hunk0nunk0hunk0cunk0cunk0hunk0hunk0cunk0hunk0nunk0cunk0ounk0cunk0hunk0hunk0hunk0cunk0hunk0hunk0hunk0cunk0hunk0nunk0cunk0cunk0hunk0cunk0cunk0hstericstericsteric hacceptorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericsterictable unsatisfied bonds in melatonin with coronavirus2 proteinspdb ids6lu76m036w63nameunk0hunk0ounk0hunk0hunk0ounk0ounk0hunk0ounk0oatomunsatisfied typehohhoohoodonoracceptordonordonoracceptoracceptordonoracceptoracceptorrenjith thomas conceptualization formal analysis investigation methodology project administration resources softwaresupervision validation visualization writing review editingdeclaration of competing interestthe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to ‚uence the work reported in this paperacknowledgementsresearchers supporting project number rsp202078 king saudcredit authorship contribution statementuniversity riyadh saudi arabianabil alzaqri cenceptualization funding acquisition tpooventhiran investegation methodology original draft alialsalme investegation original draft ismail warad resourcesreview methods athira m john methodology writing draftappendix a supplementary datasupplementary data to this article can be found online at 101016jmolliq2020114082table noncovalent interactions between melatonin and coronavirus2 proteinshydrophobicityhydrophilicityneutral groupacidic groupbasic grouppdbids6lu7ahis41 aleu141 acys145 ahis164 amet165 and aglu1666m03 ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu1676w63 ahis41 acys44 amet49 aleu50 amet165aglu166 aleu167 and agln189ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192atyr54 agly143 andaser144agly143 aser144 andapro168apro52 atyr54 aarg188 and athr190aglu166 andaasp187aglu166aglu166and aasp187ahis41 ahis164 ahis172 and aarg188ahis41 ahis163 ahis164 and ahis172ahis41 ahis164 andaarg188

cancer7515

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "melatonin is a sleepregulating hormone created by the pineal glandand is released at night it has been found to have biological activityin almost all living anisms including plants animals and microbes itcan quickly enter cells through the bilipid bilayer and exhibit scavenging activity towards oxygen free radicals as well as antioxidant properties due to its low molecular weight and amphiphilic nature peripheral tissues have been found to show a high affinity towardsthis hormone melatonin and hence act on receptors and bindingsites studies reported by tan show that melatonin can alsobe produced in the mitochondria and hence tissue melatonin levelsare more than that of serum levels hence can be used as a moleculethat targets mitochondria the main physicochemical and biologicalproperties of melatonin are sleepinducing effects antioxidant behavior [“] anti‚ammatory activity [“] antiapoptotic effects and neuroprotective effects it also regulates variousŽ corresponding authoremail address renjithsbcollegeacin r thomas101016jmolliq2020114082 elsevier bv all rights reservedphysiological functions of the brain as melatonin can diffuse quicklythrough the bloodbrain barrier it is effectively used in the treatmentof brain injuries rapid eyemovement sleepbehavior disorder patients are managed by a combination treatment of melatoninand clonazepam [“] the sensing of bacteria through tolllikereceptor4 and regulation of bacteria through altered goblet cells andantimicrobial peptides are all involved in the anticolitic effects of melatonin in ‚ammatory bowel disease melatonin is involved in theaging process growth towards puberty and modulation of blood pressure this versatile compound blocks proangiogenic andantiangiogenic effects caused by docetaxel and vinorelbine which areantitumor drugs and it enhances their tumorfighting behavior this molecule can modify the redox state of the rat pancreaticstellate cellmelatonin is an endogenous hormone that is involved in circadianrhythm control it is inexpensive and safe as it has a significant effectas an antioxidant and anti‚ammatory melatonin chemically nacetyl5methoxytryptamine is a tryptophan derivative has multiplephysiological effects and can be used to treat many diseases related tovirus infections especially respiratory diseases in covid19 patientswith digestive complications melatonin has positive effects 0cn alzaqri of molecular liquids melatonin can thus be used as an adjunctive or even as a regular therapyas no antiviral treatment is currently available electrochemical measurements of melatonin overflow demonstrate that melatonin secretiondecreases with age melatonin treatments result in the enhancement of essential oil production in salvia species in the context of the recent covid pandemic melatonin can beresearched as a potential molecule to control the dangerous effect ofthis disease rising patients' tolerance and decreasing the mortality infatal virus infections would control the innate immune response and reduce ‚ammation during this period melatonin is a molecule with respective properties as it decreases the overreaction of the innateimmune response and overshoots ‚ammation but also facilitatesadaptive immune function even though melatonin is a critical biomolecule few works havebeen reported on the electronic structure and reactivity of this moleculeexcept for a preliminary work reported by turjanski and coworkers in using semiempirical methods in this manuscript we describe a detailed investigation into the quantum mechanical propertiesof melatonin its spectral features reactivity preferences and the resultsof docking studies with three known structural protein receptors of thenovel coronavirus2 we found that melatonin docks strongly with thethree proteins we hypothesise that this compound can be used as anadjuvant medicine for the treatment of covid19 also significant restby a person peacefully sleeping in dark surroundings will enhance theproduction of this hormone which could help in the management ofcurrent patients or as a preventive measure in the vulnerablepopulation material and methodsthe melatonin molecule was optimised using the gaussian09 software package with the dftb3lyp functional and the 6311g 2dp basis set b3lyp is a commonly used functional and 63112dpbasis set is mediumsized basis set with diffused functions over heavyatoms and polarization functions to bring accuracy we performed frequency calculations to ensure that no imaginary frequency exists suchthat the geometry determined would correspond to a global minimumfor reaching the optimised geometry we used the same geometry for calculating frontier molecular analysis natural bonding orbitals and nonlinear optical studies for uv“visible spectrum simulation we usedtimedependent density functional theory tddft with longrangecorrected camb3lyp functionals with 6311g 2dp as the basis setbecause electronic transitions are timedependent phenomena tddftcalculations are done using the optimised geometry obtained fromb3lyp6311g2dp simulations the frontier molecular orbitals wereviewed from the checkpoint file generated during the optimisation calculations a wavefunction file was generated during a single point groundstate calculation job using which the subsequent analysis performedthe melatonin molecule has more than two reaction sites for examplemethoxy carbonyl“amide and purine ring reaction sites of melatonincalculated using the multiwavefunction software for calculatingtotal electrostatic potential average localised ionization energies and noncovalent interactions as it is reported that melatonincan be used as an adjuvant therapeutic material to fight covid19 we decided to dock the molecule with three ncov2019 protein's rcsb site melatonin is effective in critical care patients by decreasing vessel permeability anxiety use of sedation and increasing the quality ofsleep which may also be beneficial to covid19 patients for improvedclinical outcome melatonin especially has a high health profile significant data indicate that melatonin reduces virusrelated diseases and willpossibly also be effective in patients with covid19 the target proteinswere downloaded cleaned removed alien atoms and molecules andthen used for docking the energy received from the swissdock software and the score values received from patchdock as well as thedocked results collected from biodiscovery studio software arepresented results and discussion geometry of melatoninthe geometry of the molecule explains its rigid structure thestructure of melatonin can be explained based on its physical parametersof bond lengths and bond angles between important atoms or groups asshown in fig the bond lengths of and arebonds of 1c14n 7c14n and 12h14n respectively and the corresponding bond angles are1090343° ° and ° for 1c14n7c 1c14n12h and 7c14n12h respectively the bond angle of° for 4c25o26c having bond lengths of and for 4c\\\\25o and 25o\\\\26c respectively the bond lengths of 18c21n21n22h 21n23c 23c\\\\24o and 23c\\\\30c are and respectively with the corresponding bondangles of ° ° ° ° ° and° for 18c21c22h 18c21c23c 22h22n23c 21n23c24o21n23c30c and 24o23c30c respectively frontier molecular orbital fmo properties of melatoninthe frontier molecular orbitals are highly reactive orbitals of othermolecules and some chemical descriptors are shown in table the higher occupied molecular orbital homo lower unoccupied molecular orbital lumo and energy gap of melatonin are ˆ’ˆ’ and kcalmol respectively the energygap is significant indicating that the molecule is inherently stable theionization energy electron affinity hardness softness chemical potential electronegativity electrophilicity index and nucleophilicity indexof melatonin are ˆ’ and kcalmol respectively interactionof the melatonin with the biological target can be explained by the softness value the softness value is high kcalmol indicatingthat the molecule can positively interact with biological systems andshow the desired effect electron transition study and excitedstate properties of melatonin insolutionthe electron transition study explains electrontransfer excitedstates we used the td“dft formalism using camb3lyp functionalsand 6311g 2dp basis sets in an implicit solvation atmosphere ofmethanol using the iefpcm model as transmission occurs some energyis also emitted melatonin electron transitions to homo having a pyrrole ring and oxygen in methoxy homo1 over the pyrrole ring andethyl carbons and homo2 over acetamide oxygen nitrogen andethyl carbons with energies of ˆ’ ˆ’ and ˆ’ ev respectively melatonin electron transitions to lumo which is over the pyrrolering lumo which is over the pyrrole ring oxygen in methoxyethyl carbons and acetamide nitrogen and carbon and lumo2 having acetamide carbons acetamide nitrogen and carbons with energiesof and ev respectively the electronic spectral datausing td“dft simulations indicate a significant λmax of nm in amethanol solvent the transitions are due to the movement of electronsfrom homo1 to lumo and homo to lumo with an oscillator strength of the electronic transitions are due to chargetransfer transitions from one region of the molecule to another whichindicates its inherent stability due to electronic excitations nonlinear optical behavior of melatoninscientists and technologists working in the molecular electronics fieldare continuously searching for compounds with substantial nonlinearoptical nlo activity such compounds find immense application in electronic displays surveillance equipment and consumer electronic gadgetscomputationally the ability of a molecule to act as an nlo material can be 0cn alzaqri of molecular liquids fig geometry of melatonindetermined from the polarizability and hyperpolarizability data [“]the nlo properties of melatonin are shown in table this is an essentialbehavior of melatonin that has a light absorption nature movement ofelectrons or protons as compared with a standard nlo material such asurea the dipole moment of melatonin is d which is times greater than urea hyperpolarizability mean polarizabilityand anisotropy of the polarizability of melatonin are and esu and which are and times greater than urea respectively the compound is not centrosymmetric hence generates secondorder spherical harmonics and betahyperpolarizability functions this compound can hence be used as an anic nonlinear optically active substance in anic electronicappliances nature of nbo study of melatonina molecule especially one with profound biological activity may havemany intramolecular electron delocalisation and hyperconjugativestabilisation regions natural bond orbital analysis which is a quantummechanical method is useful for this type of study the molecular orbitaltable frontier molecular orbital properties for melatoninchemical descriptorshomolumoionization energy i ɛhomo ˆ’homoelectron affinity a ɛlumo ˆ’lumoenergy gap homo ˆ’ lumoglobal hardness η i ˆ’ a global softness s ηchemical potential μ i a electronegativity χ ˆ’μelectrophilicity index ω μ2 2ηnucleophilicity index n ωenergy in kcalmolˆ’ˆ’ˆ’properties of melatonin for the occupancy of the natural orbitals wereperformed by the nbo suite embedded in the gaussian softwarefrom donorbonding orbital σ c1c2 with occupancy is toacceptor antibonding orbitals σ c3c4 σ c5c6 and σ c7c8exhibiting the transition the energies are and kcalmol respectively from σ c3c4 with occupancy is to σ c5c6 and the rydberg orbital r c30 with the energies are and kcalmol respectively from σ c5c6 having an occupancy is to σ c3c4 r and r h33 with the energies are and kcalmol respectively from σ n21c23 has occupancy to rydberg orbital r c30 and r h33 with the energies are and kcalmol respectively from σ c23o24 having the occupancy is to σ c1c2 σ c2c3 r c23 and r h33 withthe energies are and kcalmol respectivelyfrom σ c23c30 has occupancy is to r h17 r c18 rh19 r c23 r o24 r c30 r h33 σ c1c2 σ c1c6σ c2c3 σ c26h27 σ c30h31 σ c30h32 and σ c30h33 having the energies are and kcalmol respectively from σ o25c26 having occupancy is to r c30 andσ c1c2 with the energies are and kcalmol respectivelyfrom σ c26h27 to σ c1c2 having the energy kcalmol withthe occupancy is from σ c30h31 with the occupancy is to r h17 r c18 r h19 r c23 r o24 r c30 rh33 σ c1c2 σ c2c3 σ c23o24 σ c30h31 and σc30h32 having the energies are and kcalmol respectively from σ c30h32 with occupancy is to r h17 rc18 r h19 r c23 r o24 r c30 r h33 σ c1c2 σc2c3 σ c30h32 and σ c30h33 having the energies are and kcalmol respectively and from σ c30h33 with the occupancy is to r h17 r c18 r c19 r c23 r c24 ro25 r c26 r h28 r c30 r h33 σ c1c2 σ c1c6σ c2c3 σ c2c4 σ c18h19 σ c23o24 σ c26h27 σ 0cn alzaqri of molecular liquids table nonlinear optics property for melatoninnonlinear propertydipole moment μhyperpolarizability βmean polarizability α0anisotropy of the polarizability δαmelatonin d esu esu esuurea d esu esu esucomparison of melatonin with urea times greater than urea times greater than urea times greater than urea times greater than ureac30c31 σ c30h32 and σ c30h33 having the energies are and kcalmol respectivelyfrom core bonding orbital c c23 which has the occupancy electrons move to antibonding r c23 r c30 and r h33 withthe transition energies are and kcalmol respectively from c c23 with the occupancy is to r h33 σ c2c8 and σ c26h28 having the energies are and kcalmol respectively from c o24 to σ c26h29 has the energy is kcalmol with occupancy is and from c c30 having the occupancy is to r h17 r c23 r c30 r h33 σc2c8 σ c26h28 σ c30h31 and σ c30h32 with the energies are and kcalmol respectively from lone pair orbital n n14 with the occupancy is to σ c7c8 with the energy kcalmol from nn21 has the occupancy is to σ c23o24 having the energy kcalmol and from n o24 having the occupancy is to rc23 r c30 r h33 σ c1c2 and σ c2c3 with the energiesare and kcalmol respectively fromantibonding orbital σ c1c2 having the occupancy to rc30 and σ c2c3 with the energies are and kcalmolrespectively from σ c5c6 has occupancy is to r c30 withthe energy is kcalmol and from σ c23o24 has occupancy is to r c30 with the energy is kcalmol the inherentstabilisation offrom the series ofhyperconjugative interactions presented above these interactions canalso be between the melatonin and the surrounding solvent moleculeswhich reveals its stabilisation in biological medium and also betweenthe molecule and the target proteins used in the dockingthe molecule is evident total electrostatic potentials esp and average localised ionization energy alie of melatoninthe electrostatic potential explains how reactive sites canundergo nucleophilic or electrophilic addition or substitution reactionsof melatonin shown in fig within bohr and a color changefrom blue to red indicates charges on elements from ˆ’ to the blue color appears in both methoxyoxygen and acetamideoxygen these are electronrich sites and electrophiles can quickly attack them the red color appears on all of the hydrogens these areelectronpoor sites and nucleophiles can quickly attack themthe alie clarifies the stability of any molecule based on saturatedand unsaturated bond electron movements which are localised ordelocalised the number of the resonance structure is proportionalto the stability of the molecule the alie of melatonin shown in fig iswithin the range of ± bohr color is from indigo to red and the numerical value is from to the blue color of protons in themethoxy group three protons in the sixmembered ring in the indolegroup methyl protons and both adjacent carbons in the acetamidegroup are all sites that act as electrophiles the red color ofacetamide‘carbon conjugated carbon with oxygen atoms and bothacetamideamide and methoxy groups are all sites that act as nucleophiles these blue and red regions represent saturated bonds thebluishgreen regions are on indole rings to methoxy carbons via oxygenand acetamide chains this indicates that delocalised electrons and unsaturated bonds lead to several resonance structures and explains thestability of melatonin the electrophilic and nucleophilic reactive centres identified above interact with the covid virus proteins and providevarious electrostatic and noncovalent interactions and increases drugaffinity noncovalent interaction nci properties of melatoninnoncovalent interactions are a valuable biological property of molecules and are nonbonded directly but are bound by some forces suchas hydrogen bonding van der waals bonding andor steric constraintsnoncovalent interactions of melatonin are shown in fig plotted as agraph with energy plotted versus a reduced density gradient hydrogen bonds appear from ˆ’ to ˆ’ au between oxygenand protons from the acetamide group the van der waals force rangesfig electrostatic potentials of melatoninfig average localised ionization energy for melatonin 0cn alzaqri of molecular liquids fig noncovalent interactions of melatoninfrom ˆ’ to au between acetamideoxygen and its adjacentprotons in both methyl and methoxy groups the steric force rangesfrom to au between the indole ring the methyl groupand the carbonylamide group noncovalent interactions are a groupof interactions like hydrogen bond pistacking hydrophobic interactions van der waal's forces iondipole interactions and dipoledipoleinteractions responsible for the stabilisation of the molecule and thedocking between melatonin and the covid proteins molecular dockingmolecular docking is one of the essential functions of biologically active molecules this is the theoretical evidence to design the structureand reactivity relationship of a molecule at present the covid19 pandemic caused by a new strain of the coronavirus is creating havocthroughout the world we made efforts to dock the melatonin withthe three proteins isolated from the virus represented through thepdb id 6lu7 6m03 and 6w63 were deposited in the database as mentioned in the methodology sectionwith the rapid spread of the novel coronavirus globally the designof vaccines is of great importance sarscov2 is an enveloped nonsegmented and single stranded positive sense rna virus the bestdrug target among coronaviruses is the main protease mpro also called3cl protease this is a key coronavirus enzyme and plays a vitalrole in mediating viral replication and transcription it is identified ashaving a mechanismbased inhibitor the main targeting protease protein pdb 6lu7 is widely studied a series of frontier molecular orbital based interaction analyseswere performed on the complex between the main protease ofcovid19 and the peptidelike inhibitor whose fundamental structure was obtained from the protein pdb 6lu7 anothertargeted protease protein in an apo form pdb 6m03 shows themost stable form after binding with the selected drug threonine residue with the help of several covalent bond interactionwith a ˆ’ kcalmol docking affinity lasinavir brecanavirtelinavir rotigaptide 13bis2ethoxycarbonylchromon5yloxy2lysyloxypropane and pimelautide can be consideredas the main protease inhibitors of covid19 by docking them tothe binding cavities of apo pdb 6m03 and holo pdb 6lu7 another protease protein pdb 6w63 is a reversible inhibitorthe flavonoid narcissoside is reported to have a high affinity towards the protease protein pdb 6w63 according to moleculardocking studies thus these three protease proteins pdb 6lu7pdb 6m03 and pdb 6w63 can be included in the category ofnonstructural proteins in the structure of sarscov2 from table the result from swissdock explains the biological activity of melatonin with coronavirus proteins pdb id 6lu7 6m03and 6w63 in general the total δg is more than ˆ’ kcalmol isright active luckily melatonin has a total δg of ˆ’ ˆ’ andˆ’ kcalmol with coronavirus2 proteins pdb id 6lu7 6m03 and6w63 respectively and the total δg is directly proportional to the fullfitness energy values which are ˆ’ ˆ’ and kcalmol respectively it can also be seen from this table theinterfull fitness intrafull fitness full solvent fitness full surface fitnessδg complex polar solvent δg complex nonpolar solvent δg proteinpolar solvent δg protein nonpolar solvent δg ligand polar solventδg ligand nonpolar solvent δg van der waals force and δg electricforce relationships between melatonin and coronavirus2 proteins asreferredthe result from patchdock are as follows the score values are and total surface interacting area and and the minimum atomic contact energies are ˆ’ˆ’ and ˆ’ kcalmol for melatonin with coronavirus2 proteins pdb id 6lu7 6m03 and 6w63 respectively figs and s1show the skeletal structure and protein residue interactions betweenmelatonin and coronavirus2 protein pdb id 6lu7 6m03 and 6w63table explains what protein residues are interacting with melatoninand details the residue names labels hydrophobic values pka valuesaverage isotropic displacements secondary structures residue solventaccessibility sidechain solvent accessibility percent solvent accessibility and percent sidechain solvent accessibility values of coronavirus2proteinstable and fig s2 show the residue structure of the favorable nonbond interactions between melatonin and coronavirus2 proteinstable lists favorable nonbond interactions of 6lu7 having conventional hydrogen bonds carbonhydrogen bonds pidonor hydrogenbonds pisulfur and pialkyl with melatonin 6m03 has pisigmapipi tshaped and pialkyl with melatonin while 6w63 has pipi tshaped pialkyl and pialkyl with melatonin along with the bond distance from chemistry fig s2 and table show the residue structure 0cn alzaqri of molecular liquids table swissdock result for melatonin with coronovirus2 proteins pdb id 6lu7 6m03 and 6w63energysimple fitnessfull fitnessinter full fitnessintra full fitnesssolvent full fitnesssurface full fitnessextra full fitnessδg complex solvent polarδg complex solvent nonpolarδg protein solvent polarδg protein solvent nonpolarδg ligand solvent polarδg ligand solvent nonpolarδg van der waals forceδg electric forcetotal δg6lu7ˆ’ kcalmolˆ’ kcalmolˆ’ kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol 6m03ˆ’ kcalmolˆ’ kcalmolˆ’ kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol6w63ˆ’ kcalmolˆ’ kcalmolˆ’ kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmol kcalmolˆ’ kcalmolof the unfavorable nonbond interactions between melatonin and coronavirus2 proteins protein 6lu7 does not have any unfavorablestericinteractions protein 6m03 having three unfavorable nonbond interactions and protein 6w63 having unfavorable bumpnonbond interactions fig s2 and table show unsatisfied bonds within melatonininteracting with coronavirus proteins when interacting protein 6lu7has one hydrogen donor and one oxygen acceptor protein 6m03 hastwo hydrogen donors and two oxygen acceptors and protein 6w63has one hydrogen donor and two oxygen acceptors with melatonin table shows noncovalent interactions between melatonin and coronavirus2 proteins hydrophobic groups of protein 6lu7 residues areahis41 aleu141 acys145 ahis164 amet165 and aglu166those of protein 6m03 residues are ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu167 and those ofprotein residues 6w63 are ahis41 acys44 amet49 aleu50 amet165 aglu166 aleu167 and agln189 with melatonin as shownin fig s2 and table the hydrophilic groups of protein 6lu7 residuesare ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189 those of protein 6m03 residues are ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189 andthose of protein 6w63 residues are ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192 with melatonin as shownin fig s3 and table neutral groups of protein 6lu7 residues are atyr54 agly143 and aser144 those of protein 6m03 residues are agly143 aser144 and apro168 and those of protein 6w63 residuesare apro52 atyr54 aarg188 and athr190 with melatonin asshown in fig s4 and table acidic groups of protein 6lu7 residuesare aglu166 and aasp187 that of protein 6m03 residue is aglu166 and protein 6w63 residues are aglu166 and aasp187 withmelatonin as shown in table and fig s5 basic group interactions ofprotein 6lu7 residues are ahis41 ahis164 ahis172 and aarg188those of protein 6m03 residues are ahis41 ahis163 ahis164 andahis172 and those of protein 6w63 residues are ahis41 ahis164and aarg188 as shown in table and fig s6 tan etal has shownthat melatonin and derivatives has excellent biological responses likeacting against oxidative stress and free radical scavenging [“]our studies show that melatonin molecule can interact with differentproteins present in the ncov19 virus and inhibit their proliferationthese results need further clinical follow up and could assist in the management of covid pneumonia conclusionswe conducted a detailed quantummechanical investigation of thehormone melatonin and regulation of the sleepwake cycle naturalbonding orbital studies revealed the intensity of several intramolecularinteractions the various frontier molecular orbital data explain the nature and physical parameters of melatonin and the nonlinear opticalproperties are compared with urea which is a standard materialfig skeletal structure of interactions between melatonin and 6lu7 a 6m03 b and 6w63 c coronavirus2 protein residues 0cn alzaqri of molecular liquids table interactions between melatonin and coronavirus2 protein residuesnamelabelhydrophobicitypkapdbidsavg isotropicdisplacementsecondarystructureresidue solventaccessibilitysidechain solventaccessibilitypercent solventaccessibilitypercent sidechainsolvent accessibility6m03 histidine6lu7 histidineahis41amet49methionineatyr54tyrosinealeu141leucineaasn142asparagineagly143glycineaser144serineacys145cysteineahis164histidinemethionineamet165glutamic acid aglu166ahis172histidineaasp187aspartic acidaarg188arginineglutamineagln189ahis41methionineamet49phenylalanine aphe140aleu141leucineaasn142asparagineglycineagly143aser144serineacys145cysteineahis163histidineahis164histidinemethionineamet165glutamic acid aglu166aleu167leucineapro168prolineahis172histidineagln189glutamine6w63 histidineahis41cysteineacys44methionineamet49leucinealeu50prolineapro52tyrosineatyr54histidineahis164methionineamet165glutamic acid aglu166aleu167leucineapro168prolineaasp187aspartic acidarginineaarg188agln189glutamineathr190threonineglutamineagln192ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’““““““““““““““““““““““““““helixturnhelixcoilturnturncoilturnsheetsheetsheetsheetcoilcoilcoilhelixhelixcoilcoilturnturncoilturnsheetsheetsheetsheetcoilturnsheetcoilhelixcoilcoilcoilcoilhelixsheetsheetsheetcoilturncoilcoilcoilcoilcoilwavefunction studies gave information about electrostatic potentialsaverage localised ionization and noncovalent interactions these datahelped to predict the reactivity and identify the active site of the reactivity of the molecule melatonin docks with novel coronavirus proteinsand shows a variety of interactions with an excellent docking scorewhich leads to inhibition of the virus proteins leading to its destructionhence clinicians can consider incorporating melatonin also in thecovid19 treatment regime after further studiestable favorable nonbond interactions between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63hydrogen bondhydrogen bondhydrogen bondhydrogen bond otherhydrophobichydrophobichydrophobichydrophobichydrogen bondhydrophobichydrophobichydrophobicconventional hydrogen bondconventional hydrogen bondcarbon hydrogen bondpidonor hydrogen bond pisulfurpialkylpisigmapipi tshapedpialkylconventional hydrogen bondpipi tshapedpialkylpialkylaglu166nunk0hunk0hacys145sgunk0amet165caahis41unk0unk0hahis41unk0unk0hdonorhdonorhdonorhdonor sulfurpiorbitalschpiorbitalspiorbitalshdonorpiorbitalspiorbitalspiorbitalsunk0oahis164oaglu166ounk0acys145unk0unk0acys145aglu166ounk0amet165amet165hacceptorhacceptorhacceptorpiorbitals piorbitalsalkylpiorbitalspiorbitalsalkylhacceptorpiorbitalsalkylalkyl 0cn alzaqri of molecular liquids table unfavorable nonbond between melatonin and coronavirus2 proteinspdb idsdistance categorytypefromfrom chemistrytoto chemistry6lu76m036w63nilunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorableunfavorable bumpunfavorable bumpunfavorable bump carbon hydrogen bondunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpunfavorable bumpacys145sgaglu166ounk0hagln189caagln189caagln189cbagln189cgagln189cgagln189cgagln189cgagln189cdagln189cdagln189cdagln189cdagln189oe1agln189oe1agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189ne2agln189haagln189haagln189haagln189hg1agln189hg2agln189hg2agln189he21agln189he21agln189he21agln189he21agln189he22agln189he22agln189he22stericstericsteric hdonorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericunk0cunk0caglu166ounk0cunk0hunk0cunk0cunk0hunk0nunk0hunk0cunk0cunk0hunk0hunk0cunk0hunk0nunk0cunk0ounk0cunk0hunk0hunk0hunk0cunk0hunk0hunk0hunk0cunk0hunk0nunk0cunk0cunk0hunk0cunk0cunk0hstericstericsteric hacceptorstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericstericsterictable unsatisfied bonds in melatonin with coronavirus2 proteinspdb ids6lu76m036w63nameunk0hunk0ounk0hunk0hunk0ounk0ounk0hunk0ounk0oatomunsatisfied typehohhoohoodonoracceptordonordonoracceptoracceptordonoracceptoracceptorrenjith thomas conceptualization formal analysis investigation methodology project administration resources softwaresupervision validation visualization writing review editingdeclaration of competing interestthe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to ‚uence the work reported in this paperacknowledgementsresearchers supporting project number rsp202078 king saudcredit authorship contribution statementuniversity riyadh saudi arabianabil alzaqri cenceptualization funding acquisition tpooventhiran investegation methodology original draft alialsalme investegation original draft ismail warad resourcesreview methods athira m john methodology writing draftappendix a supplementary datasupplementary data to this article can be found online at 101016jmolliq2020114082table noncovalent interactions between melatonin and coronavirus2 proteinshydrophobicityhydrophilicityneutral groupacidic groupbasic grouppdbids6lu7ahis41 aleu141 acys145 ahis164 amet165 and aglu1666m03 ahis41 amet49 aphe140 aleu141 acys145 amet165 aglu166 and aleu1676w63 ahis41 acys44 amet49 aleu50 amet165aglu166 aleu167 and agln189ahis41 aasn142 ahis164 aglu166 ahis172 aasp187 aarg188 and agln189ahis41 aasn142 ahis163 ahis164 aglu166 ahis172 and agln189ahis41 ahis164 aglu166 aasp187 aarg188 agln189 and agln192atyr54 agly143 andaser144agly143 aser144 andapro168apro52 atyr54 aarg188 and athr190aglu166 andaasp187aglu166aglu166and aasp187ahis41 ahis164 ahis172 and aarg188ahis41 ahis163 ahis164 and ahis172ahis41 ahis164 andaarg188 Answer:

Colon_Cancer

" inhibiting suchtarget protein reduces the virulence of pathogens and reduces thetherapeutic resistance in the case of cancer we first come to the basicunderstanding of the role of grp78 in healthy and diseased cells grp78 in normal versus stressed cellunder normal conditions grp78 is found bound to three essentialenzymes that regulate cell growth differentiation apoptosis and signaling [“] these enzymes are activating transcription factor atf6 protein kinase rnalike endoplasmic reticulum kinase perkand inositolrequiring enzyme ire1 which are inactivated throughbinding to grp78 in the er lumen under the pressure of unfoldedproteins in the er grp78 releases atf6 perk and ire1 and theenzymes are activated once activated the enzymes atf6 perk andire1 upregulate transcription of chaperones inhibit the translationand enhance protein folding endoplasmic reticulum assisted degradation erad and other function that have been reviewed byothers [“] see also fig if the pressure of the unfolded proteins is not relieved the upr will direct part of the er to autophagyerphagy if it is not enough the whole cell will undergo apoptosis[“]on the other hand under the stress of unfolded proteins grp78 canescape the er retention and translocates to the cytoplasm and on thecell membrane and become membraneexposed termed cellsurfaceŽ corresponding authoremail addresses abdoscicuedueg aelfikyictpit aa elfiky101016jlfs2020118317received may received in revised form july accepted august available online august elsevier inc all rights reserved 0caa elfiky life sciences fig functional aspect of grp78 in normal versus stress condition in normal state left the grp78 is located in the lumen of the endoplasmic reticulum erbound to and inactivating atf6 blue triangle perk red circle and ire1 yellow square enzymes in the stress condition right the enzymes are free to do theirjobs atf6 is translocated to golgi apparatus to be cleaved then again translocated to the nucleus and helps in upregulating chaperones such as grp78 perk inhibitsthe translation and protein synthesis while ire1 enhances the folding and erad under the pressure of the unfolded proteins the grp78 escapes the er retention andtranslocate to the cytoplasm and the cell membrane csgrp78 is subjected to the recognition of pathogenic proteins spike and envelope viral protein and coatproteins of fungi for interpretation of the references to colour in this figure legend the reader is referred to the web version of this csgrp78 fig this csgrp78 characterizes many aggressive types of cancers such as breast ovarian pancreatic and coloncancers [“] additionally csgrp78 was reported to facilitate pathogenic entry both viral and fungal infections zika viruszikv dengue virus denv hepatitis c virus hcv human papilloma virus hpv ebola virus ebov middleeast respiratorysyndrome coronavirus mers cov japanese encephalitis virus jevcoxsackievirus a9 and borna disease virus bdv are among viralinfections that reported grp78 association with viral proteins andgrp78 upregulation in infected cells [“] additionally recentstudies hypothesized the association of csgrp78 with the spike protein of sarscov2 to help in virus attachment and host cell entry the primary binding viral protein to grp78 is spike proteins in coronaviruses and envelope proteins for the other viruses [“] besidesthe spore coat protein homolog coth3 of rhizopus oryzae the causative fungus for mucormycosis is reported to bind to csgrp78 on endothelial cells and the binding is responsible for adherence and invasionof the fungus since the association of the viral or pathogen infection and elevatedlevels of csgrp78 expression researchers are focused on targetinggrp78 to prevent or even weaken the pathogenic infection reducingthe concentration of grp78 over the cell membrane would reduce thenumber of internalized pathogenic ps and hence reduce the infection additionally when we target csgrp78 the pathogen virulence would be diminished at the same time cancerassociated resistance would also be dimensioned which becomes of the highestpriorities in dual diseases such as viral or fungal infections in cancerpatients grp78 associated radio and chemoresistancechemoresistance is the resistance of a tumor to chemotherapy itwas an old observation while the mechanism of grp78inducedchemoresistance in cancer cells was not fully understood twomechanisms may be responsible for the chemoresistance the upr prosurvival branch and the receptormediated activation of the aktpi3kphosphoinositide 3kinase pathway alternatively the proapoptotic action of the upr could be compensated by the activation ofthe aktpi3k pathway resulting in cell survival the extracellular loopof cleft lip and palate transmembrane 1like clptm1l is essentialfor gemcitabine resistance and interaction with grp78 additionally natural products such as isoliquiritigenin a chalconetypeflavonoid were able to reduce the chemoresistance and colonyformingability of oral squamous cell carcinomas it is reported that theprior treatment of acidic stress protects the human dermal microvascular endothelial cells from apoptosis by reduced the cleavage ofcaspase which was supposed to be due to the presence of grp78 onthe membrane of er that suppress caspase activation nonsmallcell lung cancer nsclc and glioblastoma multiformegbm have a low survival rate the overexpressed grp78 on the cellsurface is the primary reason for the radioresistance in nsclc andgbm targeting cellsurface grp78 enhances the apoptosis andreduces cell proliferation colony formation and downregulates thecrucialintracellular phosphatidylinositol3kinaseprotein kinase bmammalian target of rapamycin pi3kaktmtor signaling essentialin the cell cycle growth and survival besides tumor growthis delayed with enhanced efficacy of the radiation treatment upon antigrp78 antibody administration in mice in breast cancer grp78 is overexpressed while the amount of cellsurface grp78 is increased upon the treatment with the antiangiogenicfactor combretastatin a4p additionally cancer cells treated withdoxorubicin showed less resistance when treated with grp78 neutralizing antibodies [“] generally elevated levels of grp78 areindicative of cancer aggressivity targeting cancer cellsurface grp78 isa successful strategy to reduce the radioresistance and chemoresistance of tumors 0caa elfiky in this review the focus is not only on some of the previous trials touse antigrp78 to treat cancer but also in the diagnosis see the peptideinhibitors section below grp78 targeting strategiesdifferent strategies are used to reduce the burden of overexpressedcsgrp78 different compounds show binding affinity to csgrp78 once bound to a substrate the csgrp78 will be internalized tothe cell hence the concentration of the membranebound grp78 willbe reduced once the level of grp78 over the cell surface is droppedthe pathogens will not be able to enter the host cell through grp78hence the virulence will be reduced the inhibitory molecules that cantarget csgrp78 include phytochemicals peptides and antibodies andwill be discussed in detail in the next sections the inhibitors competewith the pathogen recognizing proteins such as spike envelope orcoat proteins for the csgrp78 substratebinding domain this domain of the grp78 is reported to be responsible for the binding ofgrp78 to unfolded proteins inside the lumen of the er through itshydrophobic batches phytochemicalsphytochemicals are compounds found in plants and have a varietyof effects on protein function they are derived from fruits vegetables beans grains and some other plants phytochemicalshave a protective role because their antioxidant characteristics whichplay a vital role in the protection of cells against oxidative damage anddecreasing the probability of cancer propagation via the reactiveoxygen species ros which can induce stress in er apoptosis initiated by the er if there is uncontrolled damage in cells wesummarize some phytochemicals crucial in cell stress relief throughinhibiting the master of upr grp78 galangin and 6shogaolgalangin is a flavonol produced from rhizomes of alpinia officinarum which belongs to the ginger family and grows in southeast asiagalangin works as a suppressor for cell proliferation in hepatocellularcarcinoma it raises er stress through the upregulation of the uprtarget genes cebp homologous protein chop grp78 glucoseregulating protein grp94 and cytosolic ca2 er is the primary site for intercellular calcium ions hence rising cytosolic ca2disrupts the function of er chaperones which induce er stress leadingto the activation of upr and subsequent upregulation of grp78 galangin upregulates er stress which inhibits tumor progressionthrough inducing apoptosis 6shogaol is produced by dehydration of 6gingerol and generatedfrom rhizomes of ginger when treating hepatocellular carcinomahcc cellline with 6shogaol cancer cells develop apoptotic phenotypes signs such as nuclear shrinkage and condensation in chromatin activation of chop expression and perk dephosphorylationinitiates reactions of caspase cascade which induce apoptosis in hccsignificant stimulation was observed in er stressrelated proteinswhich induce apoptosis by 6shogaol through rising in the upr expression grp94 grp78 and hsp70 studies proved that exposing cancer cells to 6shogaol and the activator of the perkeif2αpathway salubrinal together for a specific time induce er stress whichleads to cell apoptosis salubrinal alone enhances the phosphorylation of eif2α in the human hepatocarcinoma cell line smmc7721with negligible toxicity this reveals the significant therapeuticeffect of antigrp78 against malignancies fungisulphureuine b is produced from laetiporus sulphureus and tested byglioma cells to detect antiproliferative properties studies revealed thatsulphureuine b provides er stress by raising the level of expression oflife sciences chop caspase12 and grp78 which prevents separation of grp78from perk atf6 and ire1 which initiates upr additionallymushrooms contain pcoumaric acid and caffeic acid that proved itsbinding affinity against grp78 sbd in silico hence suggested to be apossible inhibitor for overexpressed grp78 in cancer cells or cell infected with viruses including sarscov2 grape seeds and skinproanthocyanidins and resveratrol extracted from grapeseedsexposing colorectal cancer cell crc to grape seed extract which has ahigh amount of proanthocyanidins and resveratrol leads to a modification in grp78 and protein disulfide isomerase pdi which have asignificant role in cell apoptosis which leads crc to undergo apoptoticpathway leading to inhibition of the targeted cell to proliferation on the other hand caffeic acid and pcoumaric acid polyphenolsfound in the grape skin have a protective role against photooxidativedamage additionally it has a preexposure protective role for thehuman retinal pigment epithelial cells arpe19 against blue lightassociated apoptosis in a dosedependent manner by promoting grp78expression in contrast grp78 knockdown inhibited this protective role as mentioned before caffeic acid and pcoumaric are suitablebinders to grp78 sbd in silico phytoestrogensestrogen receptorpositive breast cancer cells are responsive tohormonal therapy by blocking the estrogen synthesis leading to estrogenstarvation it was reported that grp78 plays a vital role inresist estrogenstarvation induced apoptosis in breast cancer cellshence it was suggested to dualtarget the grp78 during treating estrogenpositive breast cancer if the expression level of the grp78 ishigh to improve the efficacy and reduce the resistance it wasreported that grp78 interacts with estrogen due to the critical role ofgrp78 in folding the hormonebinding domain of estrogen receptors additionally grp78 targeting was suggested as a therapeuticstrategy to sensitize cancer cells to chemotherapy in endometrial cancerestrogen induced grp78 expression phytoestrogens are found incicer arietinum and include daidzein genistein formononetin andbiochanin a both estrogens estriol and estradiol and the fourphytoestrogens are found to be recognized by grp78 sbd and henceare suggested as possible grp78 inhibitors in silico it was concluded that estrogens and phytoestrogens are the best binders to thegrp78 while the binding affinities range from ˆ’ down toˆ’ kcalmol this indicates an excellent binding affinity to grp78sbd even better than a selective cyclic peptide pep42 despiteits phytoestrogen activity genistein activates the apoptosis processthrough upr by upregulation of grp78 and cebp homologous proteinchop also termed growth arrest and dna damage gadd153and nuclear translation of gadd153 in hcc cells epigallocatechin3gallateepigallocatechin3gallate egcg is a polyphenol found in greentea and has an antiproliferative effect on breast cancer and melanoma besidesit has an inhibition effect against grp78 functionthrough direct interaction with the atp binding site of grp78 competing against atp binding egcg increases the therapeutic efficacy of temozolomide when exposed to glioblastoma cells in vivo byinhibition of grp78 olive leaf extract and honeybee hive propolisthe olive leaf extracts active ingredient hydroxytyrosol show goodbinding affinity to the grp78 sbd in silico hydroxytyrosolproved its role as a prophylactic agent against myocardial infarctionmediated apoptosis caffeic acid phenethyl ester cape is foundin the hive propolis of the honeybee cape shows in silico binding affinity against grp78 sbd that is comparable to that of the cyclicpep42 a selective grp78 peptide besides cape induces er stress 0caa elfiky in human shsy5y neuroblastoma in an autophagydependent manner peptidesdifferent peptides are used to target the cellsurface grp78 specifically peptides as anticancer drugs have two main types ishort naked peptides to induce apoptosis ii conjugated peptides todeliver an anticancer drug into cells for the first use grp78 serves as areceptor for the peptide and facilitates the internalization of the peptide which can then modulate various pathways peptides to induce apoptosisa gmbp1 peptidemultidrug resistance mdr is drug resistance that happens whencancer cells treated with one anticancer drug develop resistance todifferent drugs that are different from the used drug in structure andfunction an example of binding peptides that use grp78 as areceptor is gmbp1 which is used in reversing gastric cancer mdrgrp78 facilitates gmbp1 internalization into cells through the transferrinrelated pathway b gonadotropinreleasing hormone analogs gnrhagonadotropinreleasing hormone gnrha is a hypothalamus secreted hormone that affects sex hormones testosterone and estrogenmodified gnrha is more efficient than the natural form hence it is usedas a drug depending on the analog gnrha used as a drug againstendometriosis a case in which cells like that lining the inside of theuterus grow outside it in other parts of the body gnrha inhibitsproliferation and induces apoptosis of defected cells by inhibitinggrp78 thus leading to apoptosis conjugated peptidescell targeting is the solution for the nonspecific toxicity of anticancer drugs that affect cancer and healthy cells altogether and thusresulting in severe side effects peptides can target cancer cells anddeliver anticancer drugs into the cell in cancer cells the peptide canbind to the surface a membranebound form of the overexpressedchaperone grp78 to choose the peptide for a particular cancercell in vitro trials are required such as phage display phage display is atechnique for studying molecular interactions such as proteindnaproteinprotein and proteinpeptide utilizing the bacteriophages toencode peptides to genetic information a pool of cyclic peptidestested against the cancer cells and then a peptide is chosen to be usedfor drug delivery a pep42pep42 is a cyclic peptide ctvalpggyvrvc identified by thephage display technique against human melanoma cell line me66524 csgrp78 is the receptor for pep42 and facilitates it's internalization to the cell pep42taxol and pep42doxorubicin conjugates bind to grp78 in highly metastatic human melanoma cellsleading to its death in vitro leading to cancer cell death pep42selectively bind to grp78 and enter the cell and thus make it a powerfultool to deliver anticancer drugs to various cancer cells pep42 was used as a profiler for in silico predicting the csgrp78 andviral proteins of the zika virus human papillomavirus sarscov2and ebola virus b wifpwiql peptidewifpwiql peptide binds to grp78 expressed in breast cancer cellslife sciences surface in the breast and metastatic cells subtilase cytotoxin is a toxinfrom the ab5 toxins family subtilase cytotoxin composed of two subunits suba which is responsible for the toxicity and subb which isresponsible for subtilase cytotoxin internalization to the cell subatoxic effect is that it induces cell apoptosis by cleaving grp78 betweenthe amino acid residues leu416 and leu417 as indicated wifpwiql peptide binds to grp78 over cancer cells csgrp78 wifpwiqlsuba fusion resulting in an efficient anticancer agent wifpwiqlsuba works simultaneously wifpwiql is responsible for grp78recognition and internalization to the cancer cells while suba is responsible for the toxic effect on the cell by cleaving grp78 inside thecell and thus leading to apoptosis wifpwiql liposomes loadedwith doxorubicin are used to target csgrp78 overexpressed overvascular endothelial growth factor vegfactivated human umbilicalvein endothelial cells wifpwiql bound n2hydroxypropylmethacrylamide hpma copolymer aminohexylgeldanamycin conjugates were able to target csgrp78 and hence inhibit human prostatecancer cells additionally the genetic engineered mung beantrypsin inhibitor gbpti that includes the wifpwiql peptide wasable to induce apoptosis in colorectal cancer cells c bone metastasis targeting peptide bmtp78bmtp78 composed of a peptide wifpwiql conjugated withproapoptotic moiety dklaklak2 grp78 facilitates the internalization of bmtp78 into the cytoplasm in vitro trials showed thatbmtp78 induces apoptosis in human and mouse mammary cell lines dklaklak2 after internalization disrupts mitochondrial membranepermeability and thus kills the cell bmtp78 induced dosedependent cytotoxicity in human leukemia and lymphoma cell lines andacute myeloid leukemia patients additionally the grp78receptorbmtp78 system was used to image breast tumors accuratelythe adenoassociated virusm13derived phage aavp can be usedclinically to detect imaging and eradicate targeted therapy of inflammatory breast cancer utilizing csgrp78 as a target d girlrg peptidegirlrg is a peptide identified using phage display and bindsgrp78 girlrg conjugated to paclitaxelencapsulated nanopsspecifically targeted breast cancer and glioblastoma it was predicted in silico that girlrg binds to the atpase domain of grp78girlrg conjugated with poly ethylene glycol peg can efficientlytarget different tumor cell lines including heterotopic cervical ht3esophageal oe33 pancreatic bxpc3 lung a549 and glioma d54 additionally the radiolabeled 111inpeggirlrg show specificity toward cervical esophageal pancreatic lung and brain tumorsusing spect imaging e vap peptidesntrvap vap is a peptide identified using the phage displaytechnique and it binds to grp78 specifically sntrvap couplingwith a sirna for grp78 effectively downregulated its expression vap modified micelles rivap retro inverso isomer of lvapand dvap retro isomer of lvap could effectively achieve gliomatargeted drug delivery through grp78 at the same time it improvedthe therapeutic efficacy of paclitaxel for glioma binding peptides in diagnosticsas we mentioned before peptides could be used for drug delivery itcan be used as a carrier for radiolabels for imaging purposes such as inthe positron emission tomography pet utilizing the same concept oftargeting grp78 over cancer cellsradiolabeled polyethylene glycol peggirlrg is used in targetingmany cancers as heterotopic cervical esophageal pancreatic lung and 0caa elfiky glioma tumors triplenegative breast cancer tnbc resembles of breast cancer cases while the available diagnostic technologyfor its detection is by the invasive needle biopsy for example 68ga aradiolabel for pet imaging can be conjugated with dodecane tetraacetic acid dotavap grp78targeted pet imaging with [68ga]dotavap is a useful and accurate technique for imaging tnbc anddifferentiates it from other cancer types monoclonal antibodiesantibody ab also called immunoglobulin ig is a huge yshapedprotein produced mainly by plasma cells that are used by the immunesystem to neutralize pathogens such as viruses and bacteria the pathogenic molecule that is recognized by the antibody is called an antigen the antibody binds with the antigen with a keylock mechanism once the interaction established the cell bearing the antigentriggers a response such as metabolic inhibition monoclonal antibody mab159mab159 is a highly specific monoclonal antibody against the humangrp78 kd nm when administered mab159 found localized on the membranes of cancer cells but not normal celllinesupon glucose starvation stress mab159 is found more abundant on thecell membrane as the csgrp78 is pi3kakt signaling upstreamregulator through its interaction with crypto and alpha2macroglobulinover the cell membrane it is required for these factors to activate thepi3kakt signaling once bound to csgrp78 mab159 endocytosed and modulate the pi3k pathway leading to inhibition for cellproliferation tumor growth and metastasis at the same time it enhances tumor cell death both in vitro and in vivo the efficacy ofmab159 was examined in various tumor xenograft models includinght29 colon cancer h249 small cell lung carcinoma and a549 lungadenocarcinoma these cells have relatively higher “ surface grp78 expression compared to healthy cells mab159treatment led to and tumor growth inhibition in thesemodels respectively monoclonal igm antibody sam6the fully human monoclonal igm antibody sam6 was isolatedfrom a gastric cancer patient and it binds to an oglycosylated form ofgrp78 sam6 is internalized via endocytosis and is finally responsiblefor a lethal accumulation of oxidized lipoproteins followed by apoptosisin cancer cells sam6 not only bind to grp78 on the cancer cellmembrane but also it reduces the drug resistance and kills the cancercell human igm antibody patsm6patsm6 specifically binds to primary multiple myelomas cellsstaining the cells by immunohistochemistry reveals binding to grp78of the patsm6 this binding induces apoptosis and complementdependent cytotoxicity α 2macroglobulin α2mα2m is associated with the nterminal region of cellsurface grp78the binding activates akt to suppress apoptotic pathways and promotescell proliferation mouse mab c38 and c107the mouse monoclonal antibody c38 recognizes the cterminaldomain of the murine grp78 exposed on the cell membrane thebinding induces inhibition of the aktpi3k proliferative pathway inmelanoma cells a comparable experiment done on melanomamouse model shows that the antibody c107 also binds to grp78 inboth experiments the binging with the antibody decreases the tumrowth anti grp78cterminal domain ctd antibodies aretested against human prostate cancer cells it significantly reduceslife sciences tumor growth inhibits cell proliferation while promotes apoptosisbesides in the prostate cancer patients the antictd grp78 antibodybinds the cellexpressed grp78 in human prostate cancer cells conclusioncancerliketypes oftriplenegative breastgrp78 a master chaperone protein of the unfolded protein response plays an essential role in cancer chemoresistance and virulenceof the pathogenic infections targeting grp78 was utilized to defeataggressivecanceradditionally inhibiting grp78 overexpressed in viral infections issuggested as a promising strategy to reduce the virulence of manyviruses and fungal infections the present review summarizesthe up to date targeting strategies used to inhibit cellsurface grp78illuminating the potential use of these strategies to defeat both cancerchemoresistance and viral and fungal infectionsdeclaration of competing interestall the authors declare no conflict of interest for this workreferencesmol cell biol l ellgaard a helenius quality control in the endoplasmic reticulum nat rev mj gething j sambrook protein folding in the cell nature h hu m tian c ding s yu the cebp homologous protein chop transcription factor functions in endoplasmic reticulum stressinduced apoptosis andmicrobial infection front immunol im ibrahim dh abdelmalek aa elfiky grp78 a cell™s response to stress lifesci “ e little m ramakrishnan b roy g gazit as lee the glucoseregulatedproteins grp78 and grp94 functions gene regulation and applications critrev eukaryot gene expr “ l brocchieri ec de macario aj macario hsp70 genes in the human genomeconservation and differentiation patterns predict a wide array of overlapping andspecialized functions bmc evol biol am ismail aa elfiky wm elshemey recognition of the gluconeogenic enzyme pck1 via the gid4 e3 ligase an in silico perspective j mol recognit e2821 pc liao sk tan ch lieu hk jung involvement of endoplasmic reticulum inpaclitaxelinduced apoptosis j cell biochem “ i haas bip”a heat shock protein involved in immunoglobulin chain assemblycurr top microbiol immunol “ j li m ni b lee e barron d hinton a lee the unfolded protein responseregulator grp78bip is required for endoplasmic reticulum integrity and stressinduced autophagy in mammalian cells cell death differ s srivastava g jain s dang s gupta r gabrani phytochemicals targetingendoplasmic reticulum stress to inhibit cancer cell proliferation anticancerplants natural products and biotechnological implements springer pp“ r ge c kao cell surface grp78 as a death receptor and an anticancer drugtarget cancers u gopal sv pizzo the endoplasmic reticulum chaperone grp78 also functionsas a cell surface signaling receptor cell surface grp78 a new paradigm in signaltransduction biology elsevier pp “ s luo c mao b lee as lee grp78bip is required for cell proliferation andprotecting the inner cell mass from apoptosis during early mouse embryonic development mol cell biol “ kt pfaffenbach as lee the critical role of grp78 in physiologic and pathologic stress curr opin cell biol “ m wang s wey y zhang r ye as lee role of the unfolded protein responseregulator grp78bip in development cancer and neurological disordersantioxid redox signal “cancer cells by fak and jnk mol cell biochem “tissue factor procoagulant activity cell surface grp78 a new paradigm in signaltransduction biology elsevier “ as lee grp78 induction in cancer therapeutic and prognostic implicationscancer res “ m liu b spellberg qt phan y fu y fu as lee the endothelial cellreceptor grp78 is required for mucormycosis pathogenesis in diabetic mice jclin invest “ h chamolstad je yu z feng sh lee jg kim p yang p62sqstm1sequestosome1 is an nrecognin of the nend rule pathway which modulatesautophagosome biogenesis nat commun x yuan m dong x li j zhou grp78 promotes the invasion of pancreatic aa alhashimi j rak rc austin cell surface grp78 a novel regulator of 0caa elfiky life sciences f visioli y wang gn alam y ning pv rados je nör glucose a choukhi s ung c wychowski j dubuisson involvement of endoplasmic yl tsai as lee cell surface grp78 anchoring and translocation mechanisms ch ji hy kim aj heo sh lee mj lee sb kim the ndegron p mehrbod sr ande j alizadeh s rahimizadeh a shariati h malek pathway mediates erphagy mol cell “1072e9the roles of apoptosis autophagy and unfolded protein response in arbovirusinfluenza virus and hiv infections virulence “and therapeutic potential in cancer cell surface grp78 a new paradigm insignal transduction biology elsevier pp “ yj chang yp huang zl li ch chen grp78 knockdown enhancesapoptosis via the downregulation of oxidative stress and akt pathway afterepirubicin treatment in colon cancer dld1 cells plos one e35123 t chen s xu chronic exposure of cisplatin induces grp78 expression in ovariancancer proceedings of the 4th international conference on biomedical andbioinformatics engineering acm “ y kim am lillo sc steiniger y liu c ballatore a anichini targetingheat shock proteins on cancer cells selection characterization and cellpenetrating properties of a peptidic grp78 ligand biochemistry “ z li l zhang y zhao h li h xiao r fu cellsurface grp78 facilitatescolorectal cancer cell migration and invasion int j biochem cell biol “ z niu m wang l zhou l yao q liao y zhao elevated grp78 expression isassociated with poor prognosis in patients with pancreatic cancer sci rep s tian w chang h du j bai z sun q zhang the interplay betweengrp78 expression and akt activation in human colon cancer cells under celecoxibtreatment anticancer drugs “ j xie zh tao j zhao t li zh wu jf zhang glucose regulatedprotein grp78 inhibits apoptosis and attentinutes chemosensitivity of gemcitabine in breast cancer cell via aktmitochondrial apoptotic pathway biochembiophys res commun “ j zhang y jiang z jia q li w gong l wang association of elevatedgrp78 expression with increased lymph node metastasis and poor prognosis inpatients with gastric cancer clin exp metastasis “ hh chen cc chen ys lin pc chang zy lu cf lin ar12suppresses dengue virus replication by downregulation of pi3kakt and grp78antivir res “reticulum chaperones in the folding of hepatitis c virus glycoproteins j virol “ h chu cm chan x zhang y wang s yuan j zhou middle eastrespiratory syndrome coronavirus and bat coronavirus hku9 both can utilizegrp78 for attachment onto host cells j biol chem “ s das sv laxminarayana n chandra v ravi a desai heat shock protein on neuro2a cells is a putative receptor for japanese encephalitis virus virology “ t honda m horie t daito k ikuta k tomonaga molecular chaperone bipinteracts with borna disease virus glycoprotein at the cell surface j virol “ s jindadamrongwech c thepparit d smith identification of grp bip as aliver cell expressed receptor element for dengue virus serotype arch virol “ e liberman yl fong mj selby ql choo l cousens m houghton activation of the grp78 andgrp94 promoters by hepatitis c virus e2 envelopeprotein j virol “ m nain s mukherjee sp karmakar aw paton jc paton m abdin grp78 is an important hostfactor for japanese encephalitis virus entry and replication in mammalian cells j virol “ s pujhari vm macias rh nissly m nomura sv kuchipudi jl rasgon heatshock protein hsp70 is involved in the zika virus cellular infection processbiorxiv protein and heat shock protein are components of dengue virus receptorcomplex in human cells j virol “ ac shurtleff ja costantino sr tritsch c retterer kb spurgers s bavarihspa5 is an essential host factor for ebola virus infection antivir res “ im ibrahim dh abdelmalek me elshahat aa elfiky covid19 spikehostcell receptor grp78 binding site prediction j infect “ aa elfiky ebola virus glycoprotein gp1host cellsurface hspa5 binding siteprediction cell stress chaperones “ aa elfiky natural products may interfere with sarscov2 attachment to thehost cell j biomol struct dyn “ aa elfiky im ibrahim zika virus envelope “ heat shock protein a5 grp78binding site prediction j biomol struct dyn “ t gebremariam m liu g luo v bruno qt phan aj waring coth3mediates fungal invasion of host cells during mucormycosis j clin invest “ wang cy cusack jc jr liu r baldwin as jr control of inducible chemoresistance enhanced antitumor therapy through increased apoptosis by inhibitionof nfkappab nat med “ c roller d maddalo the molecular chaperone grp78bip in the development ofchemoresistance mechanism and possible treatment front pharmacol wr clarke l amundadottir ma james clptm1lcrr9 ectodomain interaction with grp78 at the cell surface signals for survival and chemoresistance upon j reyesdel valle s chávezsalinas f medina rm del angel heat shock cj dillard jb german phytochemicals nutraceuticals and human health j c hetz the unfolded protein response controlling cell fate decisions under er l su x chen j wu b lin h zhang l lan galangin inhibits pro c bailly mj waring pharmacologic

cancer7516

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " inhibiting suchtarget protein reduces the virulence of pathogens and reduces thetherapeutic resistance in the case of cancer we first come to the basicunderstanding of the role of grp78 in healthy and diseased cells grp78 in normal versus stressed cellunder normal conditions grp78 is found bound to three essentialenzymes that regulate cell growth differentiation apoptosis and signaling [“] these enzymes are activating transcription factor atf6 protein kinase rnalike endoplasmic reticulum kinase perkand inositolrequiring enzyme ire1 which are inactivated throughbinding to grp78 in the er lumen under the pressure of unfoldedproteins in the er grp78 releases atf6 perk and ire1 and theenzymes are activated once activated the enzymes atf6 perk andire1 upregulate transcription of chaperones inhibit the translationand enhance protein folding endoplasmic reticulum assisted degradation erad and other function that have been reviewed byothers [“] see also fig if the pressure of the unfolded proteins is not relieved the upr will direct part of the er to autophagyerphagy if it is not enough the whole cell will undergo apoptosis[“]on the other hand under the stress of unfolded proteins grp78 canescape the er retention and translocates to the cytoplasm and on thecell membrane and become membraneexposed termed cellsurfaceŽ corresponding authoremail addresses abdoscicuedueg aelfikyictpit aa elfiky101016jlfs2020118317received may received in revised form july accepted august available online august elsevier inc all rights reserved 0caa elfiky life sciences fig functional aspect of grp78 in normal versus stress condition in normal state left the grp78 is located in the lumen of the endoplasmic reticulum erbound to and inactivating atf6 blue triangle perk red circle and ire1 yellow square enzymes in the stress condition right the enzymes are free to do theirjobs atf6 is translocated to golgi apparatus to be cleaved then again translocated to the nucleus and helps in upregulating chaperones such as grp78 perk inhibitsthe translation and protein synthesis while ire1 enhances the folding and erad under the pressure of the unfolded proteins the grp78 escapes the er retention andtranslocate to the cytoplasm and the cell membrane csgrp78 is subjected to the recognition of pathogenic proteins spike and envelope viral protein and coatproteins of fungi for interpretation of the references to colour in this figure legend the reader is referred to the web version of this csgrp78 fig this csgrp78 characterizes many aggressive types of cancers such as breast ovarian pancreatic and coloncancers [“] additionally csgrp78 was reported to facilitate pathogenic entry both viral and fungal infections zika viruszikv dengue virus denv hepatitis c virus hcv human papilloma virus hpv ebola virus ebov middleeast respiratorysyndrome coronavirus mers cov japanese encephalitis virus jevcoxsackievirus a9 and borna disease virus bdv are among viralinfections that reported grp78 association with viral proteins andgrp78 upregulation in infected cells [“] additionally recentstudies hypothesized the association of csgrp78 with the spike protein of sarscov2 to help in virus attachment and host cell entry the primary binding viral protein to grp78 is spike proteins in coronaviruses and envelope proteins for the other viruses [“] besidesthe spore coat protein homolog coth3 of rhizopus oryzae the causative fungus for mucormycosis is reported to bind to csgrp78 on endothelial cells and the binding is responsible for adherence and invasionof the fungus since the association of the viral or pathogen infection and elevatedlevels of csgrp78 expression researchers are focused on targetinggrp78 to prevent or even weaken the pathogenic infection reducingthe concentration of grp78 over the cell membrane would reduce thenumber of internalized pathogenic ps and hence reduce the infection additionally when we target csgrp78 the pathogen virulence would be diminished at the same time cancerassociated resistance would also be dimensioned which becomes of the highestpriorities in dual diseases such as viral or fungal infections in cancerpatients grp78 associated radio and chemoresistancechemoresistance is the resistance of a tumor to chemotherapy itwas an old observation while the mechanism of grp78inducedchemoresistance in cancer cells was not fully understood twomechanisms may be responsible for the chemoresistance the upr prosurvival branch and the receptormediated activation of the aktpi3kphosphoinositide 3kinase pathway alternatively the proapoptotic action of the upr could be compensated by the activation ofthe aktpi3k pathway resulting in cell survival the extracellular loopof cleft lip and palate transmembrane 1like clptm1l is essentialfor gemcitabine resistance and interaction with grp78 additionally natural products such as isoliquiritigenin a chalconetypeflavonoid were able to reduce the chemoresistance and colonyformingability of oral squamous cell carcinomas it is reported that theprior treatment of acidic stress protects the human dermal microvascular endothelial cells from apoptosis by reduced the cleavage ofcaspase which was supposed to be due to the presence of grp78 onthe membrane of er that suppress caspase activation nonsmallcell lung cancer nsclc and glioblastoma multiformegbm have a low survival rate the overexpressed grp78 on the cellsurface is the primary reason for the radioresistance in nsclc andgbm targeting cellsurface grp78 enhances the apoptosis andreduces cell proliferation colony formation and downregulates thecrucialintracellular phosphatidylinositol3kinaseprotein kinase bmammalian target of rapamycin pi3kaktmtor signaling essentialin the cell cycle growth and survival besides tumor growthis delayed with enhanced efficacy of the radiation treatment upon antigrp78 antibody administration in mice in breast cancer grp78 is overexpressed while the amount of cellsurface grp78 is increased upon the treatment with the antiangiogenicfactor combretastatin a4p additionally cancer cells treated withdoxorubicin showed less resistance when treated with grp78 neutralizing antibodies [“] generally elevated levels of grp78 areindicative of cancer aggressivity targeting cancer cellsurface grp78 isa successful strategy to reduce the radioresistance and chemoresistance of tumors 0caa elfiky in this review the focus is not only on some of the previous trials touse antigrp78 to treat cancer but also in the diagnosis see the peptideinhibitors section below grp78 targeting strategiesdifferent strategies are used to reduce the burden of overexpressedcsgrp78 different compounds show binding affinity to csgrp78 once bound to a substrate the csgrp78 will be internalized tothe cell hence the concentration of the membranebound grp78 willbe reduced once the level of grp78 over the cell surface is droppedthe pathogens will not be able to enter the host cell through grp78hence the virulence will be reduced the inhibitory molecules that cantarget csgrp78 include phytochemicals peptides and antibodies andwill be discussed in detail in the next sections the inhibitors competewith the pathogen recognizing proteins such as spike envelope orcoat proteins for the csgrp78 substratebinding domain this domain of the grp78 is reported to be responsible for the binding ofgrp78 to unfolded proteins inside the lumen of the er through itshydrophobic batches phytochemicalsphytochemicals are compounds found in plants and have a varietyof effects on protein function they are derived from fruits vegetables beans grains and some other plants phytochemicalshave a protective role because their antioxidant characteristics whichplay a vital role in the protection of cells against oxidative damage anddecreasing the probability of cancer propagation via the reactiveoxygen species ros which can induce stress in er apoptosis initiated by the er if there is uncontrolled damage in cells wesummarize some phytochemicals crucial in cell stress relief throughinhibiting the master of upr grp78 galangin and 6shogaolgalangin is a flavonol produced from rhizomes of alpinia officinarum which belongs to the ginger family and grows in southeast asiagalangin works as a suppressor for cell proliferation in hepatocellularcarcinoma it raises er stress through the upregulation of the uprtarget genes cebp homologous protein chop grp78 glucoseregulating protein grp94 and cytosolic ca2 er is the primary site for intercellular calcium ions hence rising cytosolic ca2disrupts the function of er chaperones which induce er stress leadingto the activation of upr and subsequent upregulation of grp78 galangin upregulates er stress which inhibits tumor progressionthrough inducing apoptosis 6shogaol is produced by dehydration of 6gingerol and generatedfrom rhizomes of ginger when treating hepatocellular carcinomahcc cellline with 6shogaol cancer cells develop apoptotic phenotypes signs such as nuclear shrinkage and condensation in chromatin activation of chop expression and perk dephosphorylationinitiates reactions of caspase cascade which induce apoptosis in hccsignificant stimulation was observed in er stressrelated proteinswhich induce apoptosis by 6shogaol through rising in the upr expression grp94 grp78 and hsp70 studies proved that exposing cancer cells to 6shogaol and the activator of the perkeif2αpathway salubrinal together for a specific time induce er stress whichleads to cell apoptosis salubrinal alone enhances the phosphorylation of eif2α in the human hepatocarcinoma cell line smmc7721with negligible toxicity this reveals the significant therapeuticeffect of antigrp78 against malignancies fungisulphureuine b is produced from laetiporus sulphureus and tested byglioma cells to detect antiproliferative properties studies revealed thatsulphureuine b provides er stress by raising the level of expression oflife sciences chop caspase12 and grp78 which prevents separation of grp78from perk atf6 and ire1 which initiates upr additionallymushrooms contain pcoumaric acid and caffeic acid that proved itsbinding affinity against grp78 sbd in silico hence suggested to be apossible inhibitor for overexpressed grp78 in cancer cells or cell infected with viruses including sarscov2 grape seeds and skinproanthocyanidins and resveratrol extracted from grapeseedsexposing colorectal cancer cell crc to grape seed extract which has ahigh amount of proanthocyanidins and resveratrol leads to a modification in grp78 and protein disulfide isomerase pdi which have asignificant role in cell apoptosis which leads crc to undergo apoptoticpathway leading to inhibition of the targeted cell to proliferation on the other hand caffeic acid and pcoumaric acid polyphenolsfound in the grape skin have a protective role against photooxidativedamage additionally it has a preexposure protective role for thehuman retinal pigment epithelial cells arpe19 against blue lightassociated apoptosis in a dosedependent manner by promoting grp78expression in contrast grp78 knockdown inhibited this protective role as mentioned before caffeic acid and pcoumaric are suitablebinders to grp78 sbd in silico phytoestrogensestrogen receptorpositive breast cancer cells are responsive tohormonal therapy by blocking the estrogen synthesis leading to estrogenstarvation it was reported that grp78 plays a vital role inresist estrogenstarvation induced apoptosis in breast cancer cellshence it was suggested to dualtarget the grp78 during treating estrogenpositive breast cancer if the expression level of the grp78 ishigh to improve the efficacy and reduce the resistance it wasreported that grp78 interacts with estrogen due to the critical role ofgrp78 in folding the hormonebinding domain of estrogen receptors additionally grp78 targeting was suggested as a therapeuticstrategy to sensitize cancer cells to chemotherapy in endometrial cancerestrogen induced grp78 expression phytoestrogens are found incicer arietinum and include daidzein genistein formononetin andbiochanin a both estrogens estriol and estradiol and the fourphytoestrogens are found to be recognized by grp78 sbd and henceare suggested as possible grp78 inhibitors in silico it was concluded that estrogens and phytoestrogens are the best binders to thegrp78 while the binding affinities range from ˆ’ down toˆ’ kcalmol this indicates an excellent binding affinity to grp78sbd even better than a selective cyclic peptide pep42 despiteits phytoestrogen activity genistein activates the apoptosis processthrough upr by upregulation of grp78 and cebp homologous proteinchop also termed growth arrest and dna damage gadd153and nuclear translation of gadd153 in hcc cells epigallocatechin3gallateepigallocatechin3gallate egcg is a polyphenol found in greentea and has an antiproliferative effect on breast cancer and melanoma besidesit has an inhibition effect against grp78 functionthrough direct interaction with the atp binding site of grp78 competing against atp binding egcg increases the therapeutic efficacy of temozolomide when exposed to glioblastoma cells in vivo byinhibition of grp78 olive leaf extract and honeybee hive propolisthe olive leaf extracts active ingredient hydroxytyrosol show goodbinding affinity to the grp78 sbd in silico hydroxytyrosolproved its role as a prophylactic agent against myocardial infarctionmediated apoptosis caffeic acid phenethyl ester cape is foundin the hive propolis of the honeybee cape shows in silico binding affinity against grp78 sbd that is comparable to that of the cyclicpep42 a selective grp78 peptide besides cape induces er stress 0caa elfiky in human shsy5y neuroblastoma in an autophagydependent manner peptidesdifferent peptides are used to target the cellsurface grp78 specifically peptides as anticancer drugs have two main types ishort naked peptides to induce apoptosis ii conjugated peptides todeliver an anticancer drug into cells for the first use grp78 serves as areceptor for the peptide and facilitates the internalization of the peptide which can then modulate various pathways peptides to induce apoptosisa gmbp1 peptidemultidrug resistance mdr is drug resistance that happens whencancer cells treated with one anticancer drug develop resistance todifferent drugs that are different from the used drug in structure andfunction an example of binding peptides that use grp78 as areceptor is gmbp1 which is used in reversing gastric cancer mdrgrp78 facilitates gmbp1 internalization into cells through the transferrinrelated pathway b gonadotropinreleasing hormone analogs gnrhagonadotropinreleasing hormone gnrha is a hypothalamus secreted hormone that affects sex hormones testosterone and estrogenmodified gnrha is more efficient than the natural form hence it is usedas a drug depending on the analog gnrha used as a drug againstendometriosis a case in which cells like that lining the inside of theuterus grow outside it in other parts of the body gnrha inhibitsproliferation and induces apoptosis of defected cells by inhibitinggrp78 thus leading to apoptosis conjugated peptidescell targeting is the solution for the nonspecific toxicity of anticancer drugs that affect cancer and healthy cells altogether and thusresulting in severe side effects peptides can target cancer cells anddeliver anticancer drugs into the cell in cancer cells the peptide canbind to the surface a membranebound form of the overexpressedchaperone grp78 to choose the peptide for a particular cancercell in vitro trials are required such as phage display phage display is atechnique for studying molecular interactions such as proteindnaproteinprotein and proteinpeptide utilizing the bacteriophages toencode peptides to genetic information a pool of cyclic peptidestested against the cancer cells and then a peptide is chosen to be usedfor drug delivery a pep42pep42 is a cyclic peptide ctvalpggyvrvc identified by thephage display technique against human melanoma cell line me66524 csgrp78 is the receptor for pep42 and facilitates it's internalization to the cell pep42taxol and pep42doxorubicin conjugates bind to grp78 in highly metastatic human melanoma cellsleading to its death in vitro leading to cancer cell death pep42selectively bind to grp78 and enter the cell and thus make it a powerfultool to deliver anticancer drugs to various cancer cells pep42 was used as a profiler for in silico predicting the csgrp78 andviral proteins of the zika virus human papillomavirus sarscov2and ebola virus b wifpwiql peptidewifpwiql peptide binds to grp78 expressed in breast cancer cellslife sciences surface in the breast and metastatic cells subtilase cytotoxin is a toxinfrom the ab5 toxins family subtilase cytotoxin composed of two subunits suba which is responsible for the toxicity and subb which isresponsible for subtilase cytotoxin internalization to the cell subatoxic effect is that it induces cell apoptosis by cleaving grp78 betweenthe amino acid residues leu416 and leu417 as indicated wifpwiql peptide binds to grp78 over cancer cells csgrp78 wifpwiqlsuba fusion resulting in an efficient anticancer agent wifpwiqlsuba works simultaneously wifpwiql is responsible for grp78recognition and internalization to the cancer cells while suba is responsible for the toxic effect on the cell by cleaving grp78 inside thecell and thus leading to apoptosis wifpwiql liposomes loadedwith doxorubicin are used to target csgrp78 overexpressed overvascular endothelial growth factor vegfactivated human umbilicalvein endothelial cells wifpwiql bound n2hydroxypropylmethacrylamide hpma copolymer aminohexylgeldanamycin conjugates were able to target csgrp78 and hence inhibit human prostatecancer cells additionally the genetic engineered mung beantrypsin inhibitor gbpti that includes the wifpwiql peptide wasable to induce apoptosis in colorectal cancer cells c bone metastasis targeting peptide bmtp78bmtp78 composed of a peptide wifpwiql conjugated withproapoptotic moiety dklaklak2 grp78 facilitates the internalization of bmtp78 into the cytoplasm in vitro trials showed thatbmtp78 induces apoptosis in human and mouse mammary cell lines dklaklak2 after internalization disrupts mitochondrial membranepermeability and thus kills the cell bmtp78 induced dosedependent cytotoxicity in human leukemia and lymphoma cell lines andacute myeloid leukemia patients additionally the grp78receptorbmtp78 system was used to image breast tumors accuratelythe adenoassociated virusm13derived phage aavp can be usedclinically to detect imaging and eradicate targeted therapy of inflammatory breast cancer utilizing csgrp78 as a target d girlrg peptidegirlrg is a peptide identified using phage display and bindsgrp78 girlrg conjugated to paclitaxelencapsulated nanopsspecifically targeted breast cancer and glioblastoma it was predicted in silico that girlrg binds to the atpase domain of grp78girlrg conjugated with poly ethylene glycol peg can efficientlytarget different tumor cell lines including heterotopic cervical ht3esophageal oe33 pancreatic bxpc3 lung a549 and glioma d54 additionally the radiolabeled 111inpeggirlrg show specificity toward cervical esophageal pancreatic lung and brain tumorsusing spect imaging e vap peptidesntrvap vap is a peptide identified using the phage displaytechnique and it binds to grp78 specifically sntrvap couplingwith a sirna for grp78 effectively downregulated its expression vap modified micelles rivap retro inverso isomer of lvapand dvap retro isomer of lvap could effectively achieve gliomatargeted drug delivery through grp78 at the same time it improvedthe therapeutic efficacy of paclitaxel for glioma binding peptides in diagnosticsas we mentioned before peptides could be used for drug delivery itcan be used as a carrier for radiolabels for imaging purposes such as inthe positron emission tomography pet utilizing the same concept oftargeting grp78 over cancer cellsradiolabeled polyethylene glycol peggirlrg is used in targetingmany cancers as heterotopic cervical esophageal pancreatic lung and 0caa elfiky glioma tumors triplenegative breast cancer tnbc resembles of breast cancer cases while the available diagnostic technologyfor its detection is by the invasive needle biopsy for example 68ga aradiolabel for pet imaging can be conjugated with dodecane tetraacetic acid dotavap grp78targeted pet imaging with [68ga]dotavap is a useful and accurate technique for imaging tnbc anddifferentiates it from other cancer types monoclonal antibodiesantibody ab also called immunoglobulin ig is a huge yshapedprotein produced mainly by plasma cells that are used by the immunesystem to neutralize pathogens such as viruses and bacteria the pathogenic molecule that is recognized by the antibody is called an antigen the antibody binds with the antigen with a keylock mechanism once the interaction established the cell bearing the antigentriggers a response such as metabolic inhibition monoclonal antibody mab159mab159 is a highly specific monoclonal antibody against the humangrp78 kd nm when administered mab159 found localized on the membranes of cancer cells but not normal celllinesupon glucose starvation stress mab159 is found more abundant on thecell membrane as the csgrp78 is pi3kakt signaling upstreamregulator through its interaction with crypto and alpha2macroglobulinover the cell membrane it is required for these factors to activate thepi3kakt signaling once bound to csgrp78 mab159 endocytosed and modulate the pi3k pathway leading to inhibition for cellproliferation tumor growth and metastasis at the same time it enhances tumor cell death both in vitro and in vivo the efficacy ofmab159 was examined in various tumor xenograft models includinght29 colon cancer h249 small cell lung carcinoma and a549 lungadenocarcinoma these cells have relatively higher “ surface grp78 expression compared to healthy cells mab159treatment led to and tumor growth inhibition in thesemodels respectively monoclonal igm antibody sam6the fully human monoclonal igm antibody sam6 was isolatedfrom a gastric cancer patient and it binds to an oglycosylated form ofgrp78 sam6 is internalized via endocytosis and is finally responsiblefor a lethal accumulation of oxidized lipoproteins followed by apoptosisin cancer cells sam6 not only bind to grp78 on the cancer cellmembrane but also it reduces the drug resistance and kills the cancercell human igm antibody patsm6patsm6 specifically binds to primary multiple myelomas cellsstaining the cells by immunohistochemistry reveals binding to grp78of the patsm6 this binding induces apoptosis and complementdependent cytotoxicity α 2macroglobulin α2mα2m is associated with the nterminal region of cellsurface grp78the binding activates akt to suppress apoptotic pathways and promotescell proliferation mouse mab c38 and c107the mouse monoclonal antibody c38 recognizes the cterminaldomain of the murine grp78 exposed on the cell membrane thebinding induces inhibition of the aktpi3k proliferative pathway inmelanoma cells a comparable experiment done on melanomamouse model shows that the antibody c107 also binds to grp78 inboth experiments the binging with the antibody decreases the tumrowth anti grp78cterminal domain ctd antibodies aretested against human prostate cancer cells it significantly reduceslife sciences tumor growth inhibits cell proliferation while promotes apoptosisbesides in the prostate cancer patients the antictd grp78 antibodybinds the cellexpressed grp78 in human prostate cancer cells conclusioncancerliketypes oftriplenegative breastgrp78 a master chaperone protein of the unfolded protein response plays an essential role in cancer chemoresistance and virulenceof the pathogenic infections targeting grp78 was utilized to defeataggressivecanceradditionally inhibiting grp78 overexpressed in viral infections issuggested as a promising strategy to reduce the virulence of manyviruses and fungal infections the present review summarizesthe up to date targeting strategies used to inhibit cellsurface grp78illuminating the potential use of these strategies to defeat both cancerchemoresistance and viral and fungal infectionsdeclaration of competing interestall the authors declare no conflict of interest for this workreferencesmol cell biol l ellgaard a helenius quality control in the endoplasmic reticulum nat rev mj gething j sambrook protein folding in the cell nature h hu m tian c ding s yu the cebp homologous protein chop transcription factor functions in endoplasmic reticulum stressinduced apoptosis andmicrobial infection front immunol im ibrahim dh abdelmalek aa elfiky grp78 a cell™s response to stress lifesci “ e little m ramakrishnan b roy g gazit as lee the glucoseregulatedproteins grp78 and grp94 functions gene regulation and applications critrev eukaryot gene expr “ l brocchieri ec de macario aj macario hsp70 genes in the human genomeconservation and differentiation patterns predict a wide array of overlapping andspecialized functions bmc evol biol am ismail aa elfiky wm elshemey recognition of the gluconeogenic enzyme pck1 via the gid4 e3 ligase an in silico perspective j mol recognit e2821 pc liao sk tan ch lieu hk jung involvement of endoplasmic reticulum inpaclitaxelinduced apoptosis j cell biochem “ i haas bip”a heat shock protein involved in immunoglobulin chain assemblycurr top microbiol immunol “ j li m ni b lee e barron d hinton a lee the unfolded protein responseregulator grp78bip is required for endoplasmic reticulum integrity and stressinduced autophagy in mammalian cells cell death differ s srivastava g jain s dang s gupta r gabrani phytochemicals targetingendoplasmic reticulum stress to inhibit cancer cell proliferation anticancerplants natural products and biotechnological implements springer pp“ r ge c kao cell surface grp78 as a death receptor and an anticancer drugtarget cancers u gopal sv pizzo the endoplasmic reticulum chaperone grp78 also functionsas a cell surface signaling receptor cell surface grp78 a new paradigm in signaltransduction biology elsevier pp “ s luo c mao b lee as lee grp78bip is required for cell proliferation andprotecting the inner cell mass from apoptosis during early mouse embryonic development mol cell biol “ kt pfaffenbach as lee the critical role of grp78 in physiologic and pathologic stress curr opin cell biol “ m wang s wey y zhang r ye as lee role of the unfolded protein responseregulator grp78bip in development cancer and neurological disordersantioxid redox signal “cancer cells by fak and jnk mol cell biochem “tissue factor procoagulant activity cell surface grp78 a new paradigm in signaltransduction biology elsevier “ as lee grp78 induction in cancer therapeutic and prognostic implicationscancer res “ m liu b spellberg qt phan y fu y fu as lee the endothelial cellreceptor grp78 is required for mucormycosis pathogenesis in diabetic mice jclin invest “ h chamolstad je yu z feng sh lee jg kim p yang p62sqstm1sequestosome1 is an nrecognin of the nend rule pathway which modulatesautophagosome biogenesis nat commun x yuan m dong x li j zhou grp78 promotes the invasion of pancreatic aa alhashimi j rak rc austin cell surface grp78 a novel regulator of 0caa elfiky life sciences f visioli y wang gn alam y ning pv rados je nör glucose a choukhi s ung c wychowski j dubuisson involvement of endoplasmic yl tsai as lee cell surface grp78 anchoring and translocation mechanisms ch ji hy kim aj heo sh lee mj lee sb kim the ndegron p mehrbod sr ande j alizadeh s rahimizadeh a shariati h malek pathway mediates erphagy mol cell “1072e9the roles of apoptosis autophagy and unfolded protein response in arbovirusinfluenza virus and hiv infections virulence “and therapeutic potential in cancer cell surface grp78 a new paradigm insignal transduction biology elsevier pp “ yj chang yp huang zl li ch chen grp78 knockdown enhancesapoptosis via the downregulation of oxidative stress and akt pathway afterepirubicin treatment in colon cancer dld1 cells plos one e35123 t chen s xu chronic exposure of cisplatin induces grp78 expression in ovariancancer proceedings of the 4th international conference on biomedical andbioinformatics engineering acm “ y kim am lillo sc steiniger y liu c ballatore a anichini targetingheat shock proteins on cancer cells selection characterization and cellpenetrating properties of a peptidic grp78 ligand biochemistry “ z li l zhang y zhao h li h xiao r fu cellsurface grp78 facilitatescolorectal cancer cell migration and invasion int j biochem cell biol “ z niu m wang l zhou l yao q liao y zhao elevated grp78 expression isassociated with poor prognosis in patients with pancreatic cancer sci rep s tian w chang h du j bai z sun q zhang the interplay betweengrp78 expression and akt activation in human colon cancer cells under celecoxibtreatment anticancer drugs “ j xie zh tao j zhao t li zh wu jf zhang glucose regulatedprotein grp78 inhibits apoptosis and attentinutes chemosensitivity of gemcitabine in breast cancer cell via aktmitochondrial apoptotic pathway biochembiophys res commun “ j zhang y jiang z jia q li w gong l wang association of elevatedgrp78 expression with increased lymph node metastasis and poor prognosis inpatients with gastric cancer clin exp metastasis “ hh chen cc chen ys lin pc chang zy lu cf lin ar12suppresses dengue virus replication by downregulation of pi3kakt and grp78antivir res “reticulum chaperones in the folding of hepatitis c virus glycoproteins j virol “ h chu cm chan x zhang y wang s yuan j zhou middle eastrespiratory syndrome coronavirus and bat coronavirus hku9 both can utilizegrp78 for attachment onto host cells j biol chem “ s das sv laxminarayana n chandra v ravi a desai heat shock protein on neuro2a cells is a putative receptor for japanese encephalitis virus virology “ t honda m horie t daito k ikuta k tomonaga molecular chaperone bipinteracts with borna disease virus glycoprotein at the cell surface j virol “ s jindadamrongwech c thepparit d smith identification of grp bip as aliver cell expressed receptor element for dengue virus serotype arch virol “ e liberman yl fong mj selby ql choo l cousens m houghton activation of the grp78 andgrp94 promoters by hepatitis c virus e2 envelopeprotein j virol “ m nain s mukherjee sp karmakar aw paton jc paton m abdin grp78 is an important hostfactor for japanese encephalitis virus entry and replication in mammalian cells j virol “ s pujhari vm macias rh nissly m nomura sv kuchipudi jl rasgon heatshock protein hsp70 is involved in the zika virus cellular infection processbiorxiv protein and heat shock protein are components of dengue virus receptorcomplex in human cells j virol “ ac shurtleff ja costantino sr tritsch c retterer kb spurgers s bavarihspa5 is an essential host factor for ebola virus infection antivir res “ im ibrahim dh abdelmalek me elshahat aa elfiky covid19 spikehostcell receptor grp78 binding site prediction j infect “ aa elfiky ebola virus glycoprotein gp1host cellsurface hspa5 binding siteprediction cell stress chaperones “ aa elfiky natural products may interfere with sarscov2 attachment to thehost cell j biomol struct dyn “ aa elfiky im ibrahim zika virus envelope “ heat shock protein a5 grp78binding site prediction j biomol struct dyn “ t gebremariam m liu g luo v bruno qt phan aj waring coth3mediates fungal invasion of host cells during mucormycosis j clin invest “ wang cy cusack jc jr liu r baldwin as jr control of inducible chemoresistance enhanced antitumor therapy through increased apoptosis by inhibitionof nfkappab nat med “ c roller d maddalo the molecular chaperone grp78bip in the development ofchemoresistance mechanism and possible treatment front pharmacol wr clarke l amundadottir ma james clptm1lcrr9 ectodomain interaction with grp78 at the cell surface signals for survival and chemoresistance upon j reyesdel valle s chávezsalinas f medina rm del angel heat shock cj dillard jb german phytochemicals nutraceuticals and human health j c hetz the unfolded protein response controlling cell fate decisions under er l su x chen j wu b lin h zhang l lan galangin inhibits pro c bailly mj waring pharmacologic Answer:

Colon_Cancer

section all disclosure information for gie editors can befound online at httpwwwgie contentconflictofinterest cme editors and their disclosures are as followsprasad g iyer md associate editor for cmeconsultingadvisoryspeaking olympus research supporttakeda pharmaamit rastogi md associate editor for cmeconsultingadvisoryspeaking olympuskarthik ravi md cme editordisclosed no relevant financial relationshipswilliam ross md cme editorconsultingadvisoryspeaking boston scientific olympusara sahakian md cme editordisclosed no relevant financial relationshipsbrian weston md cme editordisclosed no relevant financial relationshipsall cme activities including their associated sare copyrighted by the asge gastrointestinal endoscopy volume no wwwgie 0ccme activitycontinuing medical education questions september question objectivedemonstrate triaging advanced gi endoscopy procedures during the covid19 pandemictriaging advanced gi endoscopy procedures during the covid19 pandemicquestion due to the resurgence of covid19 in your area healthofficials issue a mandate to cease elective proceduresbased on the findings of the current study a consensuswas reached that all of the following advanced gi procedures can be safely deferred for weeks exceptpossible answers aea ercp for asymptomatic choledocholithiasisb radiofrequency ablation for barrett™s esophagus withhighgrade dysplasiac endoscopic mucosal resection for barrett™s esophaguswith nodular highgrade dysplasiad endoscopic mucosal resection for a cm adenomatous colon polyp with highgrade dysplasiae eus for incident pancreatic and common bile duct dilation on ct or mri normal liver function testslookup sawhney ms bilal m pohl h triaging advanced gi endoscopy procedures during the covid19 pandemic consensus recommendationsusing the delphi method gastrointest endosc question objectivedescribe the learning curve for ablative therapy for dysplastic barrett™s esophagus and the effect of center volumes onoutcomeslearning curves and the ‚uence of procedural volume for the treatment of dysplastic barrett™sesophagusquestion a 53yearold man returns for surveillance endoscopy ofc3m5 nondysplastic barrett™s esophagus initially diagnosed years prior the patient has been compliant with twicedailyproton pump inhibitor therapy and denies any heartburnacid regurgitation or dysphagia he has no other significantmedical history you perform an egd which again reveals ac3m5 barrett™s segment without any nodularity biopsyspecimens are obtained and subsequently reveal multifocallowgrade dysplasia which is subsequently confirmed by anexpert gi pathologist after a subsequent discussionregarding ablative therapy with radiofrequency ablationrfa versus continued surveillance the patient wishes topursue ablation you discuss referral to a new colleague whojoined your practice several months earlier the colleague isthe first endoscopist to perform rfa at your institution andhas treated a total of patients the patient inquireswhether he would be more likely to have a successfuloutcome if he is referred to a moreexperienced endoscopistat a highvolume center which of the following is truepossible answers ada the patient is more likely to achieve complete remission of intestinal metaplasia crim if he undergoesablation at a center that has treated more than patientsb the patient is more likely to achieve complete remission of dysplasia crd if he undergoes ablation at acenter that has treated more than patientsc the patient is more likely to achieve crim if he undergoes ablation by an endoscopist who has treatedmore than patientsd the patient is more likely to achieve crd if he undergoes ablation by an endoscopist who has treatedmore than patientslookup lipman g markar s gupta a learning curves and the ‚uence of procedural volume for the treatment of dysplastic barrett™s esophagusgastrointest endosc wwwgie volume no gastrointestinal endoscopy 754e1 0ccme examquestion objectiveexplain the utility of a new gi bleeding prediction score for patients with upper gi bleedingthe horibe gi bleeding prediction score a simple score for triage decisionmaking in patients withsuspected upper gi bleedingquestion you are called to the emergency department to see apatient who has just arrived with the complaint of blacktarry stools the emergency department physician has notyet seen the patient but the nurse relays vital signs labresults and history sufficient to calculate a harbingerscore according to the study by horibe in thismonth™s issue this score is used to assess risk of which ofthe followingpossible answers ada deathb need for blood transfusionc need for endoscopic interventiond presence of highrisk stigmatalookup horibe m iwasaki e bazerbachi f horibe gi bleeding prediction score a simple score for triage decisionmaking in patients with suspectedupper gi bleeding gastrointest endosc question objectivereport the longterm efficacy and safety of eusguided hepaticogastrostomy for treatment of malignant biliary obstructionlongterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy inpatients with malignant biliary obstructionpossible answers ada success rate of eushgs is less than b adverse events occur in less than of casesc recurrent biliary obstruction occurs in about onethirdof patientsd prior biliary drainage has no impact on the likelihood ofrecurrent biliary obstructionquestion a 58yearold man is transferred to your hospitalformanagement of a pancreatic head mass causing obstructive jaundice and gastric outlet obstruction an endoscopic ultrasound with fineneedle aspiration of the massconfirmed pancreatic adenocarcinoma and a contrastenhanced computed tomography scan demonstratednumerous liver lesions compatible with metastatic disease a duodenal stent was placed which precluded anercp procedure for biliary drainage biliary drainage isnecessary before chemotherapy can be initiated variousoptions are discussed with the patientincluding eusguided hepaticogastrostomy eushgs according tothe current study by nakai which of the following isaccuratelookup nakai y sato t hakuta r longterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy in patients withmalignant biliary obstruction with video gastrointest endosc 754e2 gastrointestinal endoscopy volume no wwwgie 0ccme activitycontinuing medical education answers september question correct response erationale for correct responsethe covid19 pandemic has necessitated many elective procedures to be rescheduled in order to mitigate the spreadof infection and conserve vital resources choosing which advanced endoscopic procedures should be performed or safelydeferred during the covid19 pandemic may be a complex decision14 the aim of the current study was to provideguidance for triaging advanced endoscopic procedures using a modified delphi method the delphi method is a validatedand structured technique to obtain expert consensus which is useful in the present situation in which outcome data arelimited156 the following important patient outcomes were used avoidance of deathprolongation of life avoidance of cancercancer progression avoidance of major surgery andor hospitalization and improvement orpalliation of symptoms the following procedural timing categories were used timesensitive emergent schedulewithin week timesensitive urgent schedule within to weeks or nontime sensitive defer for weeks andthen reassess the timinga prespecified consensus threshold of was achieved in of advanced endoscopy indications reviewed by expert gastroenterologists one hundred percent consensus was achieved in of indications in of indications to consensus was achieved the only indication for which consensus could not be achieved was œincidentallyfound pancreatic duct dilation mm and common bile duct dilation mm on ct scan or mri with normal liverfunction teststhis study provides a decisionmaking framework for endoscopists to determine the timing for endoscopic proceduresduring and after the pandemic the decision to perform endoscopy during the covid19 pandemic needs to balance therisks associated with delaying the procedure in the individual patient with the risk of viral exposure to patients and healthcare providerstakehome message triaging advanced gi procedures during the covid19 pandemic may be facilitated by thecurrent consensus recommendations using the delphi method individual practice and patient factors must also beconsideredreferences sawhney ms bilal m pohl h triaging advanced gi endoscopy procedures during the covid19 pandemic consensus recommendations usingthe delphi method gastrointest endosc sultan s lim jk altayer o aga institute rapid recommendations for gastrointestinal procedures during the covid19 pandemic gastroenterology epub mar joint gi society message on covid19 available at httpsgi20200315jointgisocietymessageoncovid19 accessed march gastroenterology professional society guidance on endoscopic procedures during the covid19 pandemic available at httpswebfilesgilinksmediajoint_gi_society_guidance_on_endoscopic_procedure_during_covid19_final_impending_3312020pdf accessed april hsu cc sandford ba the delphi technique making sense of consensus pract assess res eval “ donohoe h stellefson m tennant b advantages and limitations of the edelphi technique implications for health education researchers am j healtheduc question correct response crationale for correct responseendoscopic eradication therapy for dysplastic barrett™s typically uses endoscopic mucosal resection of visible lesionswith subsequent ablation of the flat barrett™s segment with rfa this approach has demonstrated high clinical successwith estimates that crd is achieved in over and crim in over of treated patients1 however studies assessingthe learning curve associated with attaining proficiency with endoscopic therapy of dysplastic barrett™s and the effect ofcase volumes at centers on outcomes have yielded unclear results23 consequently current guidelines suggesting minimum numbers of cases to achieve competence for individual endoscopists or reflecting quality for centers are based onlimited evidence4wwwgie volume no gastrointestinal endoscopy 754e3 0ccme answersin this month™s issue of gie lipman and colleagues5 report results from a retrospective study examining patientstreated in the uk rfa registry to assess crd crim and dysplasia recurrence based on endoscopist experience andcenter case volumes5 a total of consecutive patients were identified from centers which were further divided into patients treated at lowvolume centers patients treated patients treated at mediumvolume centers patients treated and patients treated at highvolume centers patients treated rates of crd and crim at months did not differ based on center volumes however dysplasia recurrence washigher in patients treated at lowvolume centers further analysis using a riskadjustment cumulative risk sum curve wasdone to assess the effect of the learning curve on individual endoscopists a significant reduction of crd was seen at cases whereas a similar reduction of crim was seen at cases takehome message in conclusion this study suggests that the learning curve for rfa may be relatively short withfewer than cases required to achieve competency further center volume may have a very limited effect on outcomesfuture prospective studies are still required to assess the optimal number of training cases required to achieve endoscopicablative competencyreferences haidry rj dunn jm butt ma radiofrequency ablation and endoscopic mucosal resection for dysplastic barrett™s esophagus and early esophagealadenocarcinoma outcomes of the uk national rfa registry gastroenterology pasricha s cotton c hathorn ke effects of the learning curve on efficacy of radiofrequency ablation for barrett™s esophagus gastroenterology fudman di lightdale cj poneros jm positive correlation between endoscopist radiofrequency ablation volume and response rates in barrett™sesophagus gastrointest endosc fitzgerald rc di pietro m ragunath k british society of gastroenterology guidelines on the diagnosis and management of barrett™s oesophagusgut lipman g markar s gupta a learning curves and the ‚uence of procedural volume for the treatement of dysplastic barrett™s esophagus gastrointest endosc question correct response drationale for correct responsefor decades gastroenterologists have been searching for a straightforward method to determine whether a patientpresenting with an upper gastrointestinal bleed is at high risk of needing hospital admission therapeutic interventions ordeath multiple scoring systems have been proposed but systematic reviews have found them all flawed to various degrees12 however one of the betterknown scores proposed by blatchford has been recommended in recentguidelines to assess risk of requiring an intervention34 yet the quest for a more predictive model continues with somegroups turning to machine learning methods for an answer5in this month™s issue of gie horibe 6 report on a simple 3factor assessment to predict the likelihood of findinghighrisk stigmata hrs on endoscopy which they call harbinger because their median time to endoscopy was only hours the prevalence of hrs was quite high up to even in patients deemed to be low risk for stigmata predictablythe intervention rate was more than double that in a large international trial vs comparing harbinger toother scoring systems including blatchford™s the authors found their model to be superior this outperformance couldhave been predicted because the other models were designed to detect risk of other endpoints such as mortality or needfor intervention not hrsin addition the performance of the model was not consistently strong across the study institutions because auc valuesfor harbinger ranged from to between institutions with no overlap in confidence intervals between the topand low scores6 concerns about how harbinger would perform in other settings are reasonable as factors like access toppis varies across marketsin a resourceconstrained world the ability to predict need for interventions would seem more helpful than predictingendoscopic appearance although hrs are linked to the need for interventions the rapid time to endoscopy skews thelinkage by ballooning the prevalence of hrs many of these patients are likely to have far less ominous endoscopic appearances if the endoscopy was done hours after presentationreferences ramaekers r mukarram m smith cam the predictive value of preendoscopic risk scores to predict outcomes in emergency department patientswith upper gastrointestinal bleeding a systematic review acad emerg med de groot nl bosman jh siersema pd prediction scores in gastrointestinal bleeding a systematic review and quantitative analysis endoscopy blatchford o murray wr blatchford m a risk score to predict need for treatment for uppergastrointestinal hemorrhage lancet barkun an almadi m kuipers ej management of nonvariceal upper gastrointestinal bleeding guideline recommendations from the internationalconsensus group ann int med 754e4 gastrointestinal endoscopy volume no wwwgie 0ccme answers shung dl au b taylor ra validation of a machine learning model that outperforms clinical risk scoring systems for upper gastrointestinalbleeding gastroenterology horibe m iwasaki e bazerbachi f horibe gi bleeding prediction score a simple score for triage decisionmaking in patients with suspected uppergi bleeding gastrointest endosc stanley aj laine l dalton hr comparison or risk scoring systems for patients presenting with upper gastrointestinal bleeding international multicenter prospective study bmj 2017356i6432question correct response crationale for correct responsebiliary drainage options may be limited by gastric outlet obstruction or surgically altered anatomy eusguided hepaticogastrostomy eushgs provides an effective alternative to ercp and percutaneous drainage in these complex cases1additionally eushgs has been compared to ercp for primary biliary drainage and outcomes were found to be comparable2 however potential adverse events of eushgs such as stent migration and peritonitis can be severe and longterm data thus far have been lacking3in the current issue of gie nakai and colleagues4 present a retrospective study of patients who underwent eushgs for drainage of malignant biliary obstruction partially covered metallic stents ranging in length from to cm wereused to create the hepaticogastrostomy technical and functional success rates were and respectively adverseevents occurred in of patients with the most common being fever and abdominal pain peritonitis occurred in andcholangitis in of patients no cases of stent dislodgement were reported recurrent biliary obstruction rbo occurredin of patients in a median time of months short length of the intragastric portion of the stent and prior biliarydrainage were both associated with recurrent biliary obstruction of the patients who had rbo underwent successfulreintervention with eushgstakehome message eusguided hepaticogastrostomy is an effective alternative for longterm biliary drainage recurrences can be successfully managed with repeat hepaticogastrostomyreferences nakai y isayama h yamamoto n safety and effectiveness of a long partially covered metal stent for endoscopic ultrasoundguided hepaticogastrostomy in patients with malignant biliary obstruction endoscopy paik wh lee th park dh eusguided biliary drainage versus ercp for the primary palliation of malignant biliary obstruction a multicenter randomized clinical trial am j gastroenterol giovannini m eusguided hepaticogastrostomy endosc ultrasound 20198s35s9 nakai y sato t hakuta r longterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy in patients with malignant biliary obstruction with video gastrointest endosc wwwgie volume no gastrointestinal endoscopy 754e5 0c'

cancer7517

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: section all disclosure information for gie editors can befound online at httpwwwgie contentconflictofinterest cme editors and their disclosures are as followsprasad g iyer md associate editor for cmeconsultingadvisoryspeaking olympus research supporttakeda pharmaamit rastogi md associate editor for cmeconsultingadvisoryspeaking olympuskarthik ravi md cme editordisclosed no relevant financial relationshipswilliam ross md cme editorconsultingadvisoryspeaking boston scientific olympusara sahakian md cme editordisclosed no relevant financial relationshipsbrian weston md cme editordisclosed no relevant financial relationshipsall cme activities including their associated sare copyrighted by the asge gastrointestinal endoscopy volume no wwwgie 0ccme activitycontinuing medical education questions september question objectivedemonstrate triaging advanced gi endoscopy procedures during the covid19 pandemictriaging advanced gi endoscopy procedures during the covid19 pandemicquestion due to the resurgence of covid19 in your area healthofficials issue a mandate to cease elective proceduresbased on the findings of the current study a consensuswas reached that all of the following advanced gi procedures can be safely deferred for weeks exceptpossible answers aea ercp for asymptomatic choledocholithiasisb radiofrequency ablation for barrett™s esophagus withhighgrade dysplasiac endoscopic mucosal resection for barrett™s esophaguswith nodular highgrade dysplasiad endoscopic mucosal resection for a cm adenomatous colon polyp with highgrade dysplasiae eus for incident pancreatic and common bile duct dilation on ct or mri normal liver function testslookup sawhney ms bilal m pohl h triaging advanced gi endoscopy procedures during the covid19 pandemic consensus recommendationsusing the delphi method gastrointest endosc question objectivedescribe the learning curve for ablative therapy for dysplastic barrett™s esophagus and the effect of center volumes onoutcomeslearning curves and the ‚uence of procedural volume for the treatment of dysplastic barrett™sesophagusquestion a 53yearold man returns for surveillance endoscopy ofc3m5 nondysplastic barrett™s esophagus initially diagnosed years prior the patient has been compliant with twicedailyproton pump inhibitor therapy and denies any heartburnacid regurgitation or dysphagia he has no other significantmedical history you perform an egd which again reveals ac3m5 barrett™s segment without any nodularity biopsyspecimens are obtained and subsequently reveal multifocallowgrade dysplasia which is subsequently confirmed by anexpert gi pathologist after a subsequent discussionregarding ablative therapy with radiofrequency ablationrfa versus continued surveillance the patient wishes topursue ablation you discuss referral to a new colleague whojoined your practice several months earlier the colleague isthe first endoscopist to perform rfa at your institution andhas treated a total of patients the patient inquireswhether he would be more likely to have a successfuloutcome if he is referred to a moreexperienced endoscopistat a highvolume center which of the following is truepossible answers ada the patient is more likely to achieve complete remission of intestinal metaplasia crim if he undergoesablation at a center that has treated more than patientsb the patient is more likely to achieve complete remission of dysplasia crd if he undergoes ablation at acenter that has treated more than patientsc the patient is more likely to achieve crim if he undergoes ablation by an endoscopist who has treatedmore than patientsd the patient is more likely to achieve crd if he undergoes ablation by an endoscopist who has treatedmore than patientslookup lipman g markar s gupta a learning curves and the ‚uence of procedural volume for the treatment of dysplastic barrett™s esophagusgastrointest endosc wwwgie volume no gastrointestinal endoscopy 754e1 0ccme examquestion objectiveexplain the utility of a new gi bleeding prediction score for patients with upper gi bleedingthe horibe gi bleeding prediction score a simple score for triage decisionmaking in patients withsuspected upper gi bleedingquestion you are called to the emergency department to see apatient who has just arrived with the complaint of blacktarry stools the emergency department physician has notyet seen the patient but the nurse relays vital signs labresults and history sufficient to calculate a harbingerscore according to the study by horibe in thismonth™s issue this score is used to assess risk of which ofthe followingpossible answers ada deathb need for blood transfusionc need for endoscopic interventiond presence of highrisk stigmatalookup horibe m iwasaki e bazerbachi f horibe gi bleeding prediction score a simple score for triage decisionmaking in patients with suspectedupper gi bleeding gastrointest endosc question objectivereport the longterm efficacy and safety of eusguided hepaticogastrostomy for treatment of malignant biliary obstructionlongterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy inpatients with malignant biliary obstructionpossible answers ada success rate of eushgs is less than b adverse events occur in less than of casesc recurrent biliary obstruction occurs in about onethirdof patientsd prior biliary drainage has no impact on the likelihood ofrecurrent biliary obstructionquestion a 58yearold man is transferred to your hospitalformanagement of a pancreatic head mass causing obstructive jaundice and gastric outlet obstruction an endoscopic ultrasound with fineneedle aspiration of the massconfirmed pancreatic adenocarcinoma and a contrastenhanced computed tomography scan demonstratednumerous liver lesions compatible with metastatic disease a duodenal stent was placed which precluded anercp procedure for biliary drainage biliary drainage isnecessary before chemotherapy can be initiated variousoptions are discussed with the patientincluding eusguided hepaticogastrostomy eushgs according tothe current study by nakai which of the following isaccuratelookup nakai y sato t hakuta r longterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy in patients withmalignant biliary obstruction with video gastrointest endosc 754e2 gastrointestinal endoscopy volume no wwwgie 0ccme activitycontinuing medical education answers september question correct response erationale for correct responsethe covid19 pandemic has necessitated many elective procedures to be rescheduled in order to mitigate the spreadof infection and conserve vital resources choosing which advanced endoscopic procedures should be performed or safelydeferred during the covid19 pandemic may be a complex decision14 the aim of the current study was to provideguidance for triaging advanced endoscopic procedures using a modified delphi method the delphi method is a validatedand structured technique to obtain expert consensus which is useful in the present situation in which outcome data arelimited156 the following important patient outcomes were used avoidance of deathprolongation of life avoidance of cancercancer progression avoidance of major surgery andor hospitalization and improvement orpalliation of symptoms the following procedural timing categories were used timesensitive emergent schedulewithin week timesensitive urgent schedule within to weeks or nontime sensitive defer for weeks andthen reassess the timinga prespecified consensus threshold of was achieved in of advanced endoscopy indications reviewed by expert gastroenterologists one hundred percent consensus was achieved in of indications in of indications to consensus was achieved the only indication for which consensus could not be achieved was œincidentallyfound pancreatic duct dilation mm and common bile duct dilation mm on ct scan or mri with normal liverfunction teststhis study provides a decisionmaking framework for endoscopists to determine the timing for endoscopic proceduresduring and after the pandemic the decision to perform endoscopy during the covid19 pandemic needs to balance therisks associated with delaying the procedure in the individual patient with the risk of viral exposure to patients and healthcare providerstakehome message triaging advanced gi procedures during the covid19 pandemic may be facilitated by thecurrent consensus recommendations using the delphi method individual practice and patient factors must also beconsideredreferences sawhney ms bilal m pohl h triaging advanced gi endoscopy procedures during the covid19 pandemic consensus recommendations usingthe delphi method gastrointest endosc sultan s lim jk altayer o aga institute rapid recommendations for gastrointestinal procedures during the covid19 pandemic gastroenterology epub mar joint gi society message on covid19 available at httpsgi20200315jointgisocietymessageoncovid19 accessed march gastroenterology professional society guidance on endoscopic procedures during the covid19 pandemic available at httpswebfilesgilinksmediajoint_gi_society_guidance_on_endoscopic_procedure_during_covid19_final_impending_3312020pdf accessed april hsu cc sandford ba the delphi technique making sense of consensus pract assess res eval “ donohoe h stellefson m tennant b advantages and limitations of the edelphi technique implications for health education researchers am j healtheduc question correct response crationale for correct responseendoscopic eradication therapy for dysplastic barrett™s typically uses endoscopic mucosal resection of visible lesionswith subsequent ablation of the flat barrett™s segment with rfa this approach has demonstrated high clinical successwith estimates that crd is achieved in over and crim in over of treated patients1 however studies assessingthe learning curve associated with attaining proficiency with endoscopic therapy of dysplastic barrett™s and the effect ofcase volumes at centers on outcomes have yielded unclear results23 consequently current guidelines suggesting minimum numbers of cases to achieve competence for individual endoscopists or reflecting quality for centers are based onlimited evidence4wwwgie volume no gastrointestinal endoscopy 754e3 0ccme answersin this month™s issue of gie lipman and colleagues5 report results from a retrospective study examining patientstreated in the uk rfa registry to assess crd crim and dysplasia recurrence based on endoscopist experience andcenter case volumes5 a total of consecutive patients were identified from centers which were further divided into patients treated at lowvolume centers patients treated patients treated at mediumvolume centers patients treated and patients treated at highvolume centers patients treated rates of crd and crim at months did not differ based on center volumes however dysplasia recurrence washigher in patients treated at lowvolume centers further analysis using a riskadjustment cumulative risk sum curve wasdone to assess the effect of the learning curve on individual endoscopists a significant reduction of crd was seen at cases whereas a similar reduction of crim was seen at cases takehome message in conclusion this study suggests that the learning curve for rfa may be relatively short withfewer than cases required to achieve competency further center volume may have a very limited effect on outcomesfuture prospective studies are still required to assess the optimal number of training cases required to achieve endoscopicablative competencyreferences haidry rj dunn jm butt ma radiofrequency ablation and endoscopic mucosal resection for dysplastic barrett™s esophagus and early esophagealadenocarcinoma outcomes of the uk national rfa registry gastroenterology pasricha s cotton c hathorn ke effects of the learning curve on efficacy of radiofrequency ablation for barrett™s esophagus gastroenterology fudman di lightdale cj poneros jm positive correlation between endoscopist radiofrequency ablation volume and response rates in barrett™sesophagus gastrointest endosc fitzgerald rc di pietro m ragunath k british society of gastroenterology guidelines on the diagnosis and management of barrett™s oesophagusgut lipman g markar s gupta a learning curves and the ‚uence of procedural volume for the treatement of dysplastic barrett™s esophagus gastrointest endosc question correct response drationale for correct responsefor decades gastroenterologists have been searching for a straightforward method to determine whether a patientpresenting with an upper gastrointestinal bleed is at high risk of needing hospital admission therapeutic interventions ordeath multiple scoring systems have been proposed but systematic reviews have found them all flawed to various degrees12 however one of the betterknown scores proposed by blatchford has been recommended in recentguidelines to assess risk of requiring an intervention34 yet the quest for a more predictive model continues with somegroups turning to machine learning methods for an answer5in this month™s issue of gie horibe 6 report on a simple 3factor assessment to predict the likelihood of findinghighrisk stigmata hrs on endoscopy which they call harbinger because their median time to endoscopy was only hours the prevalence of hrs was quite high up to even in patients deemed to be low risk for stigmata predictablythe intervention rate was more than double that in a large international trial vs comparing harbinger toother scoring systems including blatchford™s the authors found their model to be superior this outperformance couldhave been predicted because the other models were designed to detect risk of other endpoints such as mortality or needfor intervention not hrsin addition the performance of the model was not consistently strong across the study institutions because auc valuesfor harbinger ranged from to between institutions with no overlap in confidence intervals between the topand low scores6 concerns about how harbinger would perform in other settings are reasonable as factors like access toppis varies across marketsin a resourceconstrained world the ability to predict need for interventions would seem more helpful than predictingendoscopic appearance although hrs are linked to the need for interventions the rapid time to endoscopy skews thelinkage by ballooning the prevalence of hrs many of these patients are likely to have far less ominous endoscopic appearances if the endoscopy was done hours after presentationreferences ramaekers r mukarram m smith cam the predictive value of preendoscopic risk scores to predict outcomes in emergency department patientswith upper gastrointestinal bleeding a systematic review acad emerg med de groot nl bosman jh siersema pd prediction scores in gastrointestinal bleeding a systematic review and quantitative analysis endoscopy blatchford o murray wr blatchford m a risk score to predict need for treatment for uppergastrointestinal hemorrhage lancet barkun an almadi m kuipers ej management of nonvariceal upper gastrointestinal bleeding guideline recommendations from the internationalconsensus group ann int med 754e4 gastrointestinal endoscopy volume no wwwgie 0ccme answers shung dl au b taylor ra validation of a machine learning model that outperforms clinical risk scoring systems for upper gastrointestinalbleeding gastroenterology horibe m iwasaki e bazerbachi f horibe gi bleeding prediction score a simple score for triage decisionmaking in patients with suspected uppergi bleeding gastrointest endosc stanley aj laine l dalton hr comparison or risk scoring systems for patients presenting with upper gastrointestinal bleeding international multicenter prospective study bmj 2017356i6432question correct response crationale for correct responsebiliary drainage options may be limited by gastric outlet obstruction or surgically altered anatomy eusguided hepaticogastrostomy eushgs provides an effective alternative to ercp and percutaneous drainage in these complex cases1additionally eushgs has been compared to ercp for primary biliary drainage and outcomes were found to be comparable2 however potential adverse events of eushgs such as stent migration and peritonitis can be severe and longterm data thus far have been lacking3in the current issue of gie nakai and colleagues4 present a retrospective study of patients who underwent eushgs for drainage of malignant biliary obstruction partially covered metallic stents ranging in length from to cm wereused to create the hepaticogastrostomy technical and functional success rates were and respectively adverseevents occurred in of patients with the most common being fever and abdominal pain peritonitis occurred in andcholangitis in of patients no cases of stent dislodgement were reported recurrent biliary obstruction rbo occurredin of patients in a median time of months short length of the intragastric portion of the stent and prior biliarydrainage were both associated with recurrent biliary obstruction of the patients who had rbo underwent successfulreintervention with eushgstakehome message eusguided hepaticogastrostomy is an effective alternative for longterm biliary drainage recurrences can be successfully managed with repeat hepaticogastrostomyreferences nakai y isayama h yamamoto n safety and effectiveness of a long partially covered metal stent for endoscopic ultrasoundguided hepaticogastrostomy in patients with malignant biliary obstruction endoscopy paik wh lee th park dh eusguided biliary drainage versus ercp for the primary palliation of malignant biliary obstruction a multicenter randomized clinical trial am j gastroenterol giovannini m eusguided hepaticogastrostomy endosc ultrasound 20198s35s9 nakai y sato t hakuta r longterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy in patients with malignant biliary obstruction with video gastrointest endosc wwwgie volume no gastrointestinal endoscopy 754e5 0c' Answer:

Colon_Cancer

"autophagy is an evolutionary cellular program that serves for thebreakdown of cytoplasmic components within lysosomes [“] inidentified autophagy in's electron microscopic studies firstmammalian cells but the molecular pathways were not understooduntil the discovery of autophagy genes atgs in yeast by performinggenetic screening it is a cytoprotective rather than a selfdestructive process it is extensively accepted as a main regulator of innateand adaptive immune mechanisms the change in which completelyimpact the pathogenesis of disease and the processes that are influencedby autophagy includes the regulation of inflammation antigen presentation and bacterial clearance moreover autophagy aids in themaintenance of fundamental anelle populations such as mitochondria which is necessary for cellular bioenergetics and homeostasis homeostasis of the cell is been accomplished by maintaining the biosynthesis and turnoverthere are two broader protein degrading systems in eukaryoticcells first is the ubiquitinproteasome system responsible for the selective breakdown of most shortlived proteins and second is the lysosomalsystem the primary anelle called lysosome ineukaryotes is known for degradation through its acid hydrolases inunfavourable nutrient deprivation condition autophagy arbitrates aregulated phagocytosis via lysosomes autophagy is mediated byautophagosome that is thought to be an on selective degradation systemas it engulfs some of the cytoplasmic contents the ubiquitin“proteasome system concedes only ubiquitinated proteins for degradation andit marks a remarkable contrast to autophagosome process autophagosomes a doublemembered vesicle that engulfs durableproteins impaired anelles intracellular pathogenic anisms andtransports it to the lysosomes that are fused to form autolysosome andthe inner vesicle along with its cargo is been degraded at the time ofstarvation the remaining macromolecules are again recycled to thecytosol for reuse the precise mechanism in cargo recognition isuncertain but this process involves ubiquitination autophagicprocess is separated into distinct steps which includes induction recognition selection of cargo formation of vesicle then occurs the fusion of autophagosomevacuole followed by the degradation of thecargo and release into the cytosol various atg proteins are indulged inthis process and it consists of the central autophagic machinery autophagy encompasses different process by which cells delivercytoplasmicaredegradation theylysosomalsubstratesforŽ corresponding author at department of biotechnology psg college of arts and science civil aerodrome post coimbatore indiaemail address rashmiffrajangmailcom rr rasmi all authors deserve contribution equally101016jlfs2020118308received june received in revised form august accepted august available online august elsevier inc all rights reserved 0cs vishnupriya life sciences macroautophagy chaperonemediated autophagy cma and microautophagy all the three processes of autophagy are morphologically distinct in microautophagy the lysosomal membrane invaginations or protrusions are needed to seize cargo once thecargo is captured the uptake directly happens at the limiting membrane of the lysosome that includes intact anelles cma uses chaperones to sequester cargo proteins that have a pentapeptide motifthese proteins are unfolded and translocated directly across the lysosomal membrane via lamp2a receptor macroautophagy involves requisition of cargo vesicle formation and its subsequenttransport to the lysosome in recent years deletion of the autophagy related genes atg invarious model anisms has proved that autophagy plays decisive rolein adaptive responses to stress cellular differentiation and development an oncogenic event may establish by the partial minimization inthe autophagic capacity atg6beclin one of the phylogeneticallypremeditated autophagy genes is often subdued at one locus in humancancers studies in mice have shown that beclin is a haplo insufficienttumor suppressor autophagic programmed cell death was primarily depicted in actively developing tissues several conjugationsystems comprising of the atg genes are available that take part inautophagasomal elongation one such system is the lc3 microtubule associated protein light chain and atg8 conjugation system lc3 is the mammalian conjugative protein ortholog of yeastprotein atg8 the lipid derivative phosphatidylethanolamine bindsto lc3 to form lc3ii an important molecular marker of autophagylc3ii remains on the mature autophagosome until it fuses with thelysosomes fig the beclin complex gives rise to an incipientautophagosome membrane and it assemble around cargo in a vesiclethat combines with a lysosome forming autolysosome that is degradedby acid hydrolases present in the lysosomes lung injury clinical implicationsthe primary an of gas exchange is the pair of lungs theymediate inspiration of oxygen and elimination of mono and dioxides ofcarbon lung also serves as an attractive target for the entry of thepathogens regulation of the pulmonary functions is mediated by intricate cells of endothelial and epithelial lining dendritic cells alveolarmacrophages and fibroblasts all these pulmonary cells are highlyheterogeneous in nature they together in association respond to thelung injury by provoking inflammatory and immune responses airway epithelial cells express pattern recognition receptors prrsalong with toll like receptors ctype lectins rigi and inflammasome components which are involved in the innate response against microbes microbial cell wall constituents likelipopolysaccharide are sensed by prrs and induce an inflammatoryresponse alveolar epithelium comprises type i and type ii alveolarcells apart from these the mucus layer and the physical barrier madeby the epithelium contribute to the first line of defense lung injury is described as any damage to the associated tissues and compartments of the pulmonary system the concept of lung injury influenced by the microenvironment can be demonstrated via series ofchanges in cell deformability and manifestation of intercellular adhesive molecules the major causes of lung injury are atelectasisalveolar instability volutrauma barotrauma infections and oxygentoxicity the pathogenicity of acute and chronic lung injury iscorrelated with the release of proteases free radicals and growth regulatory proteins by the alveolar macrophages neutrophil takescare of the extreme ranges of lungs searching for pathogens therebyfig mechanism of autophagy ulk1pi3kmtor signalling pathway binds to endoplasmic reticulum and activates dcedp1 that initiates beclin and atg512conjugation system which forms isolation membrane this is followed by the sequestration process that leads to autophagosomal formation the autophagosomefuses with lysosomes to form autolyososome that leads to degradation 0cs vishnupriya eliminating via phagocytosis they undergo transendothelial andtransepithelial migration primary host defense mechanism oflungs include the immunity conferred by surfactant proteins spnamely spa and spd they link both innate and adaptive immunity byregulating the responses of innate immune cells antigen presentingcells apcs and tcells they can act as potential biomarkers of lunginjury the implication of autophagy over lung injury is tortuousas its function varies highly with the cell types specific to the pulmonary disorders it provides both defensive and injurious outcomes of lung injury lung injury “ types and associated pathologies acute lung injury ali and acute respiratory distress syndromeardsacute lung injury and acute respiratory distress syndrome arecommon grievous diseases among critically illpatients and are evidential sources of mortality and morbidity they are expressed alongwith hypercapnia and hypoxemia ards is outlined as the most seriousform of ali ali is characterized by inflammation of lung surfacesresulting in the disruption of alveolarcapillary membrane followed bytransmigration of neutrophils significant event in the procession of aliand ards and outbreak of cytotoxic mediators immune responsemediated by several components like tcells macrophages naturalkiller nk cells chemokines and proinflammatory cytokines also has apredominant role in the progression of the acute injury and results inimmune reconstitution inflammatory syndrome iris immune cellsinvolved in the immune response like macrophages and monocytes arethought to be involved in the pathophysiology of iris elevatedlevels of cd14 cd16ˆ’ monocyte population and an resulting increase in the proinflammatory cytokines il6 tnfα and c reactiveprotein crp are also observed in tuberculosis tbiris patients lung endothelial biomarkers like vwf and epithelial biomarkers likespd are the diagnostic targets of ali sepsis pancreatitis pneumoniatrauma transfusions aspiration and inhalation of toxic gases are someof the clinical factors of ali and ards histological patterns ofali and ards have the chances of demonstrating diffuse alveolar damage alveolar haemorrhage eosinophilic pneumonia and acute fibrinous pneumonia associated with ards may also result frompathogens like fungal viral bacterial and parasitic most commonpathogen strains include streptococcus pneumoniae respiratory virusesstaphylococcus aureus fungal pathogens like pneumocystis jirovecii andaspergillus fumigatus legionella pneumophila and enteric gramnegativeanisms among bacteria the virulence capability of pseudomonas aeruginosa is one of the prime determinant of the severity of thelung injury disease progress takes place in three degrees namelyacute phase exudative subacute phase proliferative and chronicphase fibrotic radiological features during the acute phasesignify twosided patchy groundglass densities relating to interstitialedema and hyaline membranes the important constituent of innateimmune system the pattern recognition receptors prrs are affiliatedwith the advancement of ali and ards the ligands of prrs includepathogenassociated molecular patterns pamps which induce inflammatory signalling events and the damageassociated molecularpatterns damps which induce neutrophilmediated tissue damageincreased incidences of mitochondrial damps result in high mortalityrates a common type of ali is the transfusionrelated acute lunginjury trali trali is defined as adult respiratory distress syndromeoccurring with transfusionsis manifested by pulmonary insufficiency severe hypoxia and dyspnea it is often reported as theneutrophil pmnmediated syndrome a particular study has reported that the potential risk factors for ali and ards may be associated with larger tidal volume vt and higher airway pressure pawduring one lung ventilation olv in postpneumonectomy aliardspatients and also in patients who developed aliards longtermeffects after acute lung injury are related to neurological impairmentsitlife sciences namely neuromuscular dysfunction neurocognitive dysfunction andneuropsychological dysfunction some of the minor impacts includeheterotopic ossification stiae and frozen joints chronic lung injury cli and bronchopulmonary dysplasia bpdchronic lung injury is characterized by a condition called pleuroparenchymal fibroelastosis ppfe in which a rapid multiplication ofsubpleural intestinal elastic fibres majorly in the upper lobes predominates along with other clinicopathological conditions following chronic lung injury cells undergoing apoptosis is cleared by aprocess called efferocytosis it is a type of phagocytosis confined only tothe cells that undergo apoptosis to maintain the cellturnover in pulmonary airways carried out mainly by the macrophages while immature dendritic cells mesenchymal cells and epithelial cells can alsoperform efferocytosis recently sarscov2 manifested as severerespiratory infection resulted in number of deaths worldwide commonrisk factors associated with death in sarscov2 patients identified arehypertension diabetes cardiovascular disease or chronic lung disease the most prevailing chronic lung injury ofinfants is thebronchopulmonary dysplasia bpd occurs when preterm infants suffering from various respiratory syndromes like meconium aspirationsyndrome and neonatal pneumonia are subjected to treatment withsupplemental oxygen and extensive mechanical ventilators it is proventhat the common risk factors of bpd increases with a fall in birthweight prematurity and gestational period apart from theabove other perinatal risk factors with which bpd is associated with areintrauterine growth restrictions race or ethnicity chorioamnionitis and genetic risk factors it is characterized bysaccular formation and elastic fibre aggregation of distal air spacesinflation and edema barotrauma and pulmonary oxygen toxicity arepathologies of bpd bpd is multifactorial in nature studiesdeclare that surviving patients of bpd have established pulmonarydysfunction incidence adults with bpd history are strictly prohibitedto cigarette smoking another form of bpd called the new bpdwhen surfactants are used as treatment new bpd is depicted by pulmonary hypertension and abnormalities in vasculopulmonary development glucocorticoids and tgfbeta are the efficient modulators thatinitiate bpd injury bpd infants have significantly lower levels ofvascular endothelial growth factor and platelet endothelial cell adhesion molecules chronic obstructive pulmonary disease copd and asthmacopd is a general illness worldwide it is defined as the completeirreversible state of airflow limitation characterized by weak inflammatory response of the lungs emphysema chronic obstructivebronchiolitis fibrosis blocking and narrowing of airways deprivationof lung parenchyma and elasticity immune cells like tlymphocyteswith cd8 dominance blymphocytes macrophages and neutrophilsare the regulators of copd acute provocation of copd is definedas the continuous deterioration from steady state facilitating an alteration in typical medication for rudimentary copd systemicinflammation responses are a result of leukotriene b4 tnfalpha interleukin il8 and proteases a decrease in the ratio of cd4 to cd8is a typical feature of pulmonary inflammatory responses additionalimpacts of copd are cardiovascular diseases nervous effects and osteoskeletal effects smoking is an important cause of systemicoxidative stresses immoderate inflammatory responses and emphysema as manifested as copd implications copd being an hetergoenousdisease patients with exacerbations are found to be associated withbacterial infections like moraxella catarrhalis streptococcus pneumoniaand haemophilus influenza apart from bacteria other microbes likevirus and fungi also comtribute to the lung microbiota and pathogenesisof lung microbiota through initiation of chronic inflammation it hasbeen found that bacteria and viruses fungi can promote local andsystemic inflammation that may contribute to the pathogenesis ofcopd specific biomarkers of copd observed are creactive 0cs vishnupriya protein crp il6 il10 and ccl18parc skeletal muscle losstakes place in copd wasting of the cell mass is evidenced to be theresult of tnfα participation in the pathogenesis of copd muscleglutamate reduction is linked with lactic acidosis in copd patientsending in muscle wastage similar to copd asthma is characterized by pulmonary obstruction and similar immune responses onlyvariation from copd is that the airway obstruction in asthma is reversible and it does not affect lung parenchyma dendritic cells are themodulators of th2 cells playing an important immune response ofasthma severe asthma is equal to the effects of copd illustrated by theincrease in neutrophils tumor necrosis factor tnf cxcl8 and decreased reception to corticosteroids while reversible copd is potentially to have subsequent asthma and copd ventilatorinduced lung injury vili and ventilatorassociated lunginjury valithe prevailing lung injury cases with ards when given ventilatorassistance mechanically in a clinical setup may develop additional lunginjuries ending up in ventilatorassociated lung injury vali similarlyin experimental models lung injury can be provoked by external application of injurious ventilation procedures contributing to ventilatorinduced lung injury vili thus vali can adversely aggravate thehealth of the ards patients at low lung volumes of ventilation atelectrauma occurs at high lung volumes of ventilation barotrauma andpulmonary edema occur biomarkers studied via experimental modelsof vali include several proinflammatory molecules like tnfα il1βil8 and antiinflammatory molecules like il10 il6 and stnfr1 respectively [“] positive endrespiratory pressure peep and tidalvolume applied have direct influence on vali and are to be studiedspecifically during ali and edema it is reported that peep when provokes overinflation the extent of edema also increases mechanism ofsurfactant inactivation is seen in alveolar microvessels with an increasein fluid filtration further greater the lung volume higher is thetransmural pressure in them a retrospective cohort study revealedthat height and gender of the patients should be taken into considerations along with establishing limitations to large tidal volumes beforesetting up a ventilator experiments explain that decrease in thepeak pulmonary arterial pressure or respiratory frequency can lessenthe grimness effects of vali it is proven that hypercapnic acidosisaffords protection to vili addressable implications of vali arebarotrauma volutrauma atelectrauma biotrauma and oxytoxic effects cellular pathology includes physical disruption of cells and tissuesand activation of cytotoxic responses leucocytes are raised to likelyinteract with the endothelium as the increasing intraalveolar pressurefastens the transit time of them inferences for current medicalpractices involve lungprotective ventilation survival of patients can beencouraged by prone positioning future clinical practices involveprecisioned ventilationindividualized tidal volumes using drivingpressure individualized peep and extracorporeal strategies pulmonary fibrosis pf and cystic fibrosis cfidiopathicpulmonary fibrosis ipf is a degenerative interstitial fibrosing disease prevalent worldwide it is also termed as cryptogenicfibrosing alveolitis a typical honeycomblike structure of asymmetricairspaces covered by dense fibrosis is observed ipf is followed by interstitial pneumonia which is featured by deficit inflammation and withabsence of homogeneous participation of lung tissues studies havebeen made since ages which revealed several inherited forms of pulmonary fibrosis mutations in various genes were associated to pfnamely sftpc sftpa2 tert terc and muc5b incidence withrheumatoid arthritis and scleroderma are more likely to form pf thereis a chance of clearance of alveolar basement membrane and occurrenceof hyperplastic epithelial cells ipf ensures migration of fibroblastsinto the fibrinrich exudates thus a chemoattractant activity is createdin the airspaces after lung injury this process is proven to be regulatedthe proby lysophosphatidic acid experiment proves thatlife sciences inflammatory cytokines il1β directly regulates the initiation of acuteand chronic inflammation making it a valuable target of ipf cystic fibrosis is characterized as a most common autosomal recessivegenetic disorder caused by the mutation in cftr gene transmembraneprotein genecystic fibrosis transmembrane conductance receptor pathology of the disease involves bronchiolitis obstruction of pathways endobronchiolar infection impaired ciliary actions atelectasisfinally leading to secondary alveolar injury bacterial infections of saureus p aeruginosa and h influenzae causes cf it is the pulmonarymacrophages and polymorphonuclear neutrophils that forms the defense against infections while lymphocytemediated mucosal injury isobserved in cf patients cf patients have increased flow of tnfαil8 and il1β submucosal glands of the lungs are the crucialhosts of cftr genes as the number expressed are high hence cfcontributes to epithelial airway lining abnormalities with respect to thechanges taking place in the submucosal glands respectively the macromolecular secretions of the submucosal glands have changes in theircomposition viscosity and greatly impacts on the mucociliary clearance radiationinduced lung injury riliseveral complications of the lung malignancies require treatmentsinvolving radiation therapy rt extensive reports claim that rt maylead to a state of rili the threedimensional dosimetric predictors canbe emphasized for the risk of symptomatic rili the alveolarcapillary subunit forms the most radiosensitive complex of the lungs thusthe rili is also called as the diffuse alveolar damage radiation exposure provokes the production of reactive oxygen species creatinghigh toxicity levels in the lung parenchyma this may ultimately lead tolung fibrosis which develops one to six months after rt accompaniedby dyspnea in addition to fibrosis rili also develops radiationinduced pneumonitis gradually after months to years almost all thepatients undergoing rt have the chance of developing fibrosis radiationinduced pneumonitis is characterized by cough occasional fevernonspecific symptoms of dyspnea and chest pain with or without deformities in pulmonary functional tests while radiationinduced pulmonary fibrosis is characterized by cough differential levels of dyspnea chest pain or symptomless and stable scarring of the lung tissueswhen detected radiographically it has been stated that the incidence and occurrence of rili is directly influenced by dose and volume determinants of rt several studies provide information onrili that result in provoking inflammatory responses a biphasicmanifestation of cytokines is observed in the lung tissues once exposedto rt one such study carried out to assess the cytokine productionin c3hhen irradiated mice revealed a spatial change in the expression of proinflammatory cytokines among various cellular compartments of the lungs further it was noted that the bronchoalveolar lavage cells responded immediately while the interstitial cells contributedonly in the later stages during pneumonitis profiling of cytokinesnamely the interleukins interferons monocyte chemotactic protein1tumor necrosis factors macrophage inflammatory proteins and granulocyte colonystimulating factors can be performed to analyse the developmental risks of symptomatic radiationinduced lung injury in vitro studies experiments state that the application of melatonin andcarnosine compounds reduced the reactive oxygen species and inflammatory cytokines produced following rili regulators of autophagy in lung injurynumerous regulators of autophagy play a vital part in the development of lung injury with regard to autophagy the following are theimportant regulators fig the mechanism by which these autophagy regulators act is shown in the table lc3biithe microtubuleassociated protein light chain lc3 is the 0cs vishnupriya life sciences fig figure depicts the autophagy regulators in regard to lung injury various conditions like hypoxia starvation environmental stress and cigarette smoke leadsto autophagy that in turn causes lung injury activation of class ipi3kmtor pathway takes place under hypoxic and starvation conditions while environmentalstress results in provoking beclin 1vps34 pathway that induces the sequestration of the phagophore formation cigarette smoke induces ros accumulation which inturn causes egr1 e2f signalling to activate lc3ii along with sqtm1p62 and atg512 conjugation system the ros generated may also leads to apoptosis theautophagosome fuses with lysosomes and forms autophagolysosome that causes pulmonary arterial hypertension and lung injuryprinciple autophagic protein expressed on the doublemembraned autophagosome nearly eight homologues of lc3 proteins are studied inmammals the amino acid composition of these homologues classifiesthem into two subfamilies first one comprising of lc3a splicingvariants lc3b and lc3c taking part in autophagosomal membraneelongation while the second one comprising of gabarap gabarapl1 gabarapl2 and gabarapl3 taking part in the maturation ofautophagosome generally lc3b occurs in cytosol as lc3bi by theproteolytic cleavage of cterminal of lc3b which then unites withphosphatidylethanolamine to form lc3bii to assemble on the autophagosomal membrane conjugation of phosphatidylethanolamine withlc3bii can be revoked by the activity of atg4 thus it is clear thatoccurrence of lc3bii is crucial in the process of autophagy lc3bii levels are analysed through immunoblotting while limitationsare degradation of lc3bii by autophagy itself and the nonindication ofautophagic flux at distinct time points this can be overcome by comparison studies with lc3bi and using lysosomal protease inhibitors antilc3b antibodies can be applied to detect autophagy invarious cell types application of antilc3b to glioblastoma tissuesdemonstrated a positive detection of lc3b levels both in vitro and invivo suggesting a latent monitoring system of lc3b severalstudies state that hyperoxic conditions can result in ali and ards thisfurther triggers the morphological biomarker of autophagy lc3bii toaccumulate thereby determining the fate of cell clearance experimentsperformed in hyperoxiainduced human bronchial epithelial cells andcultured epithelial cells beas2b clearly stated that the expression oflc3bii was high mediated by the apoptotic regulators similarexperiment carried out in the hyperoxiainduced lung injury in c57bl mice inferred the involvement of apoptotic pathways in the activationof lc3bii interaction of lc3bii with fas proteins was observedmarking the importance of lc3bii in the management of ali pulmonary hypertension is a main cause of copd manifestation inlungs that affects vascular architecture when chronic hyperoxia wasinduced in the lung tissue extracts of patients with pulmonarytable regulators of autophagy in lung injury and their mechanismregulatorslc3biibeclin p62hif1bnip3mtormechanismelongation of autophagosome and its maturation requires atg8map1lc3 protein lc3i combines with phosphatidylethanolamine forming lc3ii essential for autophagosome formationthe initiation of the isolation membrane that forms autophagosome after the sequestration process is regulated by beclin and pi3kp62 along with sqstm1 is the receptor for polyubiquitinated substrates it helps in the transportation of cargo into the autophagosome by bindingwith lc3ii for degradationduring hypoxic conditions hif1 induces bnip3 that in turn brings about cell survival through autophagy and provoke cell death by apoptosisthe mammalian homologue atg13 is phosphorylated by mtor that binds ulk1 proteins fip200 phosphorylates ulk and initiates the isolationmembrane formation in autophagyreference no 0cs vishnupriya hypertension the upregulation of lc3b prevailed indicating the regulatory role of lc3b in vascular cell proliferation and mediatingadaptive cellular responses smoking results in various implications of lung injury in due course a multimeric protein complexcomprising of lc3bcaveolin1fas occurs under basal state extensivestudies reveal that smoking triggers lc3b to initiate the dissociation ofcaveolin1 from fas protein thus facilitating apoptotic pathways accordingly emphysema a destructive expression of copd isworsened by cigarette smoking in vitro studies in lung tissues of miceon exposure to cigarette smoking ensured the driving role of lc3b inregulating apoptotic mechanisms and finally developing emphysemarespectively all these experiments prove the comprehensive bridgebetween autophagy and apoptosis highly regulated by the expressionlc3b lc3bi and lc3bii bind to microtubule associated protein1b map1b wherein overexpression of map1b decreases the levels oflc3bii protein kinase c is known to cease autophagy by interferingwith autophagosomal formation both in vitro and in vivo studiesconfirmed that the lc3b phosphorylation by protein kinase c takesplace consistently in lungs the emergence of autophagy either asa protective role or maladaptive response due to sepsis was studied in acecal ligation and puncture clp induced septic mice it was ensuredautophagy to be a protective response yet an overexpression of lc3bii in the later stages of sepsis leads to ali describing a maladaptive role lung injury can be provoked by ischemiareperfusion in whichautophagy is stated as the safeguarding mechanism by moderatelymaintaining the level of lc3bii also the ischemiareperfusioninduced lung injury is positively governed by the erk12 signallingpathway that regulates the cellular expression of lc3bii respectively nanoparticles of zinc oxide on exposure to lungs may induceali further zinc oxide nanoparticles resulted in the raise of autophagosomal structures followed by the accumulation of lc3bii proteinsthus zno nanoparticlesinduced ali is autophagy dependent 3methyladenine a classical autophagy inhibitor reduced the manifestationof lc3bii and lowered the release of zinc particles thereby stoppingzno nanoparticlesrelated toxicity of lungs similarly the artificially synthesized polyamidoamine dendrimers pamam used as aneffective drugdelivery system may sometimes result in pamam nanoparticlesinduced ali the levels of lc3bii biomarkers were highindicating the autophagic responses beclin beclin was first identified by beth levine as becn1atg6 inchromosome 17q21 in the year and it is the major autophagyregulating gene it is a coiledcoil protein of molecular weight kda comprising of amino acids and acts together with bcl2an antiapoptotic protein beclin is an indispensable autophagypromoting gene that is homologue to the mammalian yeastatg6 gene which regulates cell survival of different types and is involved in the constitution of autophagosomes the initiation ofthe anization of autophagosomes is regulated by class iii phsophoinositide 3kinase pi3k and autophagy related gene beclin beclin has got a novel bcl2 homology region3 bh3 domainthe bh3 domain in beclin1 can bind to bcl family proteins that initiateapoptotic signalling and prevents the beclin 1mediated autophagy byremoving beclin from hvps34 either phosphorylation or ubiquitination of beclin or bcl2 can disunite bcl2 from beclin andincrease the activity of vps34 kinase which brings about increase infunction during autophagy autophagyassociated protein beclin1 binds to lc3i that adapts to its membranebound form lc3iiand it cooperates with the ubiquitinbinding protein p62sequestosome sqstm1 the first an that fails during sepsis is lungs thefamiliar complications of sepsis are ali and ards ali activated various autophagy related proteins like lc3ii beclin and lysosomerelated protein lamp2 and rab7 expressions in sepsisinduced ali to evaluate the function of autophagy in severe sepsis an experiment is carried out using endotoxemia that frequently uses septiclife sciences shock and clp which is a clinical polymicrobial sepsis model herebecn1ˆ’ mice was susceptible to clpinduced sepsis in cysticfibrosis transmembrane conductance regulator cftr autophagy bybeclin overexpression cystamine or antioxidants and the restorationof beclin recovers the localization of beclin to endoplasmic reticulum and regresses the cf airway phenotype both in vitro and in vivoin scnn1btransgenic and cftr f508del homozygous mice and also inhuman cf nasal biopsies in lpsinduced ali there are threedistinct complexes of beclin1vps34 have been identified the firstcomplex contains beclin1 vps34 vps15 and atg14l second complexcontains beclin1 vps34 vps15 and ultraviolet irradiation resistanceassociated gene uvarag and the final complex contains beclin1vps34 vps15 uvrag and rubicon among these complex the onethat contains atg14l is concerned in the formation of autophagosomewhile others are in the autophagosome and endosome maturationbeclin forms the bridge in the recruitment of inducers and suppressorsof autophagy and simultaneously behaves as a key modulator in autophagosome formation mesenchymal stem cells mscs increases the translation level of beclin but not its transcription ratemscs might alleviate lpsali via downregulation of mir142a5p thatpermits pulmonary epithelial cells pecs to proceed with beclin mediated cell autophagy in lung disease the bacterial stu

cancer7518

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "autophagy is an evolutionary cellular program that serves for thebreakdown of cytoplasmic components within lysosomes [“] inidentified autophagy in's electron microscopic studies firstmammalian cells but the molecular pathways were not understooduntil the discovery of autophagy genes atgs in yeast by performinggenetic screening it is a cytoprotective rather than a selfdestructive process it is extensively accepted as a main regulator of innateand adaptive immune mechanisms the change in which completelyimpact the pathogenesis of disease and the processes that are influencedby autophagy includes the regulation of inflammation antigen presentation and bacterial clearance moreover autophagy aids in themaintenance of fundamental anelle populations such as mitochondria which is necessary for cellular bioenergetics and homeostasis homeostasis of the cell is been accomplished by maintaining the biosynthesis and turnoverthere are two broader protein degrading systems in eukaryoticcells first is the ubiquitinproteasome system responsible for the selective breakdown of most shortlived proteins and second is the lysosomalsystem the primary anelle called lysosome ineukaryotes is known for degradation through its acid hydrolases inunfavourable nutrient deprivation condition autophagy arbitrates aregulated phagocytosis via lysosomes autophagy is mediated byautophagosome that is thought to be an on selective degradation systemas it engulfs some of the cytoplasmic contents the ubiquitin“proteasome system concedes only ubiquitinated proteins for degradation andit marks a remarkable contrast to autophagosome process autophagosomes a doublemembered vesicle that engulfs durableproteins impaired anelles intracellular pathogenic anisms andtransports it to the lysosomes that are fused to form autolysosome andthe inner vesicle along with its cargo is been degraded at the time ofstarvation the remaining macromolecules are again recycled to thecytosol for reuse the precise mechanism in cargo recognition isuncertain but this process involves ubiquitination autophagicprocess is separated into distinct steps which includes induction recognition selection of cargo formation of vesicle then occurs the fusion of autophagosomevacuole followed by the degradation of thecargo and release into the cytosol various atg proteins are indulged inthis process and it consists of the central autophagic machinery autophagy encompasses different process by which cells delivercytoplasmicaredegradation theylysosomalsubstratesforŽ corresponding author at department of biotechnology psg college of arts and science civil aerodrome post coimbatore indiaemail address rashmiffrajangmailcom rr rasmi all authors deserve contribution equally101016jlfs2020118308received june received in revised form august accepted august available online august elsevier inc all rights reserved 0cs vishnupriya life sciences macroautophagy chaperonemediated autophagy cma and microautophagy all the three processes of autophagy are morphologically distinct in microautophagy the lysosomal membrane invaginations or protrusions are needed to seize cargo once thecargo is captured the uptake directly happens at the limiting membrane of the lysosome that includes intact anelles cma uses chaperones to sequester cargo proteins that have a pentapeptide motifthese proteins are unfolded and translocated directly across the lysosomal membrane via lamp2a receptor macroautophagy involves requisition of cargo vesicle formation and its subsequenttransport to the lysosome in recent years deletion of the autophagy related genes atg invarious model anisms has proved that autophagy plays decisive rolein adaptive responses to stress cellular differentiation and development an oncogenic event may establish by the partial minimization inthe autophagic capacity atg6beclin one of the phylogeneticallypremeditated autophagy genes is often subdued at one locus in humancancers studies in mice have shown that beclin is a haplo insufficienttumor suppressor autophagic programmed cell death was primarily depicted in actively developing tissues several conjugationsystems comprising of the atg genes are available that take part inautophagasomal elongation one such system is the lc3 microtubule associated protein light chain and atg8 conjugation system lc3 is the mammalian conjugative protein ortholog of yeastprotein atg8 the lipid derivative phosphatidylethanolamine bindsto lc3 to form lc3ii an important molecular marker of autophagylc3ii remains on the mature autophagosome until it fuses with thelysosomes fig the beclin complex gives rise to an incipientautophagosome membrane and it assemble around cargo in a vesiclethat combines with a lysosome forming autolysosome that is degradedby acid hydrolases present in the lysosomes lung injury clinical implicationsthe primary an of gas exchange is the pair of lungs theymediate inspiration of oxygen and elimination of mono and dioxides ofcarbon lung also serves as an attractive target for the entry of thepathogens regulation of the pulmonary functions is mediated by intricate cells of endothelial and epithelial lining dendritic cells alveolarmacrophages and fibroblasts all these pulmonary cells are highlyheterogeneous in nature they together in association respond to thelung injury by provoking inflammatory and immune responses airway epithelial cells express pattern recognition receptors prrsalong with toll like receptors ctype lectins rigi and inflammasome components which are involved in the innate response against microbes microbial cell wall constituents likelipopolysaccharide are sensed by prrs and induce an inflammatoryresponse alveolar epithelium comprises type i and type ii alveolarcells apart from these the mucus layer and the physical barrier madeby the epithelium contribute to the first line of defense lung injury is described as any damage to the associated tissues and compartments of the pulmonary system the concept of lung injury influenced by the microenvironment can be demonstrated via series ofchanges in cell deformability and manifestation of intercellular adhesive molecules the major causes of lung injury are atelectasisalveolar instability volutrauma barotrauma infections and oxygentoxicity the pathogenicity of acute and chronic lung injury iscorrelated with the release of proteases free radicals and growth regulatory proteins by the alveolar macrophages neutrophil takescare of the extreme ranges of lungs searching for pathogens therebyfig mechanism of autophagy ulk1pi3kmtor signalling pathway binds to endoplasmic reticulum and activates dcedp1 that initiates beclin and atg512conjugation system which forms isolation membrane this is followed by the sequestration process that leads to autophagosomal formation the autophagosomefuses with lysosomes to form autolyososome that leads to degradation 0cs vishnupriya eliminating via phagocytosis they undergo transendothelial andtransepithelial migration primary host defense mechanism oflungs include the immunity conferred by surfactant proteins spnamely spa and spd they link both innate and adaptive immunity byregulating the responses of innate immune cells antigen presentingcells apcs and tcells they can act as potential biomarkers of lunginjury the implication of autophagy over lung injury is tortuousas its function varies highly with the cell types specific to the pulmonary disorders it provides both defensive and injurious outcomes of lung injury lung injury “ types and associated pathologies acute lung injury ali and acute respiratory distress syndromeardsacute lung injury and acute respiratory distress syndrome arecommon grievous diseases among critically illpatients and are evidential sources of mortality and morbidity they are expressed alongwith hypercapnia and hypoxemia ards is outlined as the most seriousform of ali ali is characterized by inflammation of lung surfacesresulting in the disruption of alveolarcapillary membrane followed bytransmigration of neutrophils significant event in the procession of aliand ards and outbreak of cytotoxic mediators immune responsemediated by several components like tcells macrophages naturalkiller nk cells chemokines and proinflammatory cytokines also has apredominant role in the progression of the acute injury and results inimmune reconstitution inflammatory syndrome iris immune cellsinvolved in the immune response like macrophages and monocytes arethought to be involved in the pathophysiology of iris elevatedlevels of cd14 cd16ˆ’ monocyte population and an resulting increase in the proinflammatory cytokines il6 tnfα and c reactiveprotein crp are also observed in tuberculosis tbiris patients lung endothelial biomarkers like vwf and epithelial biomarkers likespd are the diagnostic targets of ali sepsis pancreatitis pneumoniatrauma transfusions aspiration and inhalation of toxic gases are someof the clinical factors of ali and ards histological patterns ofali and ards have the chances of demonstrating diffuse alveolar damage alveolar haemorrhage eosinophilic pneumonia and acute fibrinous pneumonia associated with ards may also result frompathogens like fungal viral bacterial and parasitic most commonpathogen strains include streptococcus pneumoniae respiratory virusesstaphylococcus aureus fungal pathogens like pneumocystis jirovecii andaspergillus fumigatus legionella pneumophila and enteric gramnegativeanisms among bacteria the virulence capability of pseudomonas aeruginosa is one of the prime determinant of the severity of thelung injury disease progress takes place in three degrees namelyacute phase exudative subacute phase proliferative and chronicphase fibrotic radiological features during the acute phasesignify twosided patchy groundglass densities relating to interstitialedema and hyaline membranes the important constituent of innateimmune system the pattern recognition receptors prrs are affiliatedwith the advancement of ali and ards the ligands of prrs includepathogenassociated molecular patterns pamps which induce inflammatory signalling events and the damageassociated molecularpatterns damps which induce neutrophilmediated tissue damageincreased incidences of mitochondrial damps result in high mortalityrates a common type of ali is the transfusionrelated acute lunginjury trali trali is defined as adult respiratory distress syndromeoccurring with transfusionsis manifested by pulmonary insufficiency severe hypoxia and dyspnea it is often reported as theneutrophil pmnmediated syndrome a particular study has reported that the potential risk factors for ali and ards may be associated with larger tidal volume vt and higher airway pressure pawduring one lung ventilation olv in postpneumonectomy aliardspatients and also in patients who developed aliards longtermeffects after acute lung injury are related to neurological impairmentsitlife sciences namely neuromuscular dysfunction neurocognitive dysfunction andneuropsychological dysfunction some of the minor impacts includeheterotopic ossification stiae and frozen joints chronic lung injury cli and bronchopulmonary dysplasia bpdchronic lung injury is characterized by a condition called pleuroparenchymal fibroelastosis ppfe in which a rapid multiplication ofsubpleural intestinal elastic fibres majorly in the upper lobes predominates along with other clinicopathological conditions following chronic lung injury cells undergoing apoptosis is cleared by aprocess called efferocytosis it is a type of phagocytosis confined only tothe cells that undergo apoptosis to maintain the cellturnover in pulmonary airways carried out mainly by the macrophages while immature dendritic cells mesenchymal cells and epithelial cells can alsoperform efferocytosis recently sarscov2 manifested as severerespiratory infection resulted in number of deaths worldwide commonrisk factors associated with death in sarscov2 patients identified arehypertension diabetes cardiovascular disease or chronic lung disease the most prevailing chronic lung injury ofinfants is thebronchopulmonary dysplasia bpd occurs when preterm infants suffering from various respiratory syndromes like meconium aspirationsyndrome and neonatal pneumonia are subjected to treatment withsupplemental oxygen and extensive mechanical ventilators it is proventhat the common risk factors of bpd increases with a fall in birthweight prematurity and gestational period apart from theabove other perinatal risk factors with which bpd is associated with areintrauterine growth restrictions race or ethnicity chorioamnionitis and genetic risk factors it is characterized bysaccular formation and elastic fibre aggregation of distal air spacesinflation and edema barotrauma and pulmonary oxygen toxicity arepathologies of bpd bpd is multifactorial in nature studiesdeclare that surviving patients of bpd have established pulmonarydysfunction incidence adults with bpd history are strictly prohibitedto cigarette smoking another form of bpd called the new bpdwhen surfactants are used as treatment new bpd is depicted by pulmonary hypertension and abnormalities in vasculopulmonary development glucocorticoids and tgfbeta are the efficient modulators thatinitiate bpd injury bpd infants have significantly lower levels ofvascular endothelial growth factor and platelet endothelial cell adhesion molecules chronic obstructive pulmonary disease copd and asthmacopd is a general illness worldwide it is defined as the completeirreversible state of airflow limitation characterized by weak inflammatory response of the lungs emphysema chronic obstructivebronchiolitis fibrosis blocking and narrowing of airways deprivationof lung parenchyma and elasticity immune cells like tlymphocyteswith cd8 dominance blymphocytes macrophages and neutrophilsare the regulators of copd acute provocation of copd is definedas the continuous deterioration from steady state facilitating an alteration in typical medication for rudimentary copd systemicinflammation responses are a result of leukotriene b4 tnfalpha interleukin il8 and proteases a decrease in the ratio of cd4 to cd8is a typical feature of pulmonary inflammatory responses additionalimpacts of copd are cardiovascular diseases nervous effects and osteoskeletal effects smoking is an important cause of systemicoxidative stresses immoderate inflammatory responses and emphysema as manifested as copd implications copd being an hetergoenousdisease patients with exacerbations are found to be associated withbacterial infections like moraxella catarrhalis streptococcus pneumoniaand haemophilus influenza apart from bacteria other microbes likevirus and fungi also comtribute to the lung microbiota and pathogenesisof lung microbiota through initiation of chronic inflammation it hasbeen found that bacteria and viruses fungi can promote local andsystemic inflammation that may contribute to the pathogenesis ofcopd specific biomarkers of copd observed are creactive 0cs vishnupriya protein crp il6 il10 and ccl18parc skeletal muscle losstakes place in copd wasting of the cell mass is evidenced to be theresult of tnfα participation in the pathogenesis of copd muscleglutamate reduction is linked with lactic acidosis in copd patientsending in muscle wastage similar to copd asthma is characterized by pulmonary obstruction and similar immune responses onlyvariation from copd is that the airway obstruction in asthma is reversible and it does not affect lung parenchyma dendritic cells are themodulators of th2 cells playing an important immune response ofasthma severe asthma is equal to the effects of copd illustrated by theincrease in neutrophils tumor necrosis factor tnf cxcl8 and decreased reception to corticosteroids while reversible copd is potentially to have subsequent asthma and copd ventilatorinduced lung injury vili and ventilatorassociated lunginjury valithe prevailing lung injury cases with ards when given ventilatorassistance mechanically in a clinical setup may develop additional lunginjuries ending up in ventilatorassociated lung injury vali similarlyin experimental models lung injury can be provoked by external application of injurious ventilation procedures contributing to ventilatorinduced lung injury vili thus vali can adversely aggravate thehealth of the ards patients at low lung volumes of ventilation atelectrauma occurs at high lung volumes of ventilation barotrauma andpulmonary edema occur biomarkers studied via experimental modelsof vali include several proinflammatory molecules like tnfα il1βil8 and antiinflammatory molecules like il10 il6 and stnfr1 respectively [“] positive endrespiratory pressure peep and tidalvolume applied have direct influence on vali and are to be studiedspecifically during ali and edema it is reported that peep when provokes overinflation the extent of edema also increases mechanism ofsurfactant inactivation is seen in alveolar microvessels with an increasein fluid filtration further greater the lung volume higher is thetransmural pressure in them a retrospective cohort study revealedthat height and gender of the patients should be taken into considerations along with establishing limitations to large tidal volumes beforesetting up a ventilator experiments explain that decrease in thepeak pulmonary arterial pressure or respiratory frequency can lessenthe grimness effects of vali it is proven that hypercapnic acidosisaffords protection to vili addressable implications of vali arebarotrauma volutrauma atelectrauma biotrauma and oxytoxic effects cellular pathology includes physical disruption of cells and tissuesand activation of cytotoxic responses leucocytes are raised to likelyinteract with the endothelium as the increasing intraalveolar pressurefastens the transit time of them inferences for current medicalpractices involve lungprotective ventilation survival of patients can beencouraged by prone positioning future clinical practices involveprecisioned ventilationindividualized tidal volumes using drivingpressure individualized peep and extracorporeal strategies pulmonary fibrosis pf and cystic fibrosis cfidiopathicpulmonary fibrosis ipf is a degenerative interstitial fibrosing disease prevalent worldwide it is also termed as cryptogenicfibrosing alveolitis a typical honeycomblike structure of asymmetricairspaces covered by dense fibrosis is observed ipf is followed by interstitial pneumonia which is featured by deficit inflammation and withabsence of homogeneous participation of lung tissues studies havebeen made since ages which revealed several inherited forms of pulmonary fibrosis mutations in various genes were associated to pfnamely sftpc sftpa2 tert terc and muc5b incidence withrheumatoid arthritis and scleroderma are more likely to form pf thereis a chance of clearance of alveolar basement membrane and occurrenceof hyperplastic epithelial cells ipf ensures migration of fibroblastsinto the fibrinrich exudates thus a chemoattractant activity is createdin the airspaces after lung injury this process is proven to be regulatedthe proby lysophosphatidic acid experiment proves thatlife sciences inflammatory cytokines il1β directly regulates the initiation of acuteand chronic inflammation making it a valuable target of ipf cystic fibrosis is characterized as a most common autosomal recessivegenetic disorder caused by the mutation in cftr gene transmembraneprotein genecystic fibrosis transmembrane conductance receptor pathology of the disease involves bronchiolitis obstruction of pathways endobronchiolar infection impaired ciliary actions atelectasisfinally leading to secondary alveolar injury bacterial infections of saureus p aeruginosa and h influenzae causes cf it is the pulmonarymacrophages and polymorphonuclear neutrophils that forms the defense against infections while lymphocytemediated mucosal injury isobserved in cf patients cf patients have increased flow of tnfαil8 and il1β submucosal glands of the lungs are the crucialhosts of cftr genes as the number expressed are high hence cfcontributes to epithelial airway lining abnormalities with respect to thechanges taking place in the submucosal glands respectively the macromolecular secretions of the submucosal glands have changes in theircomposition viscosity and greatly impacts on the mucociliary clearance radiationinduced lung injury riliseveral complications of the lung malignancies require treatmentsinvolving radiation therapy rt extensive reports claim that rt maylead to a state of rili the threedimensional dosimetric predictors canbe emphasized for the risk of symptomatic rili the alveolarcapillary subunit forms the most radiosensitive complex of the lungs thusthe rili is also called as the diffuse alveolar damage radiation exposure provokes the production of reactive oxygen species creatinghigh toxicity levels in the lung parenchyma this may ultimately lead tolung fibrosis which develops one to six months after rt accompaniedby dyspnea in addition to fibrosis rili also develops radiationinduced pneumonitis gradually after months to years almost all thepatients undergoing rt have the chance of developing fibrosis radiationinduced pneumonitis is characterized by cough occasional fevernonspecific symptoms of dyspnea and chest pain with or without deformities in pulmonary functional tests while radiationinduced pulmonary fibrosis is characterized by cough differential levels of dyspnea chest pain or symptomless and stable scarring of the lung tissueswhen detected radiographically it has been stated that the incidence and occurrence of rili is directly influenced by dose and volume determinants of rt several studies provide information onrili that result in provoking inflammatory responses a biphasicmanifestation of cytokines is observed in the lung tissues once exposedto rt one such study carried out to assess the cytokine productionin c3hhen irradiated mice revealed a spatial change in the expression of proinflammatory cytokines among various cellular compartments of the lungs further it was noted that the bronchoalveolar lavage cells responded immediately while the interstitial cells contributedonly in the later stages during pneumonitis profiling of cytokinesnamely the interleukins interferons monocyte chemotactic protein1tumor necrosis factors macrophage inflammatory proteins and granulocyte colonystimulating factors can be performed to analyse the developmental risks of symptomatic radiationinduced lung injury in vitro studies experiments state that the application of melatonin andcarnosine compounds reduced the reactive oxygen species and inflammatory cytokines produced following rili regulators of autophagy in lung injurynumerous regulators of autophagy play a vital part in the development of lung injury with regard to autophagy the following are theimportant regulators fig the mechanism by which these autophagy regulators act is shown in the table lc3biithe microtubuleassociated protein light chain lc3 is the 0cs vishnupriya life sciences fig figure depicts the autophagy regulators in regard to lung injury various conditions like hypoxia starvation environmental stress and cigarette smoke leadsto autophagy that in turn causes lung injury activation of class ipi3kmtor pathway takes place under hypoxic and starvation conditions while environmentalstress results in provoking beclin 1vps34 pathway that induces the sequestration of the phagophore formation cigarette smoke induces ros accumulation which inturn causes egr1 e2f signalling to activate lc3ii along with sqtm1p62 and atg512 conjugation system the ros generated may also leads to apoptosis theautophagosome fuses with lysosomes and forms autophagolysosome that causes pulmonary arterial hypertension and lung injuryprinciple autophagic protein expressed on the doublemembraned autophagosome nearly eight homologues of lc3 proteins are studied inmammals the amino acid composition of these homologues classifiesthem into two subfamilies first one comprising of lc3a splicingvariants lc3b and lc3c taking part in autophagosomal membraneelongation while the second one comprising of gabarap gabarapl1 gabarapl2 and gabarapl3 taking part in the maturation ofautophagosome generally lc3b occurs in cytosol as lc3bi by theproteolytic cleavage of cterminal of lc3b which then unites withphosphatidylethanolamine to form lc3bii to assemble on the autophagosomal membrane conjugation of phosphatidylethanolamine withlc3bii can be revoked by the activity of atg4 thus it is clear thatoccurrence of lc3bii is crucial in the process of autophagy lc3bii levels are analysed through immunoblotting while limitationsare degradation of lc3bii by autophagy itself and the nonindication ofautophagic flux at distinct time points this can be overcome by comparison studies with lc3bi and using lysosomal protease inhibitors antilc3b antibodies can be applied to detect autophagy invarious cell types application of antilc3b to glioblastoma tissuesdemonstrated a positive detection of lc3b levels both in vitro and invivo suggesting a latent monitoring system of lc3b severalstudies state that hyperoxic conditions can result in ali and ards thisfurther triggers the morphological biomarker of autophagy lc3bii toaccumulate thereby determining the fate of cell clearance experimentsperformed in hyperoxiainduced human bronchial epithelial cells andcultured epithelial cells beas2b clearly stated that the expression oflc3bii was high mediated by the apoptotic regulators similarexperiment carried out in the hyperoxiainduced lung injury in c57bl mice inferred the involvement of apoptotic pathways in the activationof lc3bii interaction of lc3bii with fas proteins was observedmarking the importance of lc3bii in the management of ali pulmonary hypertension is a main cause of copd manifestation inlungs that affects vascular architecture when chronic hyperoxia wasinduced in the lung tissue extracts of patients with pulmonarytable regulators of autophagy in lung injury and their mechanismregulatorslc3biibeclin p62hif1bnip3mtormechanismelongation of autophagosome and its maturation requires atg8map1lc3 protein lc3i combines with phosphatidylethanolamine forming lc3ii essential for autophagosome formationthe initiation of the isolation membrane that forms autophagosome after the sequestration process is regulated by beclin and pi3kp62 along with sqstm1 is the receptor for polyubiquitinated substrates it helps in the transportation of cargo into the autophagosome by bindingwith lc3ii for degradationduring hypoxic conditions hif1 induces bnip3 that in turn brings about cell survival through autophagy and provoke cell death by apoptosisthe mammalian homologue atg13 is phosphorylated by mtor that binds ulk1 proteins fip200 phosphorylates ulk and initiates the isolationmembrane formation in autophagyreference no 0cs vishnupriya hypertension the upregulation of lc3b prevailed indicating the regulatory role of lc3b in vascular cell proliferation and mediatingadaptive cellular responses smoking results in various implications of lung injury in due course a multimeric protein complexcomprising of lc3bcaveolin1fas occurs under basal state extensivestudies reveal that smoking triggers lc3b to initiate the dissociation ofcaveolin1 from fas protein thus facilitating apoptotic pathways accordingly emphysema a destructive expression of copd isworsened by cigarette smoking in vitro studies in lung tissues of miceon exposure to cigarette smoking ensured the driving role of lc3b inregulating apoptotic mechanisms and finally developing emphysemarespectively all these experiments prove the comprehensive bridgebetween autophagy and apoptosis highly regulated by the expressionlc3b lc3bi and lc3bii bind to microtubule associated protein1b map1b wherein overexpression of map1b decreases the levels oflc3bii protein kinase c is known to cease autophagy by interferingwith autophagosomal formation both in vitro and in vivo studiesconfirmed that the lc3b phosphorylation by protein kinase c takesplace consistently in lungs the emergence of autophagy either asa protective role or maladaptive response due to sepsis was studied in acecal ligation and puncture clp induced septic mice it was ensuredautophagy to be a protective response yet an overexpression of lc3bii in the later stages of sepsis leads to ali describing a maladaptive role lung injury can be provoked by ischemiareperfusion in whichautophagy is stated as the safeguarding mechanism by moderatelymaintaining the level of lc3bii also the ischemiareperfusioninduced lung injury is positively governed by the erk12 signallingpathway that regulates the cellular expression of lc3bii respectively nanoparticles of zinc oxide on exposure to lungs may induceali further zinc oxide nanoparticles resulted in the raise of autophagosomal structures followed by the accumulation of lc3bii proteinsthus zno nanoparticlesinduced ali is autophagy dependent 3methyladenine a classical autophagy inhibitor reduced the manifestationof lc3bii and lowered the release of zinc particles thereby stoppingzno nanoparticlesrelated toxicity of lungs similarly the artificially synthesized polyamidoamine dendrimers pamam used as aneffective drugdelivery system may sometimes result in pamam nanoparticlesinduced ali the levels of lc3bii biomarkers were highindicating the autophagic responses beclin beclin was first identified by beth levine as becn1atg6 inchromosome 17q21 in the year and it is the major autophagyregulating gene it is a coiledcoil protein of molecular weight kda comprising of amino acids and acts together with bcl2an antiapoptotic protein beclin is an indispensable autophagypromoting gene that is homologue to the mammalian yeastatg6 gene which regulates cell survival of different types and is involved in the constitution of autophagosomes the initiation ofthe anization of autophagosomes is regulated by class iii phsophoinositide 3kinase pi3k and autophagy related gene beclin beclin has got a novel bcl2 homology region3 bh3 domainthe bh3 domain in beclin1 can bind to bcl family proteins that initiateapoptotic signalling and prevents the beclin 1mediated autophagy byremoving beclin from hvps34 either phosphorylation or ubiquitination of beclin or bcl2 can disunite bcl2 from beclin andincrease the activity of vps34 kinase which brings about increase infunction during autophagy autophagyassociated protein beclin1 binds to lc3i that adapts to its membranebound form lc3iiand it cooperates with the ubiquitinbinding protein p62sequestosome sqstm1 the first an that fails during sepsis is lungs thefamiliar complications of sepsis are ali and ards ali activated various autophagy related proteins like lc3ii beclin and lysosomerelated protein lamp2 and rab7 expressions in sepsisinduced ali to evaluate the function of autophagy in severe sepsis an experiment is carried out using endotoxemia that frequently uses septiclife sciences shock and clp which is a clinical polymicrobial sepsis model herebecn1ˆ’ mice was susceptible to clpinduced sepsis in cysticfibrosis transmembrane conductance regulator cftr autophagy bybeclin overexpression cystamine or antioxidants and the restorationof beclin recovers the localization of beclin to endoplasmic reticulum and regresses the cf airway phenotype both in vitro and in vivoin scnn1btransgenic and cftr f508del homozygous mice and also inhuman cf nasal biopsies in lpsinduced ali there are threedistinct complexes of beclin1vps34 have been identified the firstcomplex contains beclin1 vps34 vps15 and atg14l second complexcontains beclin1 vps34 vps15 and ultraviolet irradiation resistanceassociated gene uvarag and the final complex contains beclin1vps34 vps15 uvrag and rubicon among these complex the onethat contains atg14l is concerned in the formation of autophagosomewhile others are in the autophagosome and endosome maturationbeclin forms the bridge in the recruitment of inducers and suppressorsof autophagy and simultaneously behaves as a key modulator in autophagosome formation mesenchymal stem cells mscs increases the translation level of beclin but not its transcription ratemscs might alleviate lpsali via downregulation of mir142a5p thatpermits pulmonary epithelial cells pecs to proceed with beclin mediated cell autophagy in lung disease the bacterial stu Answer:

Colon_Cancer

" preproofsevere acute respiratory syndrome coronavirus sarscov2 is a highly contagious zoonotic pathogen that has exacted heavy public health social and economic tolls in february the world health anization acronymed the disease caused by sarscov2 as covid19 for coronavirus disease the number of confirmed covid19 infections which has been detected in at least countries has reached worldwide as of april with deaths according to the us centers for disease control and prevention cdc1 many cases of covid19 resolve quickly however the disease which like other respiratory pathogens that cause common cold symptoms is believed to be transmitted through respiratory droplets infection with covid19 can also lead to significant morbidity and death this is particularly the case for cancer patients moreover because the signs and symptoms of covid are easily misattributed to the sequelae of cancer itself such as pulmonary embolism or its treatment such as nausea and diarrhea diagnosis may be delayed or missed potential covid rule out criteria based on the wells˜ criteria for pulmonary embolism another protean disease entity are provided as a decisionmaking aid this review summarizes the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis rationale to treat the cancer or not treatment and prevention of covid19 with an emphasis on implications in cancer keywords covid19 sarscov2 cancer introduction sarscov2 the rna virus responsible for the illness which has been named covid19 for coronavirus disease2019 the year it was diagnosed has sent shockwaves and dominated the news cycle due to its pandemic spread from the point of origin in wuhan china to the rest of the world declared a public health emergency of international concern pheic by the world health anization who2 sarscov2 is the third highly pathogenic novel zoonotic bat coronavirus sonamed because of the crownlike spikes on its surface to have emerged the first was sars coronavirus now named sarscov1 in with a fatality rate of and 0c preproofthe second was middle east respiratory syndrome mers in with a fatality rate of where the camel was the intermediate host3 sarscov1 merscov and sarscov2 belong to the betacoronavirus genus which are enveloped positivestranded rna viruses whose approximately nucleotide genome serves as an mrna template for the translation of viral proteins4 the virion contains four proteins spike envelope membrane and nucleocapsid and the host receptor with which the spike surface glycoprotein of sarscov2 engages is angiotensin converting enzyme ace25 the biology of sarscov2 is described in more detail in figure a zinc metallopeptidase enzyme ace2 which is abundantly present in lung and gastrointestinal epithelial cells6 not only mediates viral entry through receptormediated endocytosis7 but also the efficiency of viral replication8 its expression is upregulated with older age smoking the antihypertensives angiotensin converting enzyme ace inhibitors and angiotensin receptor blockers arbs thiazolidinediones tzds a class of oral antidiabetic drugs and ibuprofen risk factors which may increase susceptibility to the covid19 virus infection and which are common in generally elderly multimorbid cancer patients9 preproofthe clinical spectrum of sarscov2 ranges from mild upper respiratory tract infection with fever sore throat headache cough and potentially nausea and diarrhea the majority of cases recover without serious complications to severe pneumonia with sequelae that include acute respiratory distress syndrome ards cytokine storm and death10 because sars is an acronym for severe acute respiratory syndrome digestive manifestations including inappetence nausea abdominal pain and diarrhea of covid19 resulting from binding of the virus to the ace2 receptor in the gi tract may precede respiratory symptoms11 gastrointestinal manifestations are potentially underappreciated and overlooked as sentinel symptoms that herald the onset or persistence of disease especially in cancer patients and may contribute to delayed or missed opportunities for testing diagnosis and containment unlike sarscov1 which seems to have disappeareddied out12 and mers which reappears only sporadically sarscov2 is less lethal with a fatality rate between although this number is highly uncertain and debated13 but much more infective consequently public panic and economic disruption have ensued resulting in wartimelike mobilization efforts to mitigate its spread old age smoking and comorbidities such as diabetes morbid obesity immunosuppression frailty and cardiovascular disease appear to predispose to worse outcomes possibly secondary to impaired t and b cell responses notably covid19 infection is associated with lymphopenia and delayed development of the adaptive immune response which appears to correlate with prolonged virus clearance and more severe disease progression15 the first pillar of defense against infection is hand washing avoidance of face touching and minimization of close contact ie social distancing with selfquarantine and selfisolation16 in case of exposure or evidence of covid19 symptoms respectively the second prophylactic pillar is vaccination with specific viral antigens or mrnas which are not yet publicly available although the company moderna has reportedly started testing an mrna vaccine in healthy volunteers and multiple other vaccination strategiesplatforms appear to be in progressunder development17 0c transmission and prevention according to the world health anization who21 sarscov2 is spread persontoperson mainly via aerosol inhalation from sneezing coughing or exhalation 22and via fomitetoface contact since depending on the surface material the virus may remain viable and infectious for hours to days figure fecaloral transmission has also been hypothesized because diarrhea was a common feature with sars and mers and diarrhea and other digestive issues have also been reported in patients with covid1925 26notably transmission of sarscov2 is not limited to symptomatic individuals ie those with fever cough sore throat myalgias or dyspnea but also to asymptomatic or subclinically infected carriers of the virus which is problematic from the perspective of disease control 27and highlights the importance of containment measures including isolation and quarantine the basic reproduction number r0 of sarscov2 is which means that on average for every patient an additional individuals are infected because coronaviruses may persist on inanimate surfaces like metal glass or plastic for up to days careful disinfection with or greater ethanol for small surfaces or sodium hypochlorite for larger surfaces is recommended preproofdiagnosis and clinical features preproofthe available evidence is limited but clinical courses and outcomes of covid19 are likely to be worse in patients with cancer especially given the clear association between severity of disease and older age and higher levels of comorbidity the overall case fatality rate cfr for the general covid19infected population is around but in cancer it rises to overall and to in italy19 this cfr in cancer patients compares to for no comorbid conditions for cardiovascular disease for diabetes for hypertension and for chronic respiratory disease the aim of this review is to summarize and condense the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis treatment and prevention of covid19 with a special focus on cancer in the united states the test of choice for sarscov2 is a nasopharyngeal swab specimen or sputum if a productive cough is present on which a reversetranscriptase pcr rtpcr assay or an enzymelinked immunoassay eia directed particularly at the envelope e rnadependent rna polymerase rdrp spike protein s and nucleocapsid n genes is performed the fda has also approved an antibody test29 a positive test for sarscov2 in a symptomatic patient generally confirms the diagnosis of covid19 with the caveat that false positive and false negative tests have been documented if initial testing is negative but a high index of suspicion and pretest probability for covid19 remains on the basis of patient signs and symptoms then retesting is indicated in patients with high indexes of clinical suspicion and equivocal or negative test results the who recommends that lower respiratory tract specimens which contain the highest viral loads should be obtained since nasopharyngeal swabs may miss some infections30 according to cdc guidelines disease is excluded on the basis of two 0c preproofconsecutive negative tests respiratory tests separated by ‰¥ hours however in the presence of suggestive symptoms rectal swabs may also be indicated since the ace2 enzyme to which the virus binds is abundantly present in rectal epithelia cells31 the differential diagnosis for sarscov2 in cancer is extremely broad and includes conditions such as foreign body aspiration toxicities from chemotherapy and radiation tumor progression postobstructive pneumonia malignant obstruction atelectasis pulmonary embolism pneumonitis pulmonary edemafluid overload immunotherapyrelated pneumonitis copd exacerbation q fever adenovirus bocavirus coronavirus 229e hcov 229e coronavirus hku1 hcov hku1 coronavirus nl63 hcov nl63 coronavirus oc43 hcov oc43 human metapneumovirus hmpv influenza a influenza a subtype h1n1pdm09 influenza a subtypes h1 and h3 influenza b parainfluenza virus ““ piv “ respiratory syncytial virus ab rsv ab rhinovirusenterovirus hrvev bordetella pertussis legionella pneumophila and mycoplasma pneumoniae32 the diagnosis of sarscov2 is complicated by the possibility of simultaneous coinfection with other respiratory viruses33 which is especially true for immunosuppressed cancer patients whose susceptibility to microanisms is increased the heightened infectious risk for cancer patients underscores the importance of screening them at presentation with extended viral respiratory panel testing given that coinfection may impact management decisions since conceptually at least the morbidity of covid19 and the risk of severe illness should increase in the presence of a second or third virus preproofunlike infection with influenza for example covid19 signs and symptoms may vary considerably depending on the dose of viral inoculum route of inoculation concomitant medications and underlying health status34 to include fever dry cough fatigue sputum production shortness of breath sore throat headache myalgia or arthralgia chills nausea or vomiting nasal congestion diarrhea hemoptysis and conjunctival congestion with an incubation period of to days after exposure36 presymptomatic or minimally symptomatic infection may majorly drive transmission especially since detected viral loads are similar in both symptomatic and asymptomatic patients3738 populations of concern include the elderly smokers vapers and dual users those of any age with preexisting chronic medical conditions those receiving particular medications or therapies which upregulate the ace2 receptor or suppress the immune system and those from lower socioeconomic classes a conglomeration of factors which are often present in cancer patients as depicted in figure while the surveillance focus for covid19 is on the respiratory tract enteric symptoms are a potentially underappreciated overlooked and misattributed manifestation of disease as stated earlier and this is especially the case for cancer patients where gastrointestinal toxicity occurs routinely from chemotherapy ie cisplatincarboplatinoxaliplatin irinotecan 5fluorouracil ifosfamide from targeted agents ie erlotinib imatinib bortezomib temsirolimus sunitinib regorafenibsorafenib and bevacizumab39 and from locally advanced or metastatic disease therefore abdominal complaints in cancer patients which are potentially but not automatically attributable to underlying disease justify further investigation especially if persistent worsening or new particularly because sarscov2 transmission may occur via the fecal“oral route40 0c preproofabnormal laboratory findings in covid19 include lymphopenia percent prolonged prothrombin time percent elevated lactate dehydrogenase percent elevated ast and alt “ percent elevated highly sensitive hs crp and elevated procalcitonin however because these parameters routinely fall well outside of the normal reference range in cancer patients it is difficult to confirm or refute the presence of disease on this basis alone chest radiographs and chest ct abnormalities are similarly nonspecific since the most common features multifocal groundglass opacities and consolidation mimic other pneumonias41 significant antibody production is observed after infection but it is unknown whether this helps or harms since antibodydependent enhancement ade may potentiate viral entry and the induction of a severe inflammatory response42 universal screening of cancer patients for covid19 is desirable but logistically impossible for the foreseeable future since diagnostic tests are in short supply and simply not always readily available43 hence covid19 rule out criteria are proposed in table as a potential decisionmaking aide mémoire which separates patients into low and highrisk groups by analogy to the wells˜ criteria for pulmonary embolism4445 preventive measures focus on selfisolation social distancing with a 6foot 2m separation46 frequent hand washing with soap and water andor use of hand sanitizers patient isolation during clinical care use of masks to help prevent aerosol transmission and flushing with the lid closed to control socalled •toilet plume– in an asco guidance immunocompromised cancer patients are advised to minimize exposure to sick contacts and large crowds48 for healthcare personnel the use of personal protective equipment such as n95 masks ffp3 masks gowns eye protection gloves and gowns is mandated49 preproof vaccination and immunity vaccination efforts and the related topic of whether those who have recovered from covid19 develop protective immunity have drawn great attention the latter has implications on whether people who test positive for sarscov2 antibodies can be safely assumed to be immune and at negligible risk of contracting or transmitting the disease there have been case reports of patients who have recovered from covid19 and had recurrence of rtpcr positivity approximately one month after initial diagnosis with only one patient exhibiting significant clinical symptoms and another having a mild intermittent cough50 but while not zero the risk of transmissibility or recurrence of symptomatic disease in recovered patients has yet to be quantified and the paucity of currently available reports of recurrence in the setting of a pandemic suggests that it is low a separate practical question will be whether antibodybased tests prove to have sufficient sensitivity and specificity to identify people who had asymptomatic infections developed immunity and can return to normal activities without jeopardizing disease containment efforts immunity may be due to antibodies cell mediated immunity or a combination of the two previous experience with using plasma from convalescent patients to treat severe cases of the first sars and mers as well as limited experience with covid19 suggests that antibody mediated immunity alone is clinically beneficial even during acute infection51 safety concerns about antibodies have been raised based on preclinical studies of sarscov vaccination in 0c preproofferrets showing hepatotoxicity52 and of vaccination against feline infectious peritonitis virus another coronavirus leading to more severe disease when kittens were subsequently challenged with the virus53 although animal models may not be representative of human hostpathogen interactions the nature of sarscov and sarscov2 antibodies are likely different as crossneutralization was not observed invitro54 and experience with convalescent plasma has not borne evidence of antibody mediated enhancement of infection in acutely infected patients the potential risk deserves attention if vaccination is proposed for the entire population t cell responses are also readily observable in patients who recover from coronavirus infections55 and memory t cell responses alone were protective in mice56 with the potential advantage of longer persistence of memory t cell responses compared to humoral immunity when clinical data on vaccine candidates becomes available cancer patients may face different considerations surrounding vaccination than the general population particularly patients with hematologic malignancies being treated with agents targeting b cells who would derive greater benefit from vaccines eliciting cell mediated than antibody responses preproofpathogenesis and pathology relating to ace2 and ras signaling the ace2 enzyme a key regulator of the reninangiotensin system ras57 to which the virus binds through its surface spike proteins is particularly abundant in the digestive tract lungs kidney heart and blood vessels where pathology from sarscov2 occurs58 a peptidase that catalyzes the conversion of angiotensin ii angii referred to as •the quintessential perpetrator of inflammation– to angiotensin ang ace2 mediates antiproliferative and vasodilatory functions that oppose the vasoconstrictive and inflammatory functions of angiotensin converting enzyme ace60 the binding of sarscov2 to ace2 leads to downregulation of ace2 expression potentially through increased internalization and shedding from the cell surface with decreased ang1“ generation and increased ang ii levels as a consequence61 this unfavorably shifts the balance of the renin angiotensin system ras from the vasoprotective ace2ang17 axis to the aceang iiangiotensin at1 receptor axis and drives a proinflammatory profibrotic and proliferative response62 as shown in figure fang et al63 contend that because thiazolidinediones ibuprofen and angiotensin converting enzyme ace inhibitors and angiotensin ii typei receptor blockers arbs substantially increase the expression of ace2 they facilitate sarscov2 infection and therefore the risk of severe and fatal covid19 in contrast alghatrif et al64 present a diametrically opposed hypothesis that downregulated ace2 signaling is responsible for sarscov2induced acute lung injury ali acute respiratory distress syndrome ards and cytokine storm and that aceis and arbs are beneficial precisely because they increase ace2 expression and activity furthermore according to alghatrif et al lower ace2 levels and hence higher baseline oxidative stress and inflammation6566 are present in older comorbid individuals such as cancer patients which renders them more susceptible to severe covid19 than younger noncomorbid individuals with increased ace2 levels and lower baseline inflammation as shown in figure furthermore low ace2 may promote tumor progression and conversely ace2 overexpression is associated with antiangiogenesis and tumor regression67 in summary then 0c preproofdespite the concerns and controversy68 surrounding the use and continuation of aceisarbs during the sarscov2 epidemic it is likely that the pros outweigh the cons especially in cancer patients due to their potential antitumor and anticovid19 effects69 in line with ras involvement emerging data suggest that sarscov2 infection may induce serious cardiovascular injury or exacerbate existing cardiovascular disease cardiovascular sequela includes heart failure arrythmias disseminated intravascular dissemination dic and troponin elevation which may closely correlate with disease severity and the likelihood of inhospital death70 liu et al71 propose a mechanism whereby the virus which lowers hemoglobin hb levels72 binds to the porphyrin of heme and displaces iron thereby compromising the oxygencarrying capacity of red blood cells and exacerbating the hypoxemia since chloroquine and the experimental anticancer agent rrx001 also bind to porphyrins they may competitively interfere with binding by the virus rationale for continuation or discontinuation of cancer therapy preproofthe benefitrisk calculus that informs the decision whether and how to treat with anticancer therapy falls into a •gray zone– about which no consensus exists leading to a therapeutic dilemma on the one hand zhang et al73 in annals of oncology reported a strong association in patients of them with lung cancer between antineoplastic therapy in the past days and severe effects of covid19 hr4079 ci p0037 on this basis the authors recommend treatment interruption dose reduction or substitution of cytotoxic chemotherapy with nonimmunosuppressive options eg checkpoint inhibitors if available especially in the case of lung cancer patients that are already prone to develop respiratory infections and complications74 similarly heavily immunosuppressed patients such as those who have undergone hematopoietic stem cell transplantation are also particularly susceptible to viral respiratory infections these findings are supported by a nationwide analysis of data75 in china from covid19 patients of which were diagnosed with cancer this patient cohort experienced a higher incidence of severe events vs p and the administration of chemotherapy or surgery was found to have increased the risk of death andor intensive care unit admission even after adjusting for age sex and comorbidities odds ratio or ci “ p while these studies are limited by small sample sizes the data suggests that cancer predisposes to more severe disease therefore since inperson contact increases the risk of transmission several institutions have mandated realtime video or telephone interactions alternatively referred to as telehealth77 postponed surgeries biopsies endoscopies scans and routine investigations when possible and in line with esmo guidelines78 encouraged conversion from the intravenous to the oral route eg 5fluorouracil to capecitabine etoposide and vinorelbine on the other hand the immediate existential threat of progressive disease for which death is an impending imminent certainty rather than a remote possibility in the absence of treatment likely outweighs the theoretical risk of sarscov2 infection even in lower risk disease for example in situ or localized prostate breast and head and neck cancer delayed treatment is 0c preproofpotentially conducive to tumor development and progression and thus may unfavorably impact prognosis79 hanna et al have proposed a triage strategy80 which prioritizes treatment for those patients with imminent risk of early mortality from acute leukemias aggressive lymphomas metastatic germ cell tumors oncologic emergencies such as spinal cord compression chemoradiotherapeuticresponsive cancers such as head and neck cervical and anal cancers and neoadjuvant or adjuvant therapyresponsive tumor types such as stage iii colon cancer and deprioritizes visits for surveillance and survivorship however in the absence of a •one size fits all– consensus recommendation which is unlikely since cancer is so genetically diverse and heterogeneous the decisionmaking process and the subsequent treatment plan are individualized and to be determined tbd on casebycase basis taking into account multiple factors including the risk of cancer recurrence if therapy is delayed modified or interrupted the type of therapy eg surgery radiation chemotherapy checkpoint inhibitors and stem cell transplantation extent of comorbidities concomitant medications patient preferences physicianpatient relationship race age the number of cycles of therapy completed and treatment tolerance in terms of specific cancerrelated conditions asco makes the following heavily qualified recommendations81 \uf0b7 growth factor prophylaxis for neutropenia and neutropenic fever even at lower levels of risk as well as empiric antibiotics for acute care \uf0b7 erythropoietinstimulating agents for anemia prophylaxis and transfusion when necessary depending on the patient context and underlying comorbidities preproof treatment based on the high transmissibility of the virus82 the main nonpharmacologic countermeasures to mitigate or delay the impact of covid19 include rigorous hand hygiene use of facemasks respiratory etiquette ie coughing or sneeze into the upper sleeve or elbow not the hands flushing with the lid down to prevent bioaerosolization as well as quarantine stay at home policies and workplace and school closures which have upended the social cultural political and economic status quo no specific treatment or vaccine is currently available although promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir the mainstay of medical therapy includes symptomatic care such as supplemental oxygen antibiotics and hemodynamic and mechanical ventilatory support if indicated for septic shockmultiple an failure and respiratory failure respectively83 over active clinical treatment trials are underway84 these include vaccines as well as a number of different agents some with promising preliminary data as mentioned above and also those with potential anticancer activity which will hopefully serve a double purpose first to treat covid19 and second as an adjunct to bridge the time gap until the patient is recovered and the primary antineoplastic is startedrestarted as shown in table 0c preproof conclusions the alarming spread of the covid19 pandemic has disproportionately affected cancer patients an atrisk population both from the standpoint of increased disease severity and disruption to care which includes widespread suspension of clinical trials in the united states that are already fraught with barriers to enrollment and participation85 86because the symptoms of covid19 are nonspecific underlying symptoms from the cancer eg dyspnea cough fever fatigue diarrhea etc which overlap with those from the viral infection may obscure and delay the diagnosis hence if the covid19specific rapid reverse transcriptase polymerase chain reaction rtpcr test is not readily available andor in short supply which is currently the case diagnosis will depend on the maintenance of a high index of clinical suspicion especially in advanced cancer patients who check all the boxes for risk factors such as older age frailty disability immunosuppression generalized systemic inflammation and multiple comorbidities eg hypertension diabetes and cardiorenovascular diseases that predispose to severe disease and death preproofthese comorbidities are commonly treated with renin angiotensin system blockers such as angiotensinconverting enzyme inhibitors aceis or angiotensinreceptor blockers arbs which increase levels of ace2 the continued use of aceisarbs is the centerpiece of an intense debate because on the one hand sarscov2 coopts ace2 for target cell entry but on the other ace2 overexpression may counterbalance vasoconstriction and profibrotic processes and thereby reduce the incidence or mortality associated with covid19“associated ali or acute respiratory distress syndrome another controversy involves whether or not to continue cancer treatment given the high transmissibility potential of the virus however since no expert consensus recommendations have been issued to date and prognosis stage and responses to therapy are highly heterogeneous the riskbenefit tradeoff and subsequent treatment plan are highly individualized and context dependent currently the focus of treatment is infection control appropriate symptomatic care and oxygen therapy no approved medication or vaccine has been developed but promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir and several repurposed agents with antitumor properties are under investigation including thalidomide and rrx001 which may hopefully bridge the gap from the time covid is first diagnosed until the primary anticancer therapy is restarted finally multiple comparisons have been made between the allout mobilization efforts to combat covid19 with the massive scaleup of human and material resources that occurred during world war ii8788 in the words of winston churchill prime minister of great britain from whose intrepid fighting spirit iron will and intransigent defiance of tyranny galvanized the resolve of an entire nation to fight on in the face of seemingly impossible odds oncologists on the frontlines that have answered the call should •never worry about action only inaction– 0c preproof declaration of interests the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper references sitammagari k skandhan a dahlin awhat hospitalists need to know about covid19 medscape mar httpswwwmedscapecomviewarticle924596 meo sa alhowikan am alkhlaiwi t meo im halepoto dm iqbal m usmani am hajjar w ahmed n novel coronavirus 2019ncov prevalence biological and clinical characteristics comparison with sarscov and merscov eur rev med pharmacol sci feb24420122019 chan jf kok kh zhu z et al genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan emerg microbes infect kuba k imai y rao s et al a crucial role of angiotensin converting enzyme ace2 in sars coronavirusinduced lung injury nat med zhang h kang z gong h et al the digestive system is a potential route of 2019ncov infection a bioinformatics analysis based on singlecell transcriptomes [epub ahead of print] biorxiv de wit e van doremalen n falzarano d munster vj sars and mers recent insights into emerging coronaviruses nat rev microbiol “ w li et al angiotensinconverting enzyme is a functional receptor for the sars coronavirus nature hughes s covid19 and angiotensin drugs help or harm medscape march httpswwwmedscapecomviewarticle927542 yang x yu y xu j et al clinical course and outcomes of critically ill patients with sarscov2 pneumonia in wuhan china a singlecentered retrospective observational study [published online ahead of print feb ] [published correction appears in lancet respir med feb ] lancet respir med 2020s2213 gu j han b wang j covid19 gastrointestinal manifestations and potential fecaloral transmission [published online march ] gastroenterology ruiheng x chance missed but still there memoirs at the 10th anniversary of sars outbreak j thorac dis aug 5suppl s90“s93 jiang s don't rush to deploy covid19 vaccines and drugs without sufficient safety guarantees nature mar5797799321 doi 101038d41586020007519 peiris js guan y yuen ky severe acute respiratory syndrome nat med dec suppls8897 zheng h zhang m yang c et al elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid19 patients cell mol immunol2020 tognotti e lessons from the history of quarantine from plague to influenza a emerg infect dis “ httpswwwnihgovnewseventsnewsreleasesnihclinicaltrialinvestigationalvaccinecovid19begins httpswwwascoascocoronavirusinformationcareindividualscancerduringcovid19 onder g rezza g brusaferro s casefatality rate and characteristics of patients dying in relation to covid in italy published online march doi101001jama20204683 hanna tp evans ga booth cm cancer covid19 and the precautionary principle prioritizing treatment during a global pandemic nat rev clin oncol who advice on the use of masks in the community during home care and in healthcare settings in the context of the novel coronavirus 2019ncov outbreak jan rodriguezmorales aj macgregor k kanagarajah s patel d schlagenhauf p going global travel and the novel coronavirus travel med infect dis httpsdoi101016jtmaid2020101578 httpswwwnejmdoifull101056nejmc2004973 lu cw liu xf jia zf 2019ncov transmission through the ocular surface must not be ignored the lancet preproof 0c preproof holshue ml et al first case of novel coronavirus in the united states n engl j med yeo c kaushal s yeo d enteric involvement o

cancer7519

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " preproofsevere acute respiratory syndrome coronavirus sarscov2 is a highly contagious zoonotic pathogen that has exacted heavy public health social and economic tolls in february the world health anization acronymed the disease caused by sarscov2 as covid19 for coronavirus disease the number of confirmed covid19 infections which has been detected in at least countries has reached worldwide as of april with deaths according to the us centers for disease control and prevention cdc1 many cases of covid19 resolve quickly however the disease which like other respiratory pathogens that cause common cold symptoms is believed to be transmitted through respiratory droplets infection with covid19 can also lead to significant morbidity and death this is particularly the case for cancer patients moreover because the signs and symptoms of covid are easily misattributed to the sequelae of cancer itself such as pulmonary embolism or its treatment such as nausea and diarrhea diagnosis may be delayed or missed potential covid rule out criteria based on the wells˜ criteria for pulmonary embolism another protean disease entity are provided as a decisionmaking aid this review summarizes the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis rationale to treat the cancer or not treatment and prevention of covid19 with an emphasis on implications in cancer keywords covid19 sarscov2 cancer introduction sarscov2 the rna virus responsible for the illness which has been named covid19 for coronavirus disease2019 the year it was diagnosed has sent shockwaves and dominated the news cycle due to its pandemic spread from the point of origin in wuhan china to the rest of the world declared a public health emergency of international concern pheic by the world health anization who2 sarscov2 is the third highly pathogenic novel zoonotic bat coronavirus sonamed because of the crownlike spikes on its surface to have emerged the first was sars coronavirus now named sarscov1 in with a fatality rate of and 0c preproofthe second was middle east respiratory syndrome mers in with a fatality rate of where the camel was the intermediate host3 sarscov1 merscov and sarscov2 belong to the betacoronavirus genus which are enveloped positivestranded rna viruses whose approximately nucleotide genome serves as an mrna template for the translation of viral proteins4 the virion contains four proteins spike envelope membrane and nucleocapsid and the host receptor with which the spike surface glycoprotein of sarscov2 engages is angiotensin converting enzyme ace25 the biology of sarscov2 is described in more detail in figure a zinc metallopeptidase enzyme ace2 which is abundantly present in lung and gastrointestinal epithelial cells6 not only mediates viral entry through receptormediated endocytosis7 but also the efficiency of viral replication8 its expression is upregulated with older age smoking the antihypertensives angiotensin converting enzyme ace inhibitors and angiotensin receptor blockers arbs thiazolidinediones tzds a class of oral antidiabetic drugs and ibuprofen risk factors which may increase susceptibility to the covid19 virus infection and which are common in generally elderly multimorbid cancer patients9 preproofthe clinical spectrum of sarscov2 ranges from mild upper respiratory tract infection with fever sore throat headache cough and potentially nausea and diarrhea the majority of cases recover without serious complications to severe pneumonia with sequelae that include acute respiratory distress syndrome ards cytokine storm and death10 because sars is an acronym for severe acute respiratory syndrome digestive manifestations including inappetence nausea abdominal pain and diarrhea of covid19 resulting from binding of the virus to the ace2 receptor in the gi tract may precede respiratory symptoms11 gastrointestinal manifestations are potentially underappreciated and overlooked as sentinel symptoms that herald the onset or persistence of disease especially in cancer patients and may contribute to delayed or missed opportunities for testing diagnosis and containment unlike sarscov1 which seems to have disappeareddied out12 and mers which reappears only sporadically sarscov2 is less lethal with a fatality rate between although this number is highly uncertain and debated13 but much more infective consequently public panic and economic disruption have ensued resulting in wartimelike mobilization efforts to mitigate its spread old age smoking and comorbidities such as diabetes morbid obesity immunosuppression frailty and cardiovascular disease appear to predispose to worse outcomes possibly secondary to impaired t and b cell responses notably covid19 infection is associated with lymphopenia and delayed development of the adaptive immune response which appears to correlate with prolonged virus clearance and more severe disease progression15 the first pillar of defense against infection is hand washing avoidance of face touching and minimization of close contact ie social distancing with selfquarantine and selfisolation16 in case of exposure or evidence of covid19 symptoms respectively the second prophylactic pillar is vaccination with specific viral antigens or mrnas which are not yet publicly available although the company moderna has reportedly started testing an mrna vaccine in healthy volunteers and multiple other vaccination strategiesplatforms appear to be in progressunder development17 0c transmission and prevention according to the world health anization who21 sarscov2 is spread persontoperson mainly via aerosol inhalation from sneezing coughing or exhalation 22and via fomitetoface contact since depending on the surface material the virus may remain viable and infectious for hours to days figure fecaloral transmission has also been hypothesized because diarrhea was a common feature with sars and mers and diarrhea and other digestive issues have also been reported in patients with covid1925 26notably transmission of sarscov2 is not limited to symptomatic individuals ie those with fever cough sore throat myalgias or dyspnea but also to asymptomatic or subclinically infected carriers of the virus which is problematic from the perspective of disease control 27and highlights the importance of containment measures including isolation and quarantine the basic reproduction number r0 of sarscov2 is which means that on average for every patient an additional individuals are infected because coronaviruses may persist on inanimate surfaces like metal glass or plastic for up to days careful disinfection with or greater ethanol for small surfaces or sodium hypochlorite for larger surfaces is recommended preproofdiagnosis and clinical features preproofthe available evidence is limited but clinical courses and outcomes of covid19 are likely to be worse in patients with cancer especially given the clear association between severity of disease and older age and higher levels of comorbidity the overall case fatality rate cfr for the general covid19infected population is around but in cancer it rises to overall and to in italy19 this cfr in cancer patients compares to for no comorbid conditions for cardiovascular disease for diabetes for hypertension and for chronic respiratory disease the aim of this review is to summarize and condense the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis treatment and prevention of covid19 with a special focus on cancer in the united states the test of choice for sarscov2 is a nasopharyngeal swab specimen or sputum if a productive cough is present on which a reversetranscriptase pcr rtpcr assay or an enzymelinked immunoassay eia directed particularly at the envelope e rnadependent rna polymerase rdrp spike protein s and nucleocapsid n genes is performed the fda has also approved an antibody test29 a positive test for sarscov2 in a symptomatic patient generally confirms the diagnosis of covid19 with the caveat that false positive and false negative tests have been documented if initial testing is negative but a high index of suspicion and pretest probability for covid19 remains on the basis of patient signs and symptoms then retesting is indicated in patients with high indexes of clinical suspicion and equivocal or negative test results the who recommends that lower respiratory tract specimens which contain the highest viral loads should be obtained since nasopharyngeal swabs may miss some infections30 according to cdc guidelines disease is excluded on the basis of two 0c preproofconsecutive negative tests respiratory tests separated by ‰¥ hours however in the presence of suggestive symptoms rectal swabs may also be indicated since the ace2 enzyme to which the virus binds is abundantly present in rectal epithelia cells31 the differential diagnosis for sarscov2 in cancer is extremely broad and includes conditions such as foreign body aspiration toxicities from chemotherapy and radiation tumor progression postobstructive pneumonia malignant obstruction atelectasis pulmonary embolism pneumonitis pulmonary edemafluid overload immunotherapyrelated pneumonitis copd exacerbation q fever adenovirus bocavirus coronavirus 229e hcov 229e coronavirus hku1 hcov hku1 coronavirus nl63 hcov nl63 coronavirus oc43 hcov oc43 human metapneumovirus hmpv influenza a influenza a subtype h1n1pdm09 influenza a subtypes h1 and h3 influenza b parainfluenza virus ““ piv “ respiratory syncytial virus ab rsv ab rhinovirusenterovirus hrvev bordetella pertussis legionella pneumophila and mycoplasma pneumoniae32 the diagnosis of sarscov2 is complicated by the possibility of simultaneous coinfection with other respiratory viruses33 which is especially true for immunosuppressed cancer patients whose susceptibility to microanisms is increased the heightened infectious risk for cancer patients underscores the importance of screening them at presentation with extended viral respiratory panel testing given that coinfection may impact management decisions since conceptually at least the morbidity of covid19 and the risk of severe illness should increase in the presence of a second or third virus preproofunlike infection with influenza for example covid19 signs and symptoms may vary considerably depending on the dose of viral inoculum route of inoculation concomitant medications and underlying health status34 to include fever dry cough fatigue sputum production shortness of breath sore throat headache myalgia or arthralgia chills nausea or vomiting nasal congestion diarrhea hemoptysis and conjunctival congestion with an incubation period of to days after exposure36 presymptomatic or minimally symptomatic infection may majorly drive transmission especially since detected viral loads are similar in both symptomatic and asymptomatic patients3738 populations of concern include the elderly smokers vapers and dual users those of any age with preexisting chronic medical conditions those receiving particular medications or therapies which upregulate the ace2 receptor or suppress the immune system and those from lower socioeconomic classes a conglomeration of factors which are often present in cancer patients as depicted in figure while the surveillance focus for covid19 is on the respiratory tract enteric symptoms are a potentially underappreciated overlooked and misattributed manifestation of disease as stated earlier and this is especially the case for cancer patients where gastrointestinal toxicity occurs routinely from chemotherapy ie cisplatincarboplatinoxaliplatin irinotecan 5fluorouracil ifosfamide from targeted agents ie erlotinib imatinib bortezomib temsirolimus sunitinib regorafenibsorafenib and bevacizumab39 and from locally advanced or metastatic disease therefore abdominal complaints in cancer patients which are potentially but not automatically attributable to underlying disease justify further investigation especially if persistent worsening or new particularly because sarscov2 transmission may occur via the fecal“oral route40 0c preproofabnormal laboratory findings in covid19 include lymphopenia percent prolonged prothrombin time percent elevated lactate dehydrogenase percent elevated ast and alt “ percent elevated highly sensitive hs crp and elevated procalcitonin however because these parameters routinely fall well outside of the normal reference range in cancer patients it is difficult to confirm or refute the presence of disease on this basis alone chest radiographs and chest ct abnormalities are similarly nonspecific since the most common features multifocal groundglass opacities and consolidation mimic other pneumonias41 significant antibody production is observed after infection but it is unknown whether this helps or harms since antibodydependent enhancement ade may potentiate viral entry and the induction of a severe inflammatory response42 universal screening of cancer patients for covid19 is desirable but logistically impossible for the foreseeable future since diagnostic tests are in short supply and simply not always readily available43 hence covid19 rule out criteria are proposed in table as a potential decisionmaking aide mémoire which separates patients into low and highrisk groups by analogy to the wells˜ criteria for pulmonary embolism4445 preventive measures focus on selfisolation social distancing with a 6foot 2m separation46 frequent hand washing with soap and water andor use of hand sanitizers patient isolation during clinical care use of masks to help prevent aerosol transmission and flushing with the lid closed to control socalled •toilet plume– in an asco guidance immunocompromised cancer patients are advised to minimize exposure to sick contacts and large crowds48 for healthcare personnel the use of personal protective equipment such as n95 masks ffp3 masks gowns eye protection gloves and gowns is mandated49 preproof vaccination and immunity vaccination efforts and the related topic of whether those who have recovered from covid19 develop protective immunity have drawn great attention the latter has implications on whether people who test positive for sarscov2 antibodies can be safely assumed to be immune and at negligible risk of contracting or transmitting the disease there have been case reports of patients who have recovered from covid19 and had recurrence of rtpcr positivity approximately one month after initial diagnosis with only one patient exhibiting significant clinical symptoms and another having a mild intermittent cough50 but while not zero the risk of transmissibility or recurrence of symptomatic disease in recovered patients has yet to be quantified and the paucity of currently available reports of recurrence in the setting of a pandemic suggests that it is low a separate practical question will be whether antibodybased tests prove to have sufficient sensitivity and specificity to identify people who had asymptomatic infections developed immunity and can return to normal activities without jeopardizing disease containment efforts immunity may be due to antibodies cell mediated immunity or a combination of the two previous experience with using plasma from convalescent patients to treat severe cases of the first sars and mers as well as limited experience with covid19 suggests that antibody mediated immunity alone is clinically beneficial even during acute infection51 safety concerns about antibodies have been raised based on preclinical studies of sarscov vaccination in 0c preproofferrets showing hepatotoxicity52 and of vaccination against feline infectious peritonitis virus another coronavirus leading to more severe disease when kittens were subsequently challenged with the virus53 although animal models may not be representative of human hostpathogen interactions the nature of sarscov and sarscov2 antibodies are likely different as crossneutralization was not observed invitro54 and experience with convalescent plasma has not borne evidence of antibody mediated enhancement of infection in acutely infected patients the potential risk deserves attention if vaccination is proposed for the entire population t cell responses are also readily observable in patients who recover from coronavirus infections55 and memory t cell responses alone were protective in mice56 with the potential advantage of longer persistence of memory t cell responses compared to humoral immunity when clinical data on vaccine candidates becomes available cancer patients may face different considerations surrounding vaccination than the general population particularly patients with hematologic malignancies being treated with agents targeting b cells who would derive greater benefit from vaccines eliciting cell mediated than antibody responses preproofpathogenesis and pathology relating to ace2 and ras signaling the ace2 enzyme a key regulator of the reninangiotensin system ras57 to which the virus binds through its surface spike proteins is particularly abundant in the digestive tract lungs kidney heart and blood vessels where pathology from sarscov2 occurs58 a peptidase that catalyzes the conversion of angiotensin ii angii referred to as •the quintessential perpetrator of inflammation– to angiotensin ang ace2 mediates antiproliferative and vasodilatory functions that oppose the vasoconstrictive and inflammatory functions of angiotensin converting enzyme ace60 the binding of sarscov2 to ace2 leads to downregulation of ace2 expression potentially through increased internalization and shedding from the cell surface with decreased ang1“ generation and increased ang ii levels as a consequence61 this unfavorably shifts the balance of the renin angiotensin system ras from the vasoprotective ace2ang17 axis to the aceang iiangiotensin at1 receptor axis and drives a proinflammatory profibrotic and proliferative response62 as shown in figure fang et al63 contend that because thiazolidinediones ibuprofen and angiotensin converting enzyme ace inhibitors and angiotensin ii typei receptor blockers arbs substantially increase the expression of ace2 they facilitate sarscov2 infection and therefore the risk of severe and fatal covid19 in contrast alghatrif et al64 present a diametrically opposed hypothesis that downregulated ace2 signaling is responsible for sarscov2induced acute lung injury ali acute respiratory distress syndrome ards and cytokine storm and that aceis and arbs are beneficial precisely because they increase ace2 expression and activity furthermore according to alghatrif et al lower ace2 levels and hence higher baseline oxidative stress and inflammation6566 are present in older comorbid individuals such as cancer patients which renders them more susceptible to severe covid19 than younger noncomorbid individuals with increased ace2 levels and lower baseline inflammation as shown in figure furthermore low ace2 may promote tumor progression and conversely ace2 overexpression is associated with antiangiogenesis and tumor regression67 in summary then 0c preproofdespite the concerns and controversy68 surrounding the use and continuation of aceisarbs during the sarscov2 epidemic it is likely that the pros outweigh the cons especially in cancer patients due to their potential antitumor and anticovid19 effects69 in line with ras involvement emerging data suggest that sarscov2 infection may induce serious cardiovascular injury or exacerbate existing cardiovascular disease cardiovascular sequela includes heart failure arrythmias disseminated intravascular dissemination dic and troponin elevation which may closely correlate with disease severity and the likelihood of inhospital death70 liu et al71 propose a mechanism whereby the virus which lowers hemoglobin hb levels72 binds to the porphyrin of heme and displaces iron thereby compromising the oxygencarrying capacity of red blood cells and exacerbating the hypoxemia since chloroquine and the experimental anticancer agent rrx001 also bind to porphyrins they may competitively interfere with binding by the virus rationale for continuation or discontinuation of cancer therapy preproofthe benefitrisk calculus that informs the decision whether and how to treat with anticancer therapy falls into a •gray zone– about which no consensus exists leading to a therapeutic dilemma on the one hand zhang et al73 in annals of oncology reported a strong association in patients of them with lung cancer between antineoplastic therapy in the past days and severe effects of covid19 hr4079 ci p0037 on this basis the authors recommend treatment interruption dose reduction or substitution of cytotoxic chemotherapy with nonimmunosuppressive options eg checkpoint inhibitors if available especially in the case of lung cancer patients that are already prone to develop respiratory infections and complications74 similarly heavily immunosuppressed patients such as those who have undergone hematopoietic stem cell transplantation are also particularly susceptible to viral respiratory infections these findings are supported by a nationwide analysis of data75 in china from covid19 patients of which were diagnosed with cancer this patient cohort experienced a higher incidence of severe events vs p and the administration of chemotherapy or surgery was found to have increased the risk of death andor intensive care unit admission even after adjusting for age sex and comorbidities odds ratio or ci “ p while these studies are limited by small sample sizes the data suggests that cancer predisposes to more severe disease therefore since inperson contact increases the risk of transmission several institutions have mandated realtime video or telephone interactions alternatively referred to as telehealth77 postponed surgeries biopsies endoscopies scans and routine investigations when possible and in line with esmo guidelines78 encouraged conversion from the intravenous to the oral route eg 5fluorouracil to capecitabine etoposide and vinorelbine on the other hand the immediate existential threat of progressive disease for which death is an impending imminent certainty rather than a remote possibility in the absence of treatment likely outweighs the theoretical risk of sarscov2 infection even in lower risk disease for example in situ or localized prostate breast and head and neck cancer delayed treatment is 0c preproofpotentially conducive to tumor development and progression and thus may unfavorably impact prognosis79 hanna et al have proposed a triage strategy80 which prioritizes treatment for those patients with imminent risk of early mortality from acute leukemias aggressive lymphomas metastatic germ cell tumors oncologic emergencies such as spinal cord compression chemoradiotherapeuticresponsive cancers such as head and neck cervical and anal cancers and neoadjuvant or adjuvant therapyresponsive tumor types such as stage iii colon cancer and deprioritizes visits for surveillance and survivorship however in the absence of a •one size fits all– consensus recommendation which is unlikely since cancer is so genetically diverse and heterogeneous the decisionmaking process and the subsequent treatment plan are individualized and to be determined tbd on casebycase basis taking into account multiple factors including the risk of cancer recurrence if therapy is delayed modified or interrupted the type of therapy eg surgery radiation chemotherapy checkpoint inhibitors and stem cell transplantation extent of comorbidities concomitant medications patient preferences physicianpatient relationship race age the number of cycles of therapy completed and treatment tolerance in terms of specific cancerrelated conditions asco makes the following heavily qualified recommendations81 \uf0b7 growth factor prophylaxis for neutropenia and neutropenic fever even at lower levels of risk as well as empiric antibiotics for acute care \uf0b7 erythropoietinstimulating agents for anemia prophylaxis and transfusion when necessary depending on the patient context and underlying comorbidities preproof treatment based on the high transmissibility of the virus82 the main nonpharmacologic countermeasures to mitigate or delay the impact of covid19 include rigorous hand hygiene use of facemasks respiratory etiquette ie coughing or sneeze into the upper sleeve or elbow not the hands flushing with the lid down to prevent bioaerosolization as well as quarantine stay at home policies and workplace and school closures which have upended the social cultural political and economic status quo no specific treatment or vaccine is currently available although promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir the mainstay of medical therapy includes symptomatic care such as supplemental oxygen antibiotics and hemodynamic and mechanical ventilatory support if indicated for septic shockmultiple an failure and respiratory failure respectively83 over active clinical treatment trials are underway84 these include vaccines as well as a number of different agents some with promising preliminary data as mentioned above and also those with potential anticancer activity which will hopefully serve a double purpose first to treat covid19 and second as an adjunct to bridge the time gap until the patient is recovered and the primary antineoplastic is startedrestarted as shown in table 0c preproof conclusions the alarming spread of the covid19 pandemic has disproportionately affected cancer patients an atrisk population both from the standpoint of increased disease severity and disruption to care which includes widespread suspension of clinical trials in the united states that are already fraught with barriers to enrollment and participation85 86because the symptoms of covid19 are nonspecific underlying symptoms from the cancer eg dyspnea cough fever fatigue diarrhea etc which overlap with those from the viral infection may obscure and delay the diagnosis hence if the covid19specific rapid reverse transcriptase polymerase chain reaction rtpcr test is not readily available andor in short supply which is currently the case diagnosis will depend on the maintenance of a high index of clinical suspicion especially in advanced cancer patients who check all the boxes for risk factors such as older age frailty disability immunosuppression generalized systemic inflammation and multiple comorbidities eg hypertension diabetes and cardiorenovascular diseases that predispose to severe disease and death preproofthese comorbidities are commonly treated with renin angiotensin system blockers such as angiotensinconverting enzyme inhibitors aceis or angiotensinreceptor blockers arbs which increase levels of ace2 the continued use of aceisarbs is the centerpiece of an intense debate because on the one hand sarscov2 coopts ace2 for target cell entry but on the other ace2 overexpression may counterbalance vasoconstriction and profibrotic processes and thereby reduce the incidence or mortality associated with covid19“associated ali or acute respiratory distress syndrome another controversy involves whether or not to continue cancer treatment given the high transmissibility potential of the virus however since no expert consensus recommendations have been issued to date and prognosis stage and responses to therapy are highly heterogeneous the riskbenefit tradeoff and subsequent treatment plan are highly individualized and context dependent currently the focus of treatment is infection control appropriate symptomatic care and oxygen therapy no approved medication or vaccine has been developed but promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir and several repurposed agents with antitumor properties are under investigation including thalidomide and rrx001 which may hopefully bridge the gap from the time covid is first diagnosed until the primary anticancer therapy is restarted finally multiple comparisons have been made between the allout mobilization efforts to combat covid19 with the massive scaleup of human and material resources that occurred during world war ii8788 in the words of winston churchill prime minister of great britain from whose intrepid fighting spirit iron will and intransigent defiance of tyranny galvanized the resolve of an entire nation to fight on in the face of seemingly impossible odds oncologists on the frontlines that have answered the call should •never worry about action only inaction– 0c preproof declaration of interests the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper references sitammagari k skandhan a dahlin awhat hospitalists need to know about covid19 medscape mar httpswwwmedscapecomviewarticle924596 meo sa alhowikan am alkhlaiwi t meo im halepoto dm iqbal m usmani am hajjar w ahmed n novel coronavirus 2019ncov prevalence biological and clinical characteristics comparison with sarscov and merscov eur rev med pharmacol sci feb24420122019 chan jf kok kh zhu z et al genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan emerg microbes infect kuba k imai y rao s et al a crucial role of angiotensin converting enzyme ace2 in sars coronavirusinduced lung injury nat med zhang h kang z gong h et al the digestive system is a potential route of 2019ncov infection a bioinformatics analysis based on singlecell transcriptomes [epub ahead of print] biorxiv de wit e van doremalen n falzarano d munster vj sars and mers recent insights into emerging coronaviruses nat rev microbiol “ w li et al angiotensinconverting enzyme is a functional receptor for the sars coronavirus nature hughes s covid19 and angiotensin drugs help or harm medscape march httpswwwmedscapecomviewarticle927542 yang x yu y xu j et al clinical course and outcomes of critically ill patients with sarscov2 pneumonia in wuhan china a singlecentered retrospective observational study [published online ahead of print feb ] [published correction appears in lancet respir med feb ] lancet respir med 2020s2213 gu j han b wang j covid19 gastrointestinal manifestations and potential fecaloral transmission [published online march ] gastroenterology ruiheng x chance missed but still there memoirs at the 10th anniversary of sars outbreak j thorac dis aug 5suppl s90“s93 jiang s don't rush to deploy covid19 vaccines and drugs without sufficient safety guarantees nature mar5797799321 doi 101038d41586020007519 peiris js guan y yuen ky severe acute respiratory syndrome nat med dec suppls8897 zheng h zhang m yang c et al elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid19 patients cell mol immunol2020 tognotti e lessons from the history of quarantine from plague to influenza a emerg infect dis “ httpswwwnihgovnewseventsnewsreleasesnihclinicaltrialinvestigationalvaccinecovid19begins httpswwwascoascocoronavirusinformationcareindividualscancerduringcovid19 onder g rezza g brusaferro s casefatality rate and characteristics of patients dying in relation to covid in italy published online march doi101001jama20204683 hanna tp evans ga booth cm cancer covid19 and the precautionary principle prioritizing treatment during a global pandemic nat rev clin oncol who advice on the use of masks in the community during home care and in healthcare settings in the context of the novel coronavirus 2019ncov outbreak jan rodriguezmorales aj macgregor k kanagarajah s patel d schlagenhauf p going global travel and the novel coronavirus travel med infect dis httpsdoi101016jtmaid2020101578 httpswwwnejmdoifull101056nejmc2004973 lu cw liu xf jia zf 2019ncov transmission through the ocular surface must not be ignored the lancet preproof 0c preproof holshue ml et al first case of novel coronavirus in the united states n engl j med yeo c kaushal s yeo d enteric involvement o Answer:

Colon_Cancer

"what clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials as a result medical research and care have been centred on male physiology the assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 in the us national institutes of health nih mandated the inclusion of women in nihfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy preclinical research and drug development studies have also predominantly used male animal models and cells4“ it is not surprising that a us government accountability office report found that eight of the ten prescription drugs withdrawn from the market between and œposed greater health risks for women than for men most funding agencies from europe and north america have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentthis review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences we aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom vol august biological and environmental modifiers of chronic disease ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and mensex as a genetic modifier of biology and diseasesex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an x or a y chromosome resulting in an embryo carrying either xx or xy chromosomes this fundamental difference in chromosome complement eg genes outside the testisdetermining sry gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 first the y chromosome carries genes that exhibit subtle functional differences from their xlinked homologues eg zfy vs zfx and uty vs utx and also carries genes with no homologue at all eg sry in addition in men the x chromosome carries only maternal imprints ”ie epigenetic modifications made by the parent in generating the sex cells”which alter the expression of genes in the offspring as women have x chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes random inactivation of one of the x chromosomes in female cells which prevents sex differences in x chromosome gene dosage causes another degree of sex difference in gene expression as some of these xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 sexspecific gene expression due to genomic search strategy and selection criteriawe searched pubmed for papers published in english between jan and june using œsex or œgender and the name of the disease of interest as search terms although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedlancet “diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine and southeast louisiana veterans health care system medical center new orleans la usa prof f mauvaisjarvis md barbra streisand women™s heart center cedarssinai smidt heart institute los angeles ca usa prof n bairey merz md national heart lung institute imperial college london london uk prof p j barnes md department of pharmacology and department of neurology college of medicine center for innovation in brain science university of arizona tucson az usa prof r d brinton phd department of medical epidemiology and biostatistics and center for gender medicine karolinska institutet stockholm sweden prof jj carrero phd channing division of network medicine and the division of pulmonary and critical care medicine department of medicine brigham and women™s hospital harvard medical school boston ma usa d l demeo md neuroscience institute and department of biology geia state university atlanta ga usa prof g j devries phd department of psychiatry university of colorado school of medicine anschutz medical campus aurora co usa prof c n epperson md division of oncology department of medicine washington university school of medicine st louis mo usa prof r govindan md w harry feinstone department of molecular microbiology and immunology the johns hopkins bloomberg school of public health baltimore md usa prof s l klein phd department of biomedical metabolic and neural sciences university of modena andreview 0creggio emilia azienda ospedalierouniversitaria di modena ospedale civile di baggiovara modena italy prof a lonardo md department of psychiatry department of psychology and department of obstetrics gynecology university of illinois at chicago chicago il usa prof p m maki phd department of neurology mcgovern medical school university of texas health science center houston tx usa prof l d mccullough md berlin institute of gender medicine charit”universittsmedizin berlin berlin germany prof v regitzzagrosek md department of cardiology university hospital z¼rich university of z¼rich switzerland prof v regitzzagrosek center for women™s health research divisions of general internal medicine and cardiology university of colorado school of medicine aurora co usaimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 thus fundamental sex differences deriving directly from genetic heterogeneity between the x and y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells these sex differences persist throughout life and are independent of sex hormones figure arguably the greatest source of differences between men and women comes from the y chromosomal sry gene which directs the development of a testis in men the ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 in humans the first surge occurs at the end of the first trimester of pregnancy because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood after this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations after puberty cells with androgen or afemale sexbrandom x chromosomeinactivation and escapexxxxxxxxxxxxxxxxxxxy chromosome complementmale sexsryctesticular testosterone surgefetal testistestosteroneohogenetic diï¬erences of male and female cellsepigenetic programming of male cellsfigure genetic causes of sex differencesa genetic sex differences start with cells carrying either xx or xy chromosome complement eg genes outside the testisdetermining sry gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells b random inactivation of one x chromosome in female cells causes another level of sex differences in gene expression some xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals c the y chromosomal sry gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy the testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling the combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women the combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure gender as a determinant of patients™ and doctors™ behaviour and as a modifier of health disease and medicinegender according to the global health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 gender is not a binary term it includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 in transgender people gender identity differs with the sex they were assigned at birth so far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 gender roles represent the behavioural norms applied to men and women in society which influence individuals™ everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 as such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender as such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom vol august review 0cdiseases this postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 in the genesispraxy prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 although beyond the scope of this review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20“ sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 sex influences behaviours eg towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes figure summarises how sex and gender are interrelated in biology and diseasesex and gender differences in major chronic diseaseshaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the usa as an example figure note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 in most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this review focuses on how women differ from men we discuss some key aspects regarding the dimensions of men in a dedicated sectionheart diseaseepidemiology pathogenesis manifestations and diagnosisheart disease is the leading cause of death in the usa in heart disease accounted for · of all deaths for men and for · of all deaths for women figure ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences for example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women the strength of the association with cardiovascular risk factors differ by sex biological sexsex chromosomesepigeneticeï¬ectssex hormonesohohohobehaviour of patients and doctorssocietygender constructslifestylenutritional habitsexerciseperceived stresssmokingdiseasepathophysiologymanifestationresponse to treatmentdisease perceptionhelpseeking behaviouruse of health caredecision makingtherapeutic responsesex and gender diï¬erences in health disease and medicinefigure interrelation between sex and gender in health diseases and medicinebiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment sex also influences behaviours towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex gender constructs determine patients™ perception of disease helpseeking behaviour and individual use of health care gender constructs also influence decision making and trigger different therapeutic responses from providers biased by gendersystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33“ the reasons for this disparity reflect the intersection between sex and gender first biological sex differences exist in the pathogenesis of ischaemic heart disease whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 a metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathprof j g regensteiner phd department of medicine department of paediatrics and department of neuroscience washington university school of medicine st louis mo usa prof j b rubin md center for the study of sex differences in health aging and disease geetown university washington dc usa prof k sandberg phd division of gastroenterology duke university medical center durham nc usa a suzuki md and durham va medical center durham nc usa a suzukicorrespondence to prof franck mauvaisjarvis diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine new orleans la usa fmauvaistulaneeduwwwthelancetcom vol august review 0cmale individualsother·heart disease·protection might disappear after menopause45 by contrast testosterone induces adverse cardiac remod elling in the male heart44chronic liver disease ·influenza and pneumonia ·suicide ·alzheimer™s disease ·type diabetes ·stroke ·cpd ·injuries ·female individualsother·septicaemia ·chronic kidney disease ·influenza and pneumonia ·type diabetes ·injuries ·alzheimer™s disease ·stroke ·cpd ·cancer·heart disease·cancer·figure percent distribution of the ten leading causes of death by sex usa adapted from heron25 cpdchronic pulmonary diseasesecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39heart failure affects of adults aged years and older and more women than men in absolute numbers4041 heart failure occurs at an older age and with less ischaemic causes in women than in men however hypertension and diabetes predispose older women to heart failure to a greater extent than men heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men by contrast heart failure with reduced ejection fraction affects more men than women women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction under stress premenopausal women™s hearts develop less inflammation resulting in less fibrosis than men™s hearts4243 this difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 this response to treatmentcompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33“ an stelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 this treatment disparity between women and men can be corrected by improving emergency recognition of stelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47guidelines for the treatment of heart failure are similar for women and men24 however evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41cancersepidemiology pathogenesis manifestations and diagnosiscancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure more men develop cancer than women49 with few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than a male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 survival is also shorter for men than women across multiple cancer types the higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 it is unlikely to be the only cause after appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53the universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom vol august review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology sexspecific biology includes genetic differences xx vs xy chromosomes the incomplete xinactivation in female individuals which results in biallelic expression of xencoded female cells54 y chromosomeencoded oncogenes such as the rnabinding motif on y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 these mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 a crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer in glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 after puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer for example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant take colon cancer the second leading cause of cancerrelated death for example although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 this molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksresponse to treatmentin the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches for example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 the molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 in colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968chronic pulmonary diseaseepidemiology pathogenesis manifestations and diagnosischronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure it is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 the female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 the genetic epidemiology of chronic obstructive pulmonary disease copdgene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences future studies should focus on the contribution of maternally inherited factors such as mitochondrial and x chromosome genes to understand disease pathogenesis it is important to consider gender constructs as well smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced women™s risk for developing chronic obstructive pulmonary disease73 from a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom vol august review 0casthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently asthma is more prevalent in prepubertal boys than girls regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 from puberty onwards more female than male individuals have asthma with an increased severity in middleaged women and a higher mortality rate76 this phe nomenon could be secondary to gender differences eg symptoms perception or healthseeking behaviours however biological sex plays a crucial role in asthma and sex hormones have a major impact on female asthma symptoms and severity after puberty75 worsening of asthma occurs in women before menstruation and is known as premenstrual asthma premenstrual asthma is more common in women with severe rather than mild asthma obesity rather than normal weight and a long rather than a short duration of asthma77 premenstrual asthma is hypoth esised to be due to a fall in progesterone and patients with a severe disease respond to progestogens78 during pregnancy approximately a third of asthmatic women exhibit a worsening of asthma a third show an improvement and the remainder are unaffected79response to treatmentthe menopausal transition represents a pivotal time of accelerated decline in lung function in women with chronic obstructive pulmonary disease and thus represents a sexspecific window for treatment intensification these observations also suggest that oestrogens protect from chronic obstructive pulmonary disease women exhibit greater expression of m2 over m3 muscarinic receptors and accordingly show greater improvements in lung function than men in response to the muscarinic anticholinergic bronchodilator ipatroprium80 pooled analyses of drug studies also suggest that women experience a greater improvement in quality of life than men after treatment of chronic obstructive pulmonary disease with a β2adrenoreceptor agonist combined with an anti cholinergic drug eg indacaterol and glycopyrronium81unlike chronic obstructiv

cancer7520

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "what clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials as a result medical research and care have been centred on male physiology the assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 in the us national institutes of health nih mandated the inclusion of women in nihfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy preclinical research and drug development studies have also predominantly used male animal models and cells4“ it is not surprising that a us government accountability office report found that eight of the ten prescription drugs withdrawn from the market between and œposed greater health risks for women than for men most funding agencies from europe and north america have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentthis review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences we aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom vol august biological and environmental modifiers of chronic disease ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and mensex as a genetic modifier of biology and diseasesex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an x or a y chromosome resulting in an embryo carrying either xx or xy chromosomes this fundamental difference in chromosome complement eg genes outside the testisdetermining sry gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 first the y chromosome carries genes that exhibit subtle functional differences from their xlinked homologues eg zfy vs zfx and uty vs utx and also carries genes with no homologue at all eg sry in addition in men the x chromosome carries only maternal imprints ”ie epigenetic modifications made by the parent in generating the sex cells”which alter the expression of genes in the offspring as women have x chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes random inactivation of one of the x chromosomes in female cells which prevents sex differences in x chromosome gene dosage causes another degree of sex difference in gene expression as some of these xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 sexspecific gene expression due to genomic search strategy and selection criteriawe searched pubmed for papers published in english between jan and june using œsex or œgender and the name of the disease of interest as search terms although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedlancet “diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine and southeast louisiana veterans health care system medical center new orleans la usa prof f mauvaisjarvis md barbra streisand women™s heart center cedarssinai smidt heart institute los angeles ca usa prof n bairey merz md national heart lung institute imperial college london london uk prof p j barnes md department of pharmacology and department of neurology college of medicine center for innovation in brain science university of arizona tucson az usa prof r d brinton phd department of medical epidemiology and biostatistics and center for gender medicine karolinska institutet stockholm sweden prof jj carrero phd channing division of network medicine and the division of pulmonary and critical care medicine department of medicine brigham and women™s hospital harvard medical school boston ma usa d l demeo md neuroscience institute and department of biology geia state university atlanta ga usa prof g j devries phd department of psychiatry university of colorado school of medicine anschutz medical campus aurora co usa prof c n epperson md division of oncology department of medicine washington university school of medicine st louis mo usa prof r govindan md w harry feinstone department of molecular microbiology and immunology the johns hopkins bloomberg school of public health baltimore md usa prof s l klein phd department of biomedical metabolic and neural sciences university of modena andreview 0creggio emilia azienda ospedalierouniversitaria di modena ospedale civile di baggiovara modena italy prof a lonardo md department of psychiatry department of psychology and department of obstetrics gynecology university of illinois at chicago chicago il usa prof p m maki phd department of neurology mcgovern medical school university of texas health science center houston tx usa prof l d mccullough md berlin institute of gender medicine charit”universittsmedizin berlin berlin germany prof v regitzzagrosek md department of cardiology university hospital z¼rich university of z¼rich switzerland prof v regitzzagrosek center for women™s health research divisions of general internal medicine and cardiology university of colorado school of medicine aurora co usaimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 thus fundamental sex differences deriving directly from genetic heterogeneity between the x and y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells these sex differences persist throughout life and are independent of sex hormones figure arguably the greatest source of differences between men and women comes from the y chromosomal sry gene which directs the development of a testis in men the ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 in humans the first surge occurs at the end of the first trimester of pregnancy because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood after this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations after puberty cells with androgen or afemale sexbrandom x chromosomeinactivation and escapexxxxxxxxxxxxxxxxxxxy chromosome complementmale sexsryctesticular testosterone surgefetal testistestosteroneohogenetic diï¬erences of male and female cellsepigenetic programming of male cellsfigure genetic causes of sex differencesa genetic sex differences start with cells carrying either xx or xy chromosome complement eg genes outside the testisdetermining sry gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells b random inactivation of one x chromosome in female cells causes another level of sex differences in gene expression some xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals c the y chromosomal sry gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy the testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling the combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women the combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure gender as a determinant of patients™ and doctors™ behaviour and as a modifier of health disease and medicinegender according to the global health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 gender is not a binary term it includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 in transgender people gender identity differs with the sex they were assigned at birth so far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 gender roles represent the behavioural norms applied to men and women in society which influence individuals™ everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 as such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender as such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom vol august review 0cdiseases this postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 in the genesispraxy prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 although beyond the scope of this review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20“ sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 sex influences behaviours eg towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes figure summarises how sex and gender are interrelated in biology and diseasesex and gender differences in major chronic diseaseshaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the usa as an example figure note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 in most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this review focuses on how women differ from men we discuss some key aspects regarding the dimensions of men in a dedicated sectionheart diseaseepidemiology pathogenesis manifestations and diagnosisheart disease is the leading cause of death in the usa in heart disease accounted for · of all deaths for men and for · of all deaths for women figure ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences for example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women the strength of the association with cardiovascular risk factors differ by sex biological sexsex chromosomesepigeneticeï¬ectssex hormonesohohohobehaviour of patients and doctorssocietygender constructslifestylenutritional habitsexerciseperceived stresssmokingdiseasepathophysiologymanifestationresponse to treatmentdisease perceptionhelpseeking behaviouruse of health caredecision makingtherapeutic responsesex and gender diï¬erences in health disease and medicinefigure interrelation between sex and gender in health diseases and medicinebiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment sex also influences behaviours towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex gender constructs determine patients™ perception of disease helpseeking behaviour and individual use of health care gender constructs also influence decision making and trigger different therapeutic responses from providers biased by gendersystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33“ the reasons for this disparity reflect the intersection between sex and gender first biological sex differences exist in the pathogenesis of ischaemic heart disease whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 a metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathprof j g regensteiner phd department of medicine department of paediatrics and department of neuroscience washington university school of medicine st louis mo usa prof j b rubin md center for the study of sex differences in health aging and disease geetown university washington dc usa prof k sandberg phd division of gastroenterology duke university medical center durham nc usa a suzuki md and durham va medical center durham nc usa a suzukicorrespondence to prof franck mauvaisjarvis diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine new orleans la usa fmauvaistulaneeduwwwthelancetcom vol august review 0cmale individualsother·heart disease·protection might disappear after menopause45 by contrast testosterone induces adverse cardiac remod elling in the male heart44chronic liver disease ·influenza and pneumonia ·suicide ·alzheimer™s disease ·type diabetes ·stroke ·cpd ·injuries ·female individualsother·septicaemia ·chronic kidney disease ·influenza and pneumonia ·type diabetes ·injuries ·alzheimer™s disease ·stroke ·cpd ·cancer·heart disease·cancer·figure percent distribution of the ten leading causes of death by sex usa adapted from heron25 cpdchronic pulmonary diseasesecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39heart failure affects of adults aged years and older and more women than men in absolute numbers4041 heart failure occurs at an older age and with less ischaemic causes in women than in men however hypertension and diabetes predispose older women to heart failure to a greater extent than men heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men by contrast heart failure with reduced ejection fraction affects more men than women women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction under stress premenopausal women™s hearts develop less inflammation resulting in less fibrosis than men™s hearts4243 this difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 this response to treatmentcompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33“ an stelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 this treatment disparity between women and men can be corrected by improving emergency recognition of stelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47guidelines for the treatment of heart failure are similar for women and men24 however evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41cancersepidemiology pathogenesis manifestations and diagnosiscancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure more men develop cancer than women49 with few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than a male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 survival is also shorter for men than women across multiple cancer types the higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 it is unlikely to be the only cause after appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53the universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom vol august review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology sexspecific biology includes genetic differences xx vs xy chromosomes the incomplete xinactivation in female individuals which results in biallelic expression of xencoded female cells54 y chromosomeencoded oncogenes such as the rnabinding motif on y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 these mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 a crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer in glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 after puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer for example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant take colon cancer the second leading cause of cancerrelated death for example although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 this molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksresponse to treatmentin the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches for example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 the molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 in colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968chronic pulmonary diseaseepidemiology pathogenesis manifestations and diagnosischronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure it is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 the female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 the genetic epidemiology of chronic obstructive pulmonary disease copdgene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences future studies should focus on the contribution of maternally inherited factors such as mitochondrial and x chromosome genes to understand disease pathogenesis it is important to consider gender constructs as well smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced women™s risk for developing chronic obstructive pulmonary disease73 from a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom vol august review 0casthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently asthma is more prevalent in prepubertal boys than girls regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 from puberty onwards more female than male individuals have asthma with an increased severity in middleaged women and a higher mortality rate76 this phe nomenon could be secondary to gender differences eg symptoms perception or healthseeking behaviours however biological sex plays a crucial role in asthma and sex hormones have a major impact on female asthma symptoms and severity after puberty75 worsening of asthma occurs in women before menstruation and is known as premenstrual asthma premenstrual asthma is more common in women with severe rather than mild asthma obesity rather than normal weight and a long rather than a short duration of asthma77 premenstrual asthma is hypoth esised to be due to a fall in progesterone and patients with a severe disease respond to progestogens78 during pregnancy approximately a third of asthmatic women exhibit a worsening of asthma a third show an improvement and the remainder are unaffected79response to treatmentthe menopausal transition represents a pivotal time of accelerated decline in lung function in women with chronic obstructive pulmonary disease and thus represents a sexspecific window for treatment intensification these observations also suggest that oestrogens protect from chronic obstructive pulmonary disease women exhibit greater expression of m2 over m3 muscarinic receptors and accordingly show greater improvements in lung function than men in response to the muscarinic anticholinergic bronchodilator ipatroprium80 pooled analyses of drug studies also suggest that women experience a greater improvement in quality of life than men after treatment of chronic obstructive pulmonary disease with a β2adrenoreceptor agonist combined with an anti cholinergic drug eg indacaterol and glycopyrronium81unlike chronic obstructiv Answer:

Colon_Cancer

in the uk the death toll from severe covid19 is among the highest worldwide1 severe covid19 is characterised by respiratory failure with socalled cytokine storm occurring in some patient subsets2 pathological correlates are required to understand the pathophysiology of covid19 autopsybased histopathological analysis is crucial in this respect in anticipation of the covid19 pandemic our group produced national guidelines for autopsy performance in suspected covid19 cases3covid19 is caused by infection with severe acute respiratory syndrome coronavirus sarscov245 although sarscov2 and its predecessor sarscov causing severe acute respiratory syndrome [sars] are toll similar on a molecular and clinical level covid19 has a lower death rate for covid19 vs for sars and a substantially higher death deaths worldwide from covid19 as of aug vs from sars than sars due to a higher basic reproduction number1 the postmortem findings in patients with sarscov infection included diffuse alveolar damage dad splenic and nodal lymphocyte depletion haemophagocytosis renal acute tubular injury cerebral oedema microthrombosis and adrenalitis with necrosis with intracellular sarscov detected in the lungs kidney brain and haematological ans6 various autopsy series on covid19 have begun to emerge in the literature7“ here we document the major pathological lancet microbe published online august 101016 s2666524720301154department of cellular pathology northwest london pathology b hanley mbbch prof k n naresh md c roufosse phd j weir frcpath prof r goldin md p viola md m osborn frcpath and department of hepatology p manousou phd imperial college london nhs trust london uk centre for haematology b hanley prof k n naresh and centre for inflammatory diseases c roufosse department of immunology and inflammation department of infectious disease prof g s cooke frcp a abdolrasouli phd o c swann mres l baillon bsc r penn msc prof w s barclay phd and department of metabolism prof m thursz md faculty of medicine imperial college london london uk department of histopathology royal brompton and harefield nhs foundation trust and national heart and lung institute imperial college london london uk prof a g nicholson dm renal and transplant centre hammersmith hospital imperial college healthcare nhs london uk r corbett phd department of neuropathology kings college hospital london uk prof s alsarraj frcpath death investigation committee royal college of pathologists london uk m osborn and nightingale nhs hospital london uk m osborn correspondence to dr brian hanley department of cellular pathology northwest london pathology charing cross hospital campus london w6 8na uk bhanleyimperialacukwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cresearch in contextevidence before this studycovid19 is a new disease and comprehensive descriptions of the histopathological findings at autopsy are scarce we reviewed the literature available on covid19 autopsy findings up to and including may for this we searched pubmed and google scholar databases with no language restrictions using the search terms œcovid19 œsarscov2 œhistology œautopsy and œpostmortemadded value of this studyour series focused on providing a comprehensive description of the histopathological findings in patients with severe fatal covid19 and correlating these findings with data on viral tropism the most prominent findings included diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion several novel autopsy findings in patients with covid19 were also described including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activationimplications of all the available evidenceour study supports the existing clinical and autopsy literature that identified diffuse alveolar damage thrombosis immune cell depletion and macrophage activation as the most prominent pathological features in covid19 other factors including acute kidney injury pancreatitis pericarditis secondary fungal infections and preexisting liver disease require further investigation the presence of ongoing viral replication in late stage covid19 supports the continued use of antiviral therapy even at a point in illness when immunopathology is dominantsee online for appendixfindings of ten postmortem examinations done on patients with clinically confirmed covid19methodspatient selectionfor this study eligible patients were older than years with premortem sarscov2 infection and covid19 listed clinically as the direct cause of death under part on the medical certificate of cause of death [mccd] consent was obtained for all included patients according to the human tissue authority codes of practice by a member of the trust core postmortem consent team consent rate was · ten of patients exclusion criteria included extended postmortem interval before autopsy days and patients with covid19 contributing but not directly leading to death under part of the mccd patients were from imperial college national health service nhs trust nine patients london uk and royal brompton harefield foundation nhs trust one patient london uk premortem sarscov2 infection was identified using the coronavirus typing multiplextandem pcr highplex system aus diagnostics chesham uk ethical approval for this project was provided by the imperial college healthcare tissue bank r20012autopsy proceduresfull autopsies were done on nine patients pm1“ and one patient underwent percutaneous biopsy sampling heart lungs pancreas kidneys and liver using percutaneous biopsy under ultrasound guidance pm10 full postmortem examinations included standard sampling and were done according to royal college of pathologists guidelines3 eight different regions of the brain were sampled for each full neuropathological examination all tissue samples were fixed in formalin for a minimum of h before embedding histochemical stains and immunohistochemistry were applied according to local protocols appendix p ans were reviewed by subspecialist pathologists in lung agn and pv haem ato pathology and immune pathology knn liver rg gastrointestinal mo neuropathology sas and renal pathology cr integrated interpretation was done by a subspecialty autopsy pathologist bh and mo all cases were reviewed independently by at least two pathologistspcr proceduresfresh tissue for quantitative rtpcr qrtpcr analysis was processed within the biosafety level facilities at st mary™s hospital london uk approved by the uk health and safety executive and in accordance with local rules at imperial college london total rna was obtained from fresh tissue samples by use of trizolchloroform extraction followed by precipitation and purification using the rneasy kit qiagen hilden germany qrtpcr against e gene rdrp and subgenomic rna was done as described elsewhere1617 in patient pm5 total fungal genomic dna was extracted from four to five ribbon slices of a formalinfixed paraffinembedded lung tissue block purified dna was amplified with pcr for panfungal and mucoralesspecific targetsstatistical analysisall data was analysed using spss software version and expressed using median iqr and percentagerole of the funding sourcethe funder of the study had no role in study design data collection data analysis data interpretation or writing of the the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publicationresultsbetween march and april ten patients were included in the study the median age at death was years wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ciqr “ seven of ten patients were men three were women and most patients were white or asian nine hypertension four patients and chronic obstructive pulmonary disease three were the most common contributing factors to death according to mccd all ten patients developed fever and had at least two respiratory symptoms or signs cough shortness of breath reduced oxygen saturations or pleuritic chest pain during their early presentation of eight patients assessed for inflammatory markers all had elevated inflammatory markers these features were either apparent upon presentation to hospital eight of ten patients or developed in an inpatient two patients pm8 and pm9 most patients died within weeks of symptom onset seven patients and were not intubated or ventilated six patients four patients were intubated during their presentation pm2 for days pm5 for days pm6 for less than day and pm7 for days the median bodymass index bmi was in the obese range · iqr ·“· and more patients were obese according to bmi at post mortem five of nine than indicated on the mccd one of ten the median interval between death and postmortem examination was days iqr ·“· although the limited post mortem had a shorter interval less than h after death detailed clinical case vignettes are available in the appendix p and clinical data are summarised also in the appendix p all patients had dad six showed purely exudative phase dad and four showed a mixture of exudative and anising dad appendix p figure three of four patients with anisingphase dad had spent a substantial period on a ventilator days days and days florid acute bronchopneumonia and ventilatorassociated pneumonia were not noted in this series although mild interstitial neutrophilic inflammation three of ten patients and patchy acute bronchopneumonia three patients were observed interstitial macrophages were prominent macrophages were accompanied by scattered plasma cells mild or moderate lymphocyte inflammation was present in all ten patients although focal lymphocyte cuffing of small vessels was noted in six patients we noted that lymphocytes in the lung were predominantly cd4positive t cells cd56positive natural killer cells were rarely found occasionally a patient had small aggregates of small b cells chronic bronchiolitis was seen in most patients nine of ten no granulomas or viral inclusion were seen invasive mucormycosis was noted in one patient pm5 figure and confirmed with mucoralesspecific pcr the mucormycosis was vasculocentric and disseminated involving the hilar lymph nodes heart brain and kidney in the same patientmacroscopic two of nine patients and microscopic eight of nine pulmonary thromboemboli were frequent observations appendix p figure both fibrinrich and plateletrich thrombi were identified in smallsized and mediumsized vessels and within the capillaries in alveolar septa figure external examination findings of deep venous thrombosis were not noted very focal lymphocytic vasculitis was identified in one patientthrombotic features were universal in this cohort and all nine patients who underwent a full autopsy had at least one microthrombosis or macrothrombosis in a major an one of nine patients had a macroscopic acute coronary thrombosis in the right coronary artery whereas five patients had thrombi in the microcirculation of the heart on histological analysis coronary artery disease was negligible or mild in most patients seven of nine acute myocardial ischaemic damage h old was noted in the patient with an acute coronary artery thrombus figure 2a pm1 a mottled myocardium and subendocardial contraction band necrosis was noted in a acebdf 03m µm µmfigure pulmonary pathological findings in patients with covid19a macroscopic subpleural petechial haemorrhage in a 24yearold man pm6 b hyaline membranes indicative of exudative phase diffuse alveolar damage in a 79yearold woman pm9 at 20x magnification c cd61 immunohistochemical staining indicating plateletrich microthrombosis in alveolar capillaries pm6 d squamous metaplasia in a 61yearold man pm1 with exudative phase diffuse alveolar damage at × magnification e interstitial multinucleated giant cells in a 79yearold man pm7 with anising phase diffuse alveolar damage at × magnification the top right insert is of multinucleated giant cells showing positive cd68 staining indicative of macrophage lineage the bottom left insert shows absence of staining for cytokeratins f periodic acid schiff staining indicating wide irregular aseptate and ribbonlike hyphae with openangle branching and a vasculocentric pattern indicative of mucormycosis in a 22yearold man pm5 the insert is a grocott silver stain highlighting mucormycosis at 20x magnificationwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0csecond patient pm2 whether the contraction band necrosis was related to ischaemia or inotropic medication received in the intensive care unit is uncertain appendix p pm1 and pm2 were the two patients with the highest active viral load detected in the heart a single patient had a right atrial thrombus pericarditis was acegbdfh µm µm 03m µmfigure thrombotic features identified at autopsy in patients with covid19a macroscopic right coronary artery thrombosis arrow in a 61yearold man pm1 with exudative phase diffuse alveolar damage b macroscopic pulmonary thromboembolism arrow in a 97yearold man pm8 c thrombus in the lung of a 79yearold woman pm9 on haematoxylin and eosin staining at 20x magnification the insert shows cd61 immunohistochemistry indicating moderate staining for platelets d plateletrich thrombus in the mediumsized vessels surrounding the heart in a 61yearold man pm1 the insert shows strong cd61 staining for platelets periodic acid schiff staining showing a glomerular microaneurysm arrow e and microthrombi within glomerular capillary loops arrow f at 40x magnification indicative of thrombotic microangiopathy in a 97yearold man pm8 macroscopic splenic g and hepatic h infarction in a 22year old man pm5identified in two patients one patient showed florid fibrinous pericarditis containing fungal hyphae pm5 while the other showed only microscopic acute pericarditis appendix p figure the median heart weight was high g and four of nine patients had left ventricular hypertrophy nonbacterial thrombotic marantic endocarditis was noted in one patient pm5 with no known history or autopsy findings consistent with malignancy or chronic disorder associated with nonbacterial thrombotic marantic endocarditis appendix p figure pm5 had disseminated mucormycosis and numerous other thrombotic features appendix p cardiac amyloidosis and right atrial thrombosis were identified in one of ten patients pm8 appendix p lymphocyte depletion involving specific compartments and increased phagocytosis were prominent findings appendix p figure increased phagocytosis of other cells was identified in the sinusoidal macrophages of the red pulp of the spleen in four of seven patients sinus histiocytes of hilar lymph nodes in three of six and bone marrow four of eight phagocytosis was identified in at least one of these ans in six of nine patients bone marrow haemophagocytosis was prominent in two patients pm4 and pm8 and focal in two patients pm7 and pm9 depletion of periarteriolar tcell sheaths within the white pulp was observed figure red pulp was generally congested showing reduced numbers of cd8positive t cells plasma cells were variably prominent and sinusoidal histiocytes showed phagocytosis of red blood cells and other cells to varying extents both igmpositive and iggpositive plasma cells were identified and they were polytypic for lightchain expression figure lymph nodes showed preservation of follicles and relative depletion of paracortical areas medullary areas showed prominence of plasma cells and histiocytes were prominent in the sinuses bone marrow samples showed reactive changes with trilineage hyperplasia and prominence of plasma cells and histiocytes were a common finding a necrotising granuloma was noted in a single hilar lymph node in one patient and acidfast bacilli were noted on ziehl neelson staining appendix p all spleen and lymphoid material examined with immuno histochemistry were negative for epsteinbarr virus and cytomegaloviruspancreatitis was noted in two of eight patients pm5 was a 22yearold man with frank necrotichaemorrhagic pancreatitis and secondary mucor mycosis figure no fungal hyphae were noted in the pancreas pm8 was a 97yearold man who showed no substantial macroscopic pancreatitis although micro scopic acute inflammation within the pancreas and periadrenal fat necrosis was noted figure a third of patients three of nine showed patchy areas of infarcttype adrenocortical necrosis with one patient showing anising microthrombi in adrenal vessels figure no wwwthelancetcommicrobe published online august 101016s2666524720301154s 0cadrenalitis was noted two of nine patients showed chronic inflammation in the thyroid with follicular epithelial cell disruption however the significance of this finding is uncertainmedian combined kidney weight was within normal range at g iqr “ salient renal pathology findings were acute tubular injury in all nine patients underlying moderate cortical scarring of uncertain cause in one patient glomerular microaneurysm and thrombi in one patient figure and rare thrombi in interlobular arteries in four patients pm6 24yearold man of arterial intimal thickening than expected for that age appendix p we observed no evidence of focal and segmental glomerulosclerosis diabetic glomerulopathy or glomerulonephritisdegree had a higher large droplet fatty change was seen in most patients seven of eight cirrhosis or bridging hepatic fibrosis were noted in three patients no liver thrombosis was identified histologically but one patient showed macroscopic liver infarction figure the median liver weight was g iqr “ and three of nine patients showed hepatomegaly liver weighing g two patients pm4 and pm7 showed marked autolysis and were not included in analysismoderate to intense microglial activation was the most prominent pathological feature in the cns five of five patients mild tcell infiltration was noted around blood vessels and capillaries in all five patients but b cells were absent we found ischaemic changes of variable extent in the neurons of the cortex and in the white matter detected by bapp β amyloid precursor protein stain however no necrosis of brain tissue or extensive infiltration of inflammatory cells in brain parenchyma or meninges was observed on histological examination although one of nine patients showed macroscopic haemorrhagic transformation in a large recent cerebral infarction in the distribution of the middle cerebral arterytissues from five patients were analysed for presence of viral genomes against e gene and indications of viral replication against subgenomic rna transcripts by qrtpcr viral rna was present in respiratory tract samples including lung of all five cases analysed in addition two of three patients had detectable viral rna in the nasal epithelium and four of five patients in the trachea evidence of viral genomes outside the respiratory tract was found for all five patients but the distribution and viral loads varied case by case figure 5a viral genomes were also detected using a different qrtpcr targeted at rdrp gene and patterns were consistent between the two sets of primers data not shown a third primer set that detected subgenomic rna indicated virus replication in all tissues examined with variation between patients in levels and distribution figure 5bace µmbdf µm µmfigure other notable autopsy findings in patients with covid19a contained aortic dissection green arrow and fibrinous pericarditis red arrow in a 22yearold man pm5 insert is a haematoxylin and eosin stain image of the pericardium showing fibrinous pericarditis 10x magnification b adrenocortical microinfarcts in a 79yearold woman pm9 with reendothelialising thrombus in small adrenal vessels highlighted by cd34 insert bottom left and haematoxylin and eosin insert top right marantic endocarditis c highlight with haematoxylin and eosin staining bottom left at 10x magnification and necrotising haemorrhagic pancreatitis d in a 22yearold man pm5 with covid19 and a secondary fungal lung infection e periodic acid schiff staining showing a granular cast arrow indicative of acute tubular injury in a 24yearold man pm6 20x magnification f microscopic acute pancreatitis on haematoxylin and eosin staining in a 97yearold man pm8 20x magnificationdiscussionin this series we have described the major pathological findings identified at autopsy in ten patients who died of severe covid19 the most consistent findings were dad thrombosis haemophagocytosis and immune cell depletion although unexpected pathologies that are probably related to sarscov2 infection were also identifieddad was the most consistent and prominent feature in our series and others78 the specific phase of dad probably represents the degree and chronicity of the offending insult sarscov2 infection in relation to the time of death this is similar to previous coronavirus epidemics6 the conclusion by copin and colleagues7 that covid19related lung injury œis not diffuse alveolar damage might relate to their sampling strategy and wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cadbc µm µm µm µm µmef µm µm µmfigure pathological findings in haematological ans in patients with covid19tcell depletion in the spleen of a 79yearold woman pm9 with covid19 haematoxylin and eosin staining of the spleen at 10x magnification a cd20 staining of spleen indicating presence of b cells b 10x magnification with the insert showing the same region at higher power 20x magnification and cd3 staining of spleen indicating depletion of t cells c 10x magnification with the insert showing the same region at higher power 20x magnification bone marrow phagocytosis in a 97yearold man pm8 with covid19 haematoxylin and eosin staining of a well preserved bone marrow with an arrow indicating presence of phagocytosis d 40x magnification and cd68pgm1 staining of bone marrow indicating presence of phagocytosis 20x [e] and 40x [f] magnificationchronicity five patients had spent approximately weeks on a ventilator barton and colleagues8 described prominent acute bronchopneumonia as the major finding in one of two patients although the authors acknowledge that this was probably affected by aspiration in their patient with muscular dystrophy reports of lung histology in early covid19 also suggest a degree of lymphocytic pneumonia although dad is probably superimposed on this over time in the majority of fatal cases7 pulmonary macrophage infiltration and multinucleated giant cell reactions are prominent similar to other series8“ definite evidence of in covid19 will require quantitative analysis comparing tissues from covid19 patients with dad associated with other conditions and unaffected tissues several cases of invasive pulmonary aspergillosis have been reported in patients with severe covid19 pneumonia18 to our knowledge this is the first description of histologically proven mucormycosis in patients with covid19 and suggests that other human fungal pathogens including members of mucoromycotina can complicate covid19associated infectionslymphocyte depletion tissuerelated numerous clinical features including raised serum ddimer concentrations raised procalcitonin concentrations and imaging findings suggest thrombosis is prominent in patients with covid192 thrombotic features were universal among patients who underwent full autopsies all nine patients had thrombi in at least one major an and have been noted to be prominent in other covid19 autopsy series15 in a retrospective study of autopsies in patients with acute respiratory distress syndrome and dad of various causes only showed thrombi within the small vessels of the lung despite sampling of every lobe of the lung19 another study used postmortem angiography and identified thrombi in nearly all cases of acute respiratory distress syndrome from various causes20 whether thrombosis in covid19 is more common than in other causes of dad remains uncertain however our data support thrombosis as being a striking feature in these patients a study suggested endotheliitis as a prominent feature in patients with severe covid1910 but this was not a prominent feature in our patients importantly limited post mortem or postmortem crosssectional imaging are likely to underrepresent the true extent of thrombosis particularly microthrombosis and its impact on patient death the extent of cardiomegaly fibrointimal thickening of renal blood vessels and obesity in our series supports a contribution of hypertension beyond that noted clinically only four patients had hypertension documented on the mccda raised cytokine profile has been documented in a subset of patients with severe covid192 consistent with this haematological ans in our series showed prominent phagocytosis in several patients which has not been documented in previous series21 of the four patients with bone marrow haemophagocytosis one patient pm7 showed mild transaminitis hyperbilirubinaemia elevated serum ferritin concentrations and fever of ·°c however most clinical data were insufficient to assess the presence of haemophagocytic lympho histiocytosis wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ca substantial feature in covid19 is lymphocyte depletion and this is supported in our series by the spleen and lymph node findings when compared with those with mild disease patients with severe covid19 tend to have a higher neutrophil to lymphocyte ratio and higher cd4positive to cd8positive tcell ratio22 additionally a negative correlation exists between peripheral blood lymphocyte count and viral copy number22 we have corroborated this evidence by documenting a low number of t cells especially cd8positive t cells and foxp3positive regulatory t cells in the spleen and lymph nodes in severe fatal covid19 notably normal plasma cell both igm and igg positive response was present in haematological ans in most patientsthe extent to which anspecific pathologies relate to direct viral replication or consequent immunological and cardiovascular complications is of clinical relevance we report here evidence of viral genomic rna outside the respiratory tract this finding is in agreement with several previous studies that have identified viral genomes by qrtpcr in postmortem tissues including the colon14 spleen14 liver1423 skin24 heart23 and brain25 we also report detection of subgenomic rna a product that is only produced in actively infected cells a report identified low viral load in the brain of three of patients with covid19 but could not detect the virus in subsequent immunohistochemistry and concluded that the viral genomes might have been present in the blood25 although we cannot exclude that the rnas detected in our series were similarly carried to the site of sampling in blood the distribution of rna in different tissues varied widely between postmortem casespm3 and pm4 appear to have died earlier in the disease course days after symptom onset and had higher viral loads in the respiratory tract than other patients whereas pm3 and pm4 died after long stays in intensive care units and had either lower overall viral rna pm5 or higher viral rna outside the respiratory tract pm2 pm1 and pm2 were the only patients in whom we detected viral rna and subgenomic rna in the heart and are the only two patients with evidence of acute myocardial injury moreover unlike the previously mentioned study where virus detected in the brain was times less than that detected in the lung the number of viral genomes detected in external tissues in our series was frequently of similar or even higher levels than that found in the respiratory tree one study has detected sarscov2 infection of the endothelium in the vasculature of the skin and lung by immunofluorescent staining for viral antigens24 it is not obvious what determines spread and tropism of sarscov2 outside the respiratory tract several studies have reported ace2 expression levels that were higher in some ans than in others26 the sites of highest expression did not correlate with areas of most severe pathological involvement in our series we should note that cellular receptor status is one of many things that determines the degree of anr latot g 03 rep seipocenege larivpm1pm2pm3pm4pm5abtcbone marrowbrainheartileumkidneyliverspleentonguetrachealungnasal epitheliumfigure tissue tropism of sarscov2 in postmortem samplesfresh tissues were collected from a subset of postmortem examinations and viral load quantified by use of qrtpcr targeting the viral e gene a detection of viral rna was verified by use of qrtpcr against the viral polymerase gene data not shown tissues were additionally tested for subgenomic viral rna transcripts b dotted lines indicate the limit of detection as ascertained by negative control data are included for a 61yearold man pm1 a 64yearold man pm2 a 69yearold woman pm3 a 78yearold man pm4 and a 22yearold man pm5 qrtpcrquantitative rtpcr sarscov2severe acute respiratory syndrome coronavirus pathological involvement other aspects are likely to play a role such as route of transmission acid lability in the stomach coreceptors expression of proteases required to activate entry of virus into the cell eg tmprss2 possibly other yet unidentified host factors and invivo routes of dissemination the evidence of ongoing replication late in disease supports the use of antiviral therapy even at a point in illness when immunopathology is dominantpancreatic pathology was a major unexpected pathology in this series which had not been previously reported in covid19 autopsies or large clinical covid19 series2 a 22yearold man had haemorrhagic necrotic pancreatitis pm5 although this patient also had disseminated mucormycosis he had evidence of viral persistence in the lung and no fungal hyphae were identified in the pancreas on histological sections the other patient with wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cpancreatic pathology had microscopic evidence of pancreatitis that could be missed at autopsy without adequate sampling the pancreas is a classic site of unexpected pathology identified at autopsy27 serum amylase is not part of the routine care bundle for covid19 in our trust whether the pancreatitis was related to sarscov2 iatrogenic comorbidities or secondary infection is not clear in our study most cases had evidence of hepatic steatosis which is consistent with clinical findings that obesity is a risk factor for poor outcome in covid19 and liver cirrhosis or bridging fibrosis was prominent in this cohort28 from these data nothing suggests direct viral inflammation of the liverinfection or other causes the interval between time of death and autopsy impaired histological interpretation in many ans and affected antigen preservation postmortem endothelial stripping did affect the endothelial interpretation in our study however a subs

cancer7521

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: in the uk the death toll from severe covid19 is among the highest worldwide1 severe covid19 is characterised by respiratory failure with socalled cytokine storm occurring in some patient subsets2 pathological correlates are required to understand the pathophysiology of covid19 autopsybased histopathological analysis is crucial in this respect in anticipation of the covid19 pandemic our group produced national guidelines for autopsy performance in suspected covid19 cases3covid19 is caused by infection with severe acute respiratory syndrome coronavirus sarscov245 although sarscov2 and its predecessor sarscov causing severe acute respiratory syndrome [sars] are toll similar on a molecular and clinical level covid19 has a lower death rate for covid19 vs for sars and a substantially higher death deaths worldwide from covid19 as of aug vs from sars than sars due to a higher basic reproduction number1 the postmortem findings in patients with sarscov infection included diffuse alveolar damage dad splenic and nodal lymphocyte depletion haemophagocytosis renal acute tubular injury cerebral oedema microthrombosis and adrenalitis with necrosis with intracellular sarscov detected in the lungs kidney brain and haematological ans6 various autopsy series on covid19 have begun to emerge in the literature7“ here we document the major pathological lancet microbe published online august 101016 s2666524720301154department of cellular pathology northwest london pathology b hanley mbbch prof k n naresh md c roufosse phd j weir frcpath prof r goldin md p viola md m osborn frcpath and department of hepatology p manousou phd imperial college london nhs trust london uk centre for haematology b hanley prof k n naresh and centre for inflammatory diseases c roufosse department of immunology and inflammation department of infectious disease prof g s cooke frcp a abdolrasouli phd o c swann mres l baillon bsc r penn msc prof w s barclay phd and department of metabolism prof m thursz md faculty of medicine imperial college london london uk department of histopathology royal brompton and harefield nhs foundation trust and national heart and lung institute imperial college london london uk prof a g nicholson dm renal and transplant centre hammersmith hospital imperial college healthcare nhs london uk r corbett phd department of neuropathology kings college hospital london uk prof s alsarraj frcpath death investigation committee royal college of pathologists london uk m osborn and nightingale nhs hospital london uk m osborn correspondence to dr brian hanley department of cellular pathology northwest london pathology charing cross hospital campus london w6 8na uk bhanleyimperialacukwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cresearch in contextevidence before this studycovid19 is a new disease and comprehensive descriptions of the histopathological findings at autopsy are scarce we reviewed the literature available on covid19 autopsy findings up to and including may for this we searched pubmed and google scholar databases with no language restrictions using the search terms œcovid19 œsarscov2 œhistology œautopsy and œpostmortemadded value of this studyour series focused on providing a comprehensive description of the histopathological findings in patients with severe fatal covid19 and correlating these findings with data on viral tropism the most prominent findings included diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion several novel autopsy findings in patients with covid19 were also described including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activationimplications of all the available evidenceour study supports the existing clinical and autopsy literature that identified diffuse alveolar damage thrombosis immune cell depletion and macrophage activation as the most prominent pathological features in covid19 other factors including acute kidney injury pancreatitis pericarditis secondary fungal infections and preexisting liver disease require further investigation the presence of ongoing viral replication in late stage covid19 supports the continued use of antiviral therapy even at a point in illness when immunopathology is dominantsee online for appendixfindings of ten postmortem examinations done on patients with clinically confirmed covid19methodspatient selectionfor this study eligible patients were older than years with premortem sarscov2 infection and covid19 listed clinically as the direct cause of death under part on the medical certificate of cause of death [mccd] consent was obtained for all included patients according to the human tissue authority codes of practice by a member of the trust core postmortem consent team consent rate was · ten of patients exclusion criteria included extended postmortem interval before autopsy days and patients with covid19 contributing but not directly leading to death under part of the mccd patients were from imperial college national health service nhs trust nine patients london uk and royal brompton harefield foundation nhs trust one patient london uk premortem sarscov2 infection was identified using the coronavirus typing multiplextandem pcr highplex system aus diagnostics chesham uk ethical approval for this project was provided by the imperial college healthcare tissue bank r20012autopsy proceduresfull autopsies were done on nine patients pm1“ and one patient underwent percutaneous biopsy sampling heart lungs pancreas kidneys and liver using percutaneous biopsy under ultrasound guidance pm10 full postmortem examinations included standard sampling and were done according to royal college of pathologists guidelines3 eight different regions of the brain were sampled for each full neuropathological examination all tissue samples were fixed in formalin for a minimum of h before embedding histochemical stains and immunohistochemistry were applied according to local protocols appendix p ans were reviewed by subspecialist pathologists in lung agn and pv haem ato pathology and immune pathology knn liver rg gastrointestinal mo neuropathology sas and renal pathology cr integrated interpretation was done by a subspecialty autopsy pathologist bh and mo all cases were reviewed independently by at least two pathologistspcr proceduresfresh tissue for quantitative rtpcr qrtpcr analysis was processed within the biosafety level facilities at st mary™s hospital london uk approved by the uk health and safety executive and in accordance with local rules at imperial college london total rna was obtained from fresh tissue samples by use of trizolchloroform extraction followed by precipitation and purification using the rneasy kit qiagen hilden germany qrtpcr against e gene rdrp and subgenomic rna was done as described elsewhere1617 in patient pm5 total fungal genomic dna was extracted from four to five ribbon slices of a formalinfixed paraffinembedded lung tissue block purified dna was amplified with pcr for panfungal and mucoralesspecific targetsstatistical analysisall data was analysed using spss software version and expressed using median iqr and percentagerole of the funding sourcethe funder of the study had no role in study design data collection data analysis data interpretation or writing of the the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publicationresultsbetween march and april ten patients were included in the study the median age at death was years wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ciqr “ seven of ten patients were men three were women and most patients were white or asian nine hypertension four patients and chronic obstructive pulmonary disease three were the most common contributing factors to death according to mccd all ten patients developed fever and had at least two respiratory symptoms or signs cough shortness of breath reduced oxygen saturations or pleuritic chest pain during their early presentation of eight patients assessed for inflammatory markers all had elevated inflammatory markers these features were either apparent upon presentation to hospital eight of ten patients or developed in an inpatient two patients pm8 and pm9 most patients died within weeks of symptom onset seven patients and were not intubated or ventilated six patients four patients were intubated during their presentation pm2 for days pm5 for days pm6 for less than day and pm7 for days the median bodymass index bmi was in the obese range · iqr ·“· and more patients were obese according to bmi at post mortem five of nine than indicated on the mccd one of ten the median interval between death and postmortem examination was days iqr ·“· although the limited post mortem had a shorter interval less than h after death detailed clinical case vignettes are available in the appendix p and clinical data are summarised also in the appendix p all patients had dad six showed purely exudative phase dad and four showed a mixture of exudative and anising dad appendix p figure three of four patients with anisingphase dad had spent a substantial period on a ventilator days days and days florid acute bronchopneumonia and ventilatorassociated pneumonia were not noted in this series although mild interstitial neutrophilic inflammation three of ten patients and patchy acute bronchopneumonia three patients were observed interstitial macrophages were prominent macrophages were accompanied by scattered plasma cells mild or moderate lymphocyte inflammation was present in all ten patients although focal lymphocyte cuffing of small vessels was noted in six patients we noted that lymphocytes in the lung were predominantly cd4positive t cells cd56positive natural killer cells were rarely found occasionally a patient had small aggregates of small b cells chronic bronchiolitis was seen in most patients nine of ten no granulomas or viral inclusion were seen invasive mucormycosis was noted in one patient pm5 figure and confirmed with mucoralesspecific pcr the mucormycosis was vasculocentric and disseminated involving the hilar lymph nodes heart brain and kidney in the same patientmacroscopic two of nine patients and microscopic eight of nine pulmonary thromboemboli were frequent observations appendix p figure both fibrinrich and plateletrich thrombi were identified in smallsized and mediumsized vessels and within the capillaries in alveolar septa figure external examination findings of deep venous thrombosis were not noted very focal lymphocytic vasculitis was identified in one patientthrombotic features were universal in this cohort and all nine patients who underwent a full autopsy had at least one microthrombosis or macrothrombosis in a major an one of nine patients had a macroscopic acute coronary thrombosis in the right coronary artery whereas five patients had thrombi in the microcirculation of the heart on histological analysis coronary artery disease was negligible or mild in most patients seven of nine acute myocardial ischaemic damage h old was noted in the patient with an acute coronary artery thrombus figure 2a pm1 a mottled myocardium and subendocardial contraction band necrosis was noted in a acebdf 03m µm µmfigure pulmonary pathological findings in patients with covid19a macroscopic subpleural petechial haemorrhage in a 24yearold man pm6 b hyaline membranes indicative of exudative phase diffuse alveolar damage in a 79yearold woman pm9 at 20x magnification c cd61 immunohistochemical staining indicating plateletrich microthrombosis in alveolar capillaries pm6 d squamous metaplasia in a 61yearold man pm1 with exudative phase diffuse alveolar damage at × magnification e interstitial multinucleated giant cells in a 79yearold man pm7 with anising phase diffuse alveolar damage at × magnification the top right insert is of multinucleated giant cells showing positive cd68 staining indicative of macrophage lineage the bottom left insert shows absence of staining for cytokeratins f periodic acid schiff staining indicating wide irregular aseptate and ribbonlike hyphae with openangle branching and a vasculocentric pattern indicative of mucormycosis in a 22yearold man pm5 the insert is a grocott silver stain highlighting mucormycosis at 20x magnificationwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0csecond patient pm2 whether the contraction band necrosis was related to ischaemia or inotropic medication received in the intensive care unit is uncertain appendix p pm1 and pm2 were the two patients with the highest active viral load detected in the heart a single patient had a right atrial thrombus pericarditis was acegbdfh µm µm 03m µmfigure thrombotic features identified at autopsy in patients with covid19a macroscopic right coronary artery thrombosis arrow in a 61yearold man pm1 with exudative phase diffuse alveolar damage b macroscopic pulmonary thromboembolism arrow in a 97yearold man pm8 c thrombus in the lung of a 79yearold woman pm9 on haematoxylin and eosin staining at 20x magnification the insert shows cd61 immunohistochemistry indicating moderate staining for platelets d plateletrich thrombus in the mediumsized vessels surrounding the heart in a 61yearold man pm1 the insert shows strong cd61 staining for platelets periodic acid schiff staining showing a glomerular microaneurysm arrow e and microthrombi within glomerular capillary loops arrow f at 40x magnification indicative of thrombotic microangiopathy in a 97yearold man pm8 macroscopic splenic g and hepatic h infarction in a 22year old man pm5identified in two patients one patient showed florid fibrinous pericarditis containing fungal hyphae pm5 while the other showed only microscopic acute pericarditis appendix p figure the median heart weight was high g and four of nine patients had left ventricular hypertrophy nonbacterial thrombotic marantic endocarditis was noted in one patient pm5 with no known history or autopsy findings consistent with malignancy or chronic disorder associated with nonbacterial thrombotic marantic endocarditis appendix p figure pm5 had disseminated mucormycosis and numerous other thrombotic features appendix p cardiac amyloidosis and right atrial thrombosis were identified in one of ten patients pm8 appendix p lymphocyte depletion involving specific compartments and increased phagocytosis were prominent findings appendix p figure increased phagocytosis of other cells was identified in the sinusoidal macrophages of the red pulp of the spleen in four of seven patients sinus histiocytes of hilar lymph nodes in three of six and bone marrow four of eight phagocytosis was identified in at least one of these ans in six of nine patients bone marrow haemophagocytosis was prominent in two patients pm4 and pm8 and focal in two patients pm7 and pm9 depletion of periarteriolar tcell sheaths within the white pulp was observed figure red pulp was generally congested showing reduced numbers of cd8positive t cells plasma cells were variably prominent and sinusoidal histiocytes showed phagocytosis of red blood cells and other cells to varying extents both igmpositive and iggpositive plasma cells were identified and they were polytypic for lightchain expression figure lymph nodes showed preservation of follicles and relative depletion of paracortical areas medullary areas showed prominence of plasma cells and histiocytes were prominent in the sinuses bone marrow samples showed reactive changes with trilineage hyperplasia and prominence of plasma cells and histiocytes were a common finding a necrotising granuloma was noted in a single hilar lymph node in one patient and acidfast bacilli were noted on ziehl neelson staining appendix p all spleen and lymphoid material examined with immuno histochemistry were negative for epsteinbarr virus and cytomegaloviruspancreatitis was noted in two of eight patients pm5 was a 22yearold man with frank necrotichaemorrhagic pancreatitis and secondary mucor mycosis figure no fungal hyphae were noted in the pancreas pm8 was a 97yearold man who showed no substantial macroscopic pancreatitis although micro scopic acute inflammation within the pancreas and periadrenal fat necrosis was noted figure a third of patients three of nine showed patchy areas of infarcttype adrenocortical necrosis with one patient showing anising microthrombi in adrenal vessels figure no wwwthelancetcommicrobe published online august 101016s2666524720301154s 0cadrenalitis was noted two of nine patients showed chronic inflammation in the thyroid with follicular epithelial cell disruption however the significance of this finding is uncertainmedian combined kidney weight was within normal range at g iqr “ salient renal pathology findings were acute tubular injury in all nine patients underlying moderate cortical scarring of uncertain cause in one patient glomerular microaneurysm and thrombi in one patient figure and rare thrombi in interlobular arteries in four patients pm6 24yearold man of arterial intimal thickening than expected for that age appendix p we observed no evidence of focal and segmental glomerulosclerosis diabetic glomerulopathy or glomerulonephritisdegree had a higher large droplet fatty change was seen in most patients seven of eight cirrhosis or bridging hepatic fibrosis were noted in three patients no liver thrombosis was identified histologically but one patient showed macroscopic liver infarction figure the median liver weight was g iqr “ and three of nine patients showed hepatomegaly liver weighing g two patients pm4 and pm7 showed marked autolysis and were not included in analysismoderate to intense microglial activation was the most prominent pathological feature in the cns five of five patients mild tcell infiltration was noted around blood vessels and capillaries in all five patients but b cells were absent we found ischaemic changes of variable extent in the neurons of the cortex and in the white matter detected by bapp β amyloid precursor protein stain however no necrosis of brain tissue or extensive infiltration of inflammatory cells in brain parenchyma or meninges was observed on histological examination although one of nine patients showed macroscopic haemorrhagic transformation in a large recent cerebral infarction in the distribution of the middle cerebral arterytissues from five patients were analysed for presence of viral genomes against e gene and indications of viral replication against subgenomic rna transcripts by qrtpcr viral rna was present in respiratory tract samples including lung of all five cases analysed in addition two of three patients had detectable viral rna in the nasal epithelium and four of five patients in the trachea evidence of viral genomes outside the respiratory tract was found for all five patients but the distribution and viral loads varied case by case figure 5a viral genomes were also detected using a different qrtpcr targeted at rdrp gene and patterns were consistent between the two sets of primers data not shown a third primer set that detected subgenomic rna indicated virus replication in all tissues examined with variation between patients in levels and distribution figure 5bace µmbdf µm µmfigure other notable autopsy findings in patients with covid19a contained aortic dissection green arrow and fibrinous pericarditis red arrow in a 22yearold man pm5 insert is a haematoxylin and eosin stain image of the pericardium showing fibrinous pericarditis 10x magnification b adrenocortical microinfarcts in a 79yearold woman pm9 with reendothelialising thrombus in small adrenal vessels highlighted by cd34 insert bottom left and haematoxylin and eosin insert top right marantic endocarditis c highlight with haematoxylin and eosin staining bottom left at 10x magnification and necrotising haemorrhagic pancreatitis d in a 22yearold man pm5 with covid19 and a secondary fungal lung infection e periodic acid schiff staining showing a granular cast arrow indicative of acute tubular injury in a 24yearold man pm6 20x magnification f microscopic acute pancreatitis on haematoxylin and eosin staining in a 97yearold man pm8 20x magnificationdiscussionin this series we have described the major pathological findings identified at autopsy in ten patients who died of severe covid19 the most consistent findings were dad thrombosis haemophagocytosis and immune cell depletion although unexpected pathologies that are probably related to sarscov2 infection were also identifieddad was the most consistent and prominent feature in our series and others78 the specific phase of dad probably represents the degree and chronicity of the offending insult sarscov2 infection in relation to the time of death this is similar to previous coronavirus epidemics6 the conclusion by copin and colleagues7 that covid19related lung injury œis not diffuse alveolar damage might relate to their sampling strategy and wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cadbc µm µm µm µm µmef µm µm µmfigure pathological findings in haematological ans in patients with covid19tcell depletion in the spleen of a 79yearold woman pm9 with covid19 haematoxylin and eosin staining of the spleen at 10x magnification a cd20 staining of spleen indicating presence of b cells b 10x magnification with the insert showing the same region at higher power 20x magnification and cd3 staining of spleen indicating depletion of t cells c 10x magnification with the insert showing the same region at higher power 20x magnification bone marrow phagocytosis in a 97yearold man pm8 with covid19 haematoxylin and eosin staining of a well preserved bone marrow with an arrow indicating presence of phagocytosis d 40x magnification and cd68pgm1 staining of bone marrow indicating presence of phagocytosis 20x [e] and 40x [f] magnificationchronicity five patients had spent approximately weeks on a ventilator barton and colleagues8 described prominent acute bronchopneumonia as the major finding in one of two patients although the authors acknowledge that this was probably affected by aspiration in their patient with muscular dystrophy reports of lung histology in early covid19 also suggest a degree of lymphocytic pneumonia although dad is probably superimposed on this over time in the majority of fatal cases7 pulmonary macrophage infiltration and multinucleated giant cell reactions are prominent similar to other series8“ definite evidence of in covid19 will require quantitative analysis comparing tissues from covid19 patients with dad associated with other conditions and unaffected tissues several cases of invasive pulmonary aspergillosis have been reported in patients with severe covid19 pneumonia18 to our knowledge this is the first description of histologically proven mucormycosis in patients with covid19 and suggests that other human fungal pathogens including members of mucoromycotina can complicate covid19associated infectionslymphocyte depletion tissuerelated numerous clinical features including raised serum ddimer concentrations raised procalcitonin concentrations and imaging findings suggest thrombosis is prominent in patients with covid192 thrombotic features were universal among patients who underwent full autopsies all nine patients had thrombi in at least one major an and have been noted to be prominent in other covid19 autopsy series15 in a retrospective study of autopsies in patients with acute respiratory distress syndrome and dad of various causes only showed thrombi within the small vessels of the lung despite sampling of every lobe of the lung19 another study used postmortem angiography and identified thrombi in nearly all cases of acute respiratory distress syndrome from various causes20 whether thrombosis in covid19 is more common than in other causes of dad remains uncertain however our data support thrombosis as being a striking feature in these patients a study suggested endotheliitis as a prominent feature in patients with severe covid1910 but this was not a prominent feature in our patients importantly limited post mortem or postmortem crosssectional imaging are likely to underrepresent the true extent of thrombosis particularly microthrombosis and its impact on patient death the extent of cardiomegaly fibrointimal thickening of renal blood vessels and obesity in our series supports a contribution of hypertension beyond that noted clinically only four patients had hypertension documented on the mccda raised cytokine profile has been documented in a subset of patients with severe covid192 consistent with this haematological ans in our series showed prominent phagocytosis in several patients which has not been documented in previous series21 of the four patients with bone marrow haemophagocytosis one patient pm7 showed mild transaminitis hyperbilirubinaemia elevated serum ferritin concentrations and fever of ·°c however most clinical data were insufficient to assess the presence of haemophagocytic lympho histiocytosis wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ca substantial feature in covid19 is lymphocyte depletion and this is supported in our series by the spleen and lymph node findings when compared with those with mild disease patients with severe covid19 tend to have a higher neutrophil to lymphocyte ratio and higher cd4positive to cd8positive tcell ratio22 additionally a negative correlation exists between peripheral blood lymphocyte count and viral copy number22 we have corroborated this evidence by documenting a low number of t cells especially cd8positive t cells and foxp3positive regulatory t cells in the spleen and lymph nodes in severe fatal covid19 notably normal plasma cell both igm and igg positive response was present in haematological ans in most patientsthe extent to which anspecific pathologies relate to direct viral replication or consequent immunological and cardiovascular complications is of clinical relevance we report here evidence of viral genomic rna outside the respiratory tract this finding is in agreement with several previous studies that have identified viral genomes by qrtpcr in postmortem tissues including the colon14 spleen14 liver1423 skin24 heart23 and brain25 we also report detection of subgenomic rna a product that is only produced in actively infected cells a report identified low viral load in the brain of three of patients with covid19 but could not detect the virus in subsequent immunohistochemistry and concluded that the viral genomes might have been present in the blood25 although we cannot exclude that the rnas detected in our series were similarly carried to the site of sampling in blood the distribution of rna in different tissues varied widely between postmortem casespm3 and pm4 appear to have died earlier in the disease course days after symptom onset and had higher viral loads in the respiratory tract than other patients whereas pm3 and pm4 died after long stays in intensive care units and had either lower overall viral rna pm5 or higher viral rna outside the respiratory tract pm2 pm1 and pm2 were the only patients in whom we detected viral rna and subgenomic rna in the heart and are the only two patients with evidence of acute myocardial injury moreover unlike the previously mentioned study where virus detected in the brain was times less than that detected in the lung the number of viral genomes detected in external tissues in our series was frequently of similar or even higher levels than that found in the respiratory tree one study has detected sarscov2 infection of the endothelium in the vasculature of the skin and lung by immunofluorescent staining for viral antigens24 it is not obvious what determines spread and tropism of sarscov2 outside the respiratory tract several studies have reported ace2 expression levels that were higher in some ans than in others26 the sites of highest expression did not correlate with areas of most severe pathological involvement in our series we should note that cellular receptor status is one of many things that determines the degree of anr latot g 03 rep seipocenege larivpm1pm2pm3pm4pm5abtcbone marrowbrainheartileumkidneyliverspleentonguetrachealungnasal epitheliumfigure tissue tropism of sarscov2 in postmortem samplesfresh tissues were collected from a subset of postmortem examinations and viral load quantified by use of qrtpcr targeting the viral e gene a detection of viral rna was verified by use of qrtpcr against the viral polymerase gene data not shown tissues were additionally tested for subgenomic viral rna transcripts b dotted lines indicate the limit of detection as ascertained by negative control data are included for a 61yearold man pm1 a 64yearold man pm2 a 69yearold woman pm3 a 78yearold man pm4 and a 22yearold man pm5 qrtpcrquantitative rtpcr sarscov2severe acute respiratory syndrome coronavirus pathological involvement other aspects are likely to play a role such as route of transmission acid lability in the stomach coreceptors expression of proteases required to activate entry of virus into the cell eg tmprss2 possibly other yet unidentified host factors and invivo routes of dissemination the evidence of ongoing replication late in disease supports the use of antiviral therapy even at a point in illness when immunopathology is dominantpancreatic pathology was a major unexpected pathology in this series which had not been previously reported in covid19 autopsies or large clinical covid19 series2 a 22yearold man had haemorrhagic necrotic pancreatitis pm5 although this patient also had disseminated mucormycosis he had evidence of viral persistence in the lung and no fungal hyphae were identified in the pancreas on histological sections the other patient with wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cpancreatic pathology had microscopic evidence of pancreatitis that could be missed at autopsy without adequate sampling the pancreas is a classic site of unexpected pathology identified at autopsy27 serum amylase is not part of the routine care bundle for covid19 in our trust whether the pancreatitis was related to sarscov2 iatrogenic comorbidities or secondary infection is not clear in our study most cases had evidence of hepatic steatosis which is consistent with clinical findings that obesity is a risk factor for poor outcome in covid19 and liver cirrhosis or bridging fibrosis was prominent in this cohort28 from these data nothing suggests direct viral inflammation of the liverinfection or other causes the interval between time of death and autopsy impaired histological interpretation in many ans and affected antigen preservation postmortem endothelial stripping did affect the endothelial interpretation in our study however a subs Answer:

Colon_Cancer

" the severe acute respiratory syndrome coronavirus sarscov2 has caused the greatest worldwide pandemic since the flu the consequences of the coronavirus disease covid19 are devastating and represent the current major public health issue across the globe at the onset sarscov2 primarily attacks the respiratory system as it represents the main point of entry in the host but it also can affect multiple ans although most of the patients do not present symptoms or are mildly symptomatic some people infected with sarscov2 that experience more severe multian dysfunction the severity of covid19 is typically combined with a set of comorbidities such as hypertension diabetes obesity andor advanced age that seriously exacerbates the consequences of the infection also sarscov2 can cause gastrointestinal symptoms such as vomiting diarrhea or abdominal pain during the early phases of the disease intestinal dysfunction induces changes in intestinal microbes and an increase in inflammatory cytokines thus diagnosing gastrointestinal symptoms that precede respiratory problems during covid19 may be necessary for improved early detection and treatment uncovering the composition of the microbiota and its metabolic products in the context of covid19 can help determine novel biomarkers of the disease and help identify new therapeutic targets elucidating changes to the microbiome as reliable biomarkers in the context of covid19 represent an overlooked piece of the disease puzzle and requires further investigation 0c introduction severe acute respiratory syndrome coronavirus sarscov2 also called 2019ncov arose in the province of wuhan china in december sarscov2 causes severe respiratory disease coronavirus disease covid19 on march the world health anization determined that the outbreak of a novel coronavirus had reached spreading pandemic as of august there are over million confirmed cases worldwide with more than associated global deaths according to johns hopkins university over the last months the global population has been facing problems that impact both world health and global socioeconomics covid19 has been and continues to be the focus of concern in a society threatened by the most destructive pandemic since the flu transmission symptoms vaccines and treatments for covid19 continue to be investigated hiding too many unknowns the immediate strategy to curb the spread of this virus has been to recommend that people carry out a social vaccination which involves restricting social gatherings minimizing public appearances telecommuting implementing social distancing or wear masks as much as possible this is based on the high transmission ability of the sarscov2 via large respiratory droplets and by airborne routes unlike other viruses in the coronavirus family sarscov2 infects people who then have little or no symptoms despite all this asymptomatic covid19 positive individuals can spread the virus this silent transmission capacity is the main reason this virus continues to transmit and infect the global population in an accelerated and uncontrollable way in spite of best efforts to control and curb spread asymptomatic people manage to eliminate the virus without developing typical covid19 symptoms and this suggests to us that the immune system is relevant and may have the key to beat this virus covid19 affects not only the respiratory and cardiovascular systems but also to the central nervous system cns and gastrointestinal system the objective of this review is to examine whether the sarscov2 infection linked to gastrointestinal changes may be linked to 0c specific phases of covid19 associated to inflammation we will explore the studies on microbiome profiling intestinal immune system in the patients with covid19 this review will provide insights for microbiota modification therapy in the early stages of covid19 beyond antiviral and antiinflammatory approaches new therapeutic interventions might be possible through the restoration of the gut microbiome of individuals infected by sarscov2 to mitigate systemic inflammation intestinal damage and limit the effects caused in the cns through the braingut axis early and late stage of the infection of sarscov2 a multitude of epidemiological studies describe different phases in the development of covid19 the early or viral phase shortly after infection marked by a high viral load and a reduced inflammatory activity with hardly any symptoms and which is also associated with gastrointestinal illness figure besides patients with covid19 who had the highest viral load levels at the beginning of the infection and subsequently decreased over time could explain the rapid spread of the disease [ ] during the progressive or late phase of infection covid19 positive individuals develop most severe symptoms like respiratory problems and fever immune cells like neutrophils infiltrating monocytes and macrophages produce inflammatory cytokines and reactive oxygen species also respiratory manifestations such as cough sputum production and shortness of breath remain the most common symptoms following fever or pneumonia [ ] in a study with covid19 patients serum level of cytokines as interleukin il6 and il10 were indicators of disease severity showing that high levels of proinflammatory cytokine storm were associated with more severe disease development some patients with covid19 had severe complications including acute respiratory distress syndrome ards cardiovascular conditions multiple an dysfunction syndromes or septic shock these complications are related to the release of cytokines and hyperinflammation or cytokine storm syndrome figure a person is more infectious in 0c the initial phase when the symptomatology of the disease does not manifest itself with respiratory symptoms and however the presymptomatic transmission may play an important role nevertheless detecting sarscov2 infection in those without fever andor respiratory symptoms is difficult because sarscov2 tests are not usually performed in infected people without these symptoms in summary a subset of the symptoms associated with covid19 during the initial phase are intestinal complications such as vomiting or diarrhea detecting these symptoms might not only lead to slowdown in transmission but also open the door to novel treatments that could reduce the severity of covid19 more studies are necessary to interpret the phases of the disease correctly and especially if the viral load detected is considered infectious and how it is related to respiratory or gastrointestinal symptoms clinical characteristics of covid19 patients with digestive symptoms and intestinal inflammation covid19 is an emerging infection that causes great concern about respiratory manifestations furthermore diarrhea and other gastrointestinal symptoms are frequently observed in patients with covid19 however the significance remains undetermined a viral infection causes an alteration of intestinal permeability resulting in enterocyte dysfunction when we investigate what happened with another coronavirus we found that diarrhea was a frequent symptom in sars patients occurring in intestinal problems were also associated with the severity of the infection patients with diarrhea had an increased need for respiratory assistance and intensive care sarscov was also identified in ileal and terminal colonic biopsies regarding mers several studies showed the presence of diarrhea between to of known cases however a less severe prognosis of the disease was observed in mers patients with diarrhea 0c the first results linking intestinal problems with covid19 were obtained from patients in wuhan china two hundred four patients with covid19 presented at three hospitals indicate that the majority of covid19 patients had typical respiratory symptoms however many patients infected with the coronavirus complained of digestive symptoms such as diarrhea there is no evidence on the efficacy of antidiarrheal drugs but adequate rehydration and potassium monitoring should be performed as in all covid19 patients diarrhea should generate awareness of a possible sarscov2 infection and should be investigated to reach an early diagnosis of covid19 this factor should be considered when suspecting whether the patients are infected instead of waiting for respiratory symptoms to appear which would give us an earlier diagnosis unless the coronavirus tests are also performed on asymptomatic individuals patients with covid19 specifically those with digestive symptoms remained a long time from onset to hospital admission and a worse clinical outcome compared to patients who do not suffer from these symptoms likewise patients with digestive symptoms had an average time of days from the onset of symptoms until admission while patients with respiratory symptoms had an admission time of days this may indicate that those with digestive symptoms waited longer to be diagnosed in the hospital as they did not suspect they were sarscov2 positive in the absence of respiratory symptoms authors also state that patients with digestive symptoms presented other associated clinical manifestations such as anorexia diarrhea vomiting or abdominal pain as the severity of the disease increased gi symptoms became more pronounced but especially the high percentage of patients admitted with symptoms of anorexia one study indicated that covid19 patients experience gastrointestinal gi symptoms such as diarrhea anorexia and nausea however the underlying pathophysiology gastrointestinal is not well understood another study analyzed hospitalized patients and some of them showed mild initial gi symptoms such as diarrhea nausea vomiting and abdominal pain which preceded the characteristic fever and respiratory problems such as dry cough in another study diarrhea 0c was reported in patients and the fecal test remained positive until days after the disease onset in patients the stool test was still positive despite negative respiratory tests another study with patients with low severity of covid19 including presenting with digestive symptoms alone shows that patients with gastrointestinal symptoms had a longer duration between symptom onset and viral clearance and fecal viruspositive than those with respiratory symptoms a systematic metaanalysis of shows results from clinical studies with an incidence rate of diarrhea from as low as and up to of the cases how is this disparity in the data possible more clinical studies are required to elucidate the percentage of covid19 patients that develop intestinal symptoms and if these depend on other factors such as age gender or other comorbidities gastrointestinal symptoms are accompanied by inflammation or intestinal damage there is a loss of intestinal barrier integrity and gut microbes that can activate innate and adaptive immune cells to release proinflammatory cytokines into the circulatory system leading to systemic inflammation some intestinal signaling pathways can regulate inflammation through dendritic cells figure immunomodulation of the innate host immunity through the activation of epithelial receptors could represent a novel therapeutic target to eliminate sarscov2 in the early stage of the infection the viral load of coronavirus appeared in the feces of of the infected patients an interaction between viral shedding of sarscov2 in stool has been reported but its association with infection was yet to be determined identifying fecal coronavirus rna may also lead researchers to ask new questions is there transmission of the coronavirus found in fecal samples what these researchers found is the presence of the coronavirus in the stool but there is controversial studies about if viral load was infectious nor examples of transmission previously a study involving patients with covid19 reported that the median duration of viral shedding was days in survivors range – days cultivable sarscov2 was detected in stool or urine specimens for longer than weeks in three convalescent patients days suggesting that it may remain viable 0c contradictorily high sarscov2 titres detectable in the first week of illness with an early peak observed at symptom onset to day of disease [ ] although sarscov2 rna shedding in respiratory and stool can be prolonged the duration of a viable virus is relatively shortlived the presence of sarscov2 including live virus with infectious capacity in the feces of asymptomatic individuals implies that covid19 could be transmitted through the fecal route sarscov2 shedding in stool samples is detectable over a longer period than in nasopharyngeal swabs donors for fecal microbiota transplant for sarscov2 must be strict and validated to prevent the potential risk of transmission the results show that the elevated levels of fecal calprotectin in patients with covid19 add to the growing evidence that sarscov2 infection causes an inflammatory response in the intestine calprotectin concentrations were significantly higher in covid19 patients who had suffered from diarrhea and with more elevated serum il6 levels in the diagnosis and especially in the followup of covid19related diarrhea the calprotectin measurement could play a potential role in monitoring the disease also diarrhea may be secondary to virusinduced inflammation which in turn is due to the entry of inflammatory cells into the intestinal mucosa including neutrophils and lymphocytes and thus disruption of the gut microbiota viral sarscov2 ps were detected in feces during the second phase of covid19 accompanied by a decrease in the peak of inflammation therefore covid19related inflammatory diarrhea was associated with reduced levels of fecal sarscov2 rna however intestinal damage can manifest after respiratory symptoms as reported in a clinical case there may be a pathogenic role of sarscov2 on the gi in zheijiang china it was detected that half of the covid19 patients tested positive for rtpcr in their feces and intestinal microbial dysbiosis was also identified viral strains alive from feces were isolated indicating potential infectiousness of feces [ ] another analysis conducted from studies in china usa and singapore had also detected 0c sarscov2 rna in fecal specimens of patients at an average of this percentage is according to their clinical forecast intestinal problems or with more severe disease furthermore the fecal test for sarscov2 rna also bears a potential implication for physicians to decide the subsequent isolation strategies for those with positive fecal samples the possibility of the fecaloral route of transmission of sarscov2 should be investigated all candidates for fecal microbiota transplantation and healthy donors should be screened for the virus one could consider studies on the gut microbiome and its therapeutic role in transplanting feces in healthy donors to critically ill covid19 patients still precautions should be taken because infectious sarscov2 ps are known to have been found in covid19 positive feces the use of fecal transplants could be one of the immediate solutions in critically ill patients with a weakened immune system sarscov2 interaction with intestinal ace2 receptors sarscov2 enters cells primarily through binding of protein s to angiotensinconverting enzyme ii ace2 receptors to infected cells the central role of ace2 in the cleavage of angiotensin i to angiotensin ii a peptide involved in vascular homeostasis vasomotor tone and blood pressure regulation ace2 receptors are expressed in various human cells susceptible to viral infection including epithelial cells in the lungs small intestine and colon tubular cells of the kidney neuronal and glial cells in the brain enterocytes vascular endothelial cells smooth muscle cells and cardiomyocytes a very recent study has reported on the expression pattern of ace2 across more than different cell types corresponding to all major human tissues and ans based on rigorous immunohistochemical analysis sarscov2 also uses the receptors for transmembrane protease serine tmrpss2 an enzyme that is expressed in the small intestinal epithelial cells to entry to the infected cells figure the sarscov2 activity could cause ace2 modifications in the gut that increase susceptibility to 0c intestinal inflammation and diarrhea a high coexpression of ace2 and tmprss2 was detected in enterocytes and the esophagus and lungs the coexpression of ace2 and tmprss2 transcripts was highest in the small intestine with expressed in enterocytes and in colon cells as demonstrated by a singlecell rna sequencing study in gi ace2 is not only playing an essential role in intestinal inflammation but also have a significant impact on the composition of the intestinal microbiota ace2 ko mice have been shown to have decreased expression of antimicrobial peptides and exhibited altered intestinal microbial composition which is restored by the administration of tryptophan likewise it is known that there is a connection of transport of ace2 amino acid with microbial ecology in the gut during sarscov2 infection in another recent study using gnotobiotic germfree rats was observed a decrease in ace2 expression in the colon that contributed to the pathology during covid19 there are numerous studies that have shown that by regulating the reninangiotensin system ras which includes ace2 one can modulate systemic inflammation angiotensin ii receptor blockers are widely used compounds that are therapeutically effective in cardiovascular disorders renal disease metabolic syndrome and diabetes under normal healthy conditions homeostasis of rasace2 occurs while a perturbation to this system is observed in cardiovascular disease hypertension and diabetes mellitus a reduction of ace2 is associated with hypertension diabetes and cardiovascular problems which represent the significant comorbidities of covid19 sarscov2 may potentially further upregulate ras in cardiovascular patients and deplete ace2 downregulation of ace2 levels in tissues has been linked to viral replication efficiency and pathogenicity leading to the imbalance of positive and negative regulation of ras rasace2 imbalance in covid19 may greatly exacerbate tissue inflammation and may contribute to more adverse covid19 outcomes in patients with preexisting cardiovascular disease and other comorbidities ace2 receptors may represent a target for covid19 treatments in patients with cardiovascular risk burden susceptible to conditions that worsen asymmetries in rasace2 however suppression of 0c ace2protective roles due to ace2 depletion upon sarscov2 infection is very likely to uphold the poor outcomes observed in covid19 patients especially in those with preexisting conditions ace2 also displays nonrasrelated roles linked with neutral amino acids transport and gut homeostasis ace2 has been implicated in the intestinal epithelium regulation of gut microbiota composition and function in summary ace2 imbalance is likely a key player for the poor outcomes in the covid19 patients with preexisting comorbidities and addresses a possible link for gut microbiota dysbiosis in this interplay the effects of covid19 on the gut microbiome primary inflammatory stimuli trigger the release of microbial products and cytokines which can cause microbial dysbiosis that can induce an inflammatory environment releasing intestinal cytokines into the circulatory system increasing the systemic inflammation of covid19 taken together a combined inflammation can potentially initiate an immune reaction that can create even more harm than the virus itself it is necessary to know the host cytokine pathways and microbiota interactions with cytokine responses in sarscov2 infection to develop novel treatment approaches it is essential to investigate how intestinal bacteria interact in response to sarscov2 infection among covid19 patients an increase of a blood proteomic risk score prs was associated with a risk of becoming a clinically severe infection a recent study has shown more than proteins in bloodrelated to the severity of covid19 these proteins included immune factors that are elevated during systemic inflammation such as creactive protein crp correlations between prs and the intestinal microbiome might be associated with the severity of covid19 this new analysis would allow us to predict if the patient would develop more severe symptoms in the next days or weeks after the infection ruminococcus gnavus was identified in covid19 patients and positively correlated with inflammatory markers while 0c clostridia was negatively correlated another small study of patients hospitalized in hong kong has served to establish a gut microbiome profile in association with covid19 severity and changes in fecal shedding of sarscov2 through the application of indepth shotgun metagenomics analysis the authors investigated longitudinal changes of the gut microbiome in covid19 the abundance of coprobacillus clostridium ramosum and clostridium hathewayi correlated with covid19 severity and it was observed an inverse correlation between the abundance of faecalibacterium prausnitzii an antiinflammatory bacterium during the hospitalization time were detected in covid19 patients bacteroides dorei bacteroides thetaiotaomicron bacteroides massiliensis and bacteroides ovatus which downregulate the expression of ace2 in the murine gut correlating inversely with sarscov2 load in fecal samples this study opens the doors to possible interventions for gut microbiota in hospitalized patients to reduce the severity of covid19 another study of just patients showed that the gut bacteria was associated with fecal sarscov2 load the gut microbiome remained substantially different in hospitalized patients from that of healthy controls a total of bacterial species were identified to be significantly associated with the fecal viral load of sarscov2 across all fecal samples this does not indicate that the gut microbiome is affected for long periods after recovery and future therapeutic interventions would be necessary sarscov2 was detected in feces of hospitalized patients with covid19 and the viral transcriptional activity was analyzed to determine the infectivity range associated with the gut microbiome fecal samples with a signature of high sarscov2 infectivity had higher abundances of bacterial species collinsella aerofaciens collinsella tanakaei streptococcus infantis and manella manii in contrast fecal samples with a signature of lowto no sarscov2 infectivity had higher abundances of scfas producing bacteria parabacteroides merdae bacteroides stercoris alistipes onderdonkii and lachnospiraceae bacterium however live sarscov2 was not isolated from the feces covid19 patients another study 0c of covid19 patients showed a significantly reduced bacterial diversity and a higher relative abundance they compared these results with h1n1 patients who showed lower bacteria diversity compared with covid19 patients specific microbial signatures were identified in covid19 patients which could help identify biomarkers that differentiate them from influenza a h1n1 patients when there is a coinfection despite this the phases of the disease were not established in this study furthermore in another study with covid19 patients was observed a reduced alpha diversity in microbes the metatranscriptional analysis revealed that there were differentially genes associated with immune pathways and cytokine signaling related to the diagnosis and severity of covid19 such as interferongamma signaling despite reporting interesting patterns the studies mentioned above are not without limitations the bioinformatics analyses employed in these studies does not guarantee specieslevel nor strainlevel bacterial identification furthermore no longitudinal analysis has been performed with the same patients to determine if these bacteria change after recovery from covid19 more studies that prospectively include infected but asymptomatic subjects positive for sarscov2 but with different degrees of symptoms would be necessary to establish a correlation with microbial markers or their products monitoring early in the disease during early onset of covid19 and over the longterm will help to delineate the role of changes in the microbiome will be critically important to elucidating this connection further another major limitation is the small number of patients and the absence of information about microbial changes in the context of covid19 that define broad groups of the population stratified by geography ethnicity gender andor age to date there are no metabolomics and metaproteomic analysis studies that are needed to explore the products of these bacteria and their function by identifying microbial metabolites associated with covid19 we can understand what components these bacteria produce during covid19 that help us understand the influence of the communication pathways of the microbiotagutperiphery axis relevant to the association between sarscov2 and 0c hyperinflammation moreover more studies investigating the correlation between sarscov2 and gut inflammation are necessary including histopathology and molecular diagnostic assessments comorbidities as risk factors for covid19 associated with loss of microbial diversity sarscov2 can infect people of all ages but older adults and people with preexisting medical conditions appear to be more vulnerable to becoming seriously ill with the virus there are many hypotheses as to why this occurs still one of the factors could be the loss of microbial diversity associated with aging and with it higher susceptibility to inflammation comorbidities play an essential role in determining the risk of severe complications and death after covid19 infection covid19 comorbidities and risk factors include asthma hypertension smoking male gender or alzheimer's disease or dementia changes in gut microbiota have been previously linked to all of these comorbidities and this deregulation could also be associated with changes in the immune system and the susceptibility to suffer more severe consequences of covid19 and genderrelated differences in vulnerability to complications of covid19 our microbiome changes as we age which favors less diversity and a more significant inflammatory state covid19 appears to be more dangerous in older people men and with comorbidities the gut microbiota evolves during human life the infant microbiota shows reduced diversity and will remain unstable for the first few years of experience until it becomes an adultlike microbiota the gut microbiota within an individual is considered stable throughout adult life in the elderly gut microbiota diversity decreases and dysbiosis increases and is associated with cognitive deficits depression and inflammatory markers a change that is found repeatedly in the microbiota of the elderly is the decrease in the ratio of firmicutes to bacteroidetes [ ] decreased bacterial diversity as well as lower levels in specific bacterial groups have also been observed in very elderly [ ] at the genus and species 0c level the findings vary significantly between studies although bacteroides clostridium and lactobacillus appear recurrently altered in elderly individuals tragically a high rate of covid19 fatalities is associated with groups of people over years old the causes may be the inability to overcome the infection the weakness of the immune system and the reduced microbiome diversity causing the coronavirus to strongly attack this group of the population causing a higher mortality rate another recent study has found significant associations between dietary patterns and measures of gut microbial composition in older men the group of the population with the highest mortality rate from covid19 obesity is also associated with changes in the intestinal flora [ ] and is another risk factor in the severity of covid19 therefore another comorbidity in adults and children in the united states at least of patients who die from covid19 have obesity which is similar to the reported rates of cardiovascular disease in the same highrisk group it is necessary to study the relationship between obesity and the severity of the covid19 disease adipose tissue can serve as a reservoir for the spread of sarscov2 virus clearance and systemic immune activation adipocytes in obese patients express higher levels of ace2 and a reduction or elimination of already inflamed adipose tissue can reduce systemic viral spread viral entry and prolongation in obese individuals there is marked dysregulation of myeloid and lymphoid responses within adipose tissue associated with dysregulation of cytokine profiles obese patients also have heightened levels of proinflammatory adipokines leukotrienes chemerin among others which may exacerbate their risk for cytokine storm syndrome and death the alteration of the immune system causes changes in the intestinal flora and it remains to be seen whether the coronavirus also induces changes in the bacteria that modulate many of these functions of the intestinal brainmicrobiota axis the bacterial intestinal flora plays a critical role in the regulation of neurological functions such as depression or anxiety [ ] surely the results of these studies allow us to know 0c what the role of intestinal flora is in covid19 and its relationship with neurological problems at long term furthermore diabetes is another disease associated with increased severity of symptoms and complications of covid19 and this can be attributed to systemic inflammation and gutmetabolite dysfunction individuals suffering from cardiovascular disease who become infected with sarscov2 are at increased risk of developing a worse prognosis of covid19 and also develop cardiovascular complications including myocardial infarction arrhythmias stroke or heart feature or myocardial suppression cardiovascular disease is accompanied by an imbalance of gut microbiota and a decreased microbiome diversity hypertension is likely to be influenced by diet lifestyle factors and microbiome notably an increase in shortchain fatty acids scfa was previously associated with decreased blood pressure and improved arterial compliance changes in the gut microbiota composition through diet to deal with covid19 the impact of dietary patterns on susceptibility to and severity of infection with the sarscov2 virus has been largely ignored to date the commensal microbiome forms a dynamic environment that can be altered and cause dysbiosis from virus infection but can be positively modulated by diet components and probiotic treatments several studies show than an optimal immune response depends on proper diet and nutrition to control sarscov2 infection [ ] in general malnutrition can compromise the immune response therefore affecting the vulnerability of the response to covid19 consideration of the dietary and nutritional components the factors during viral infection can serve to strengthen the immune system for the prevention of infections and a meaningful and balanced basis for an immune response is an adequate and balanced diet intake of a sufficient amount of protein is crucial for the production of antibodies also a low level of vitamin a or zinc has been associated with an increased risk of infection branchedchain amino acids can maintain the hairy morphology of 0c the intestines and increase intestinal immunoglobulin levels thereby improving the intestinal barrier therefore highquality proteins are an essential component of an antiinflammatory diet nutritional dietary components known to exert antiinflammatory and antioxidant properties include omega3 fatty acids with high antiinflammatory and antioxidant capacity including vitamin c vitamin e and phytochemicals such as carotenoids and polyphenols that are widely present in plantbased foods undoubtedly omega3 fatty acids appear to have the most potent antiinflammatory capability several of these components can interact with cellular signaling components related to antiinflammatory and antioxidant effects an

cancer7522

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " the severe acute respiratory syndrome coronavirus sarscov2 has caused the greatest worldwide pandemic since the flu the consequences of the coronavirus disease covid19 are devastating and represent the current major public health issue across the globe at the onset sarscov2 primarily attacks the respiratory system as it represents the main point of entry in the host but it also can affect multiple ans although most of the patients do not present symptoms or are mildly symptomatic some people infected with sarscov2 that experience more severe multian dysfunction the severity of covid19 is typically combined with a set of comorbidities such as hypertension diabetes obesity andor advanced age that seriously exacerbates the consequences of the infection also sarscov2 can cause gastrointestinal symptoms such as vomiting diarrhea or abdominal pain during the early phases of the disease intestinal dysfunction induces changes in intestinal microbes and an increase in inflammatory cytokines thus diagnosing gastrointestinal symptoms that precede respiratory problems during covid19 may be necessary for improved early detection and treatment uncovering the composition of the microbiota and its metabolic products in the context of covid19 can help determine novel biomarkers of the disease and help identify new therapeutic targets elucidating changes to the microbiome as reliable biomarkers in the context of covid19 represent an overlooked piece of the disease puzzle and requires further investigation 0c introduction severe acute respiratory syndrome coronavirus sarscov2 also called 2019ncov arose in the province of wuhan china in december sarscov2 causes severe respiratory disease coronavirus disease covid19 on march the world health anization determined that the outbreak of a novel coronavirus had reached spreading pandemic as of august there are over million confirmed cases worldwide with more than associated global deaths according to johns hopkins university over the last months the global population has been facing problems that impact both world health and global socioeconomics covid19 has been and continues to be the focus of concern in a society threatened by the most destructive pandemic since the flu transmission symptoms vaccines and treatments for covid19 continue to be investigated hiding too many unknowns the immediate strategy to curb the spread of this virus has been to recommend that people carry out a social vaccination which involves restricting social gatherings minimizing public appearances telecommuting implementing social distancing or wear masks as much as possible this is based on the high transmission ability of the sarscov2 via large respiratory droplets and by airborne routes unlike other viruses in the coronavirus family sarscov2 infects people who then have little or no symptoms despite all this asymptomatic covid19 positive individuals can spread the virus this silent transmission capacity is the main reason this virus continues to transmit and infect the global population in an accelerated and uncontrollable way in spite of best efforts to control and curb spread asymptomatic people manage to eliminate the virus without developing typical covid19 symptoms and this suggests to us that the immune system is relevant and may have the key to beat this virus covid19 affects not only the respiratory and cardiovascular systems but also to the central nervous system cns and gastrointestinal system the objective of this review is to examine whether the sarscov2 infection linked to gastrointestinal changes may be linked to 0c specific phases of covid19 associated to inflammation we will explore the studies on microbiome profiling intestinal immune system in the patients with covid19 this review will provide insights for microbiota modification therapy in the early stages of covid19 beyond antiviral and antiinflammatory approaches new therapeutic interventions might be possible through the restoration of the gut microbiome of individuals infected by sarscov2 to mitigate systemic inflammation intestinal damage and limit the effects caused in the cns through the braingut axis early and late stage of the infection of sarscov2 a multitude of epidemiological studies describe different phases in the development of covid19 the early or viral phase shortly after infection marked by a high viral load and a reduced inflammatory activity with hardly any symptoms and which is also associated with gastrointestinal illness figure besides patients with covid19 who had the highest viral load levels at the beginning of the infection and subsequently decreased over time could explain the rapid spread of the disease [ ] during the progressive or late phase of infection covid19 positive individuals develop most severe symptoms like respiratory problems and fever immune cells like neutrophils infiltrating monocytes and macrophages produce inflammatory cytokines and reactive oxygen species also respiratory manifestations such as cough sputum production and shortness of breath remain the most common symptoms following fever or pneumonia [ ] in a study with covid19 patients serum level of cytokines as interleukin il6 and il10 were indicators of disease severity showing that high levels of proinflammatory cytokine storm were associated with more severe disease development some patients with covid19 had severe complications including acute respiratory distress syndrome ards cardiovascular conditions multiple an dysfunction syndromes or septic shock these complications are related to the release of cytokines and hyperinflammation or cytokine storm syndrome figure a person is more infectious in 0c the initial phase when the symptomatology of the disease does not manifest itself with respiratory symptoms and however the presymptomatic transmission may play an important role nevertheless detecting sarscov2 infection in those without fever andor respiratory symptoms is difficult because sarscov2 tests are not usually performed in infected people without these symptoms in summary a subset of the symptoms associated with covid19 during the initial phase are intestinal complications such as vomiting or diarrhea detecting these symptoms might not only lead to slowdown in transmission but also open the door to novel treatments that could reduce the severity of covid19 more studies are necessary to interpret the phases of the disease correctly and especially if the viral load detected is considered infectious and how it is related to respiratory or gastrointestinal symptoms clinical characteristics of covid19 patients with digestive symptoms and intestinal inflammation covid19 is an emerging infection that causes great concern about respiratory manifestations furthermore diarrhea and other gastrointestinal symptoms are frequently observed in patients with covid19 however the significance remains undetermined a viral infection causes an alteration of intestinal permeability resulting in enterocyte dysfunction when we investigate what happened with another coronavirus we found that diarrhea was a frequent symptom in sars patients occurring in intestinal problems were also associated with the severity of the infection patients with diarrhea had an increased need for respiratory assistance and intensive care sarscov was also identified in ileal and terminal colonic biopsies regarding mers several studies showed the presence of diarrhea between to of known cases however a less severe prognosis of the disease was observed in mers patients with diarrhea 0c the first results linking intestinal problems with covid19 were obtained from patients in wuhan china two hundred four patients with covid19 presented at three hospitals indicate that the majority of covid19 patients had typical respiratory symptoms however many patients infected with the coronavirus complained of digestive symptoms such as diarrhea there is no evidence on the efficacy of antidiarrheal drugs but adequate rehydration and potassium monitoring should be performed as in all covid19 patients diarrhea should generate awareness of a possible sarscov2 infection and should be investigated to reach an early diagnosis of covid19 this factor should be considered when suspecting whether the patients are infected instead of waiting for respiratory symptoms to appear which would give us an earlier diagnosis unless the coronavirus tests are also performed on asymptomatic individuals patients with covid19 specifically those with digestive symptoms remained a long time from onset to hospital admission and a worse clinical outcome compared to patients who do not suffer from these symptoms likewise patients with digestive symptoms had an average time of days from the onset of symptoms until admission while patients with respiratory symptoms had an admission time of days this may indicate that those with digestive symptoms waited longer to be diagnosed in the hospital as they did not suspect they were sarscov2 positive in the absence of respiratory symptoms authors also state that patients with digestive symptoms presented other associated clinical manifestations such as anorexia diarrhea vomiting or abdominal pain as the severity of the disease increased gi symptoms became more pronounced but especially the high percentage of patients admitted with symptoms of anorexia one study indicated that covid19 patients experience gastrointestinal gi symptoms such as diarrhea anorexia and nausea however the underlying pathophysiology gastrointestinal is not well understood another study analyzed hospitalized patients and some of them showed mild initial gi symptoms such as diarrhea nausea vomiting and abdominal pain which preceded the characteristic fever and respiratory problems such as dry cough in another study diarrhea 0c was reported in patients and the fecal test remained positive until days after the disease onset in patients the stool test was still positive despite negative respiratory tests another study with patients with low severity of covid19 including presenting with digestive symptoms alone shows that patients with gastrointestinal symptoms had a longer duration between symptom onset and viral clearance and fecal viruspositive than those with respiratory symptoms a systematic metaanalysis of shows results from clinical studies with an incidence rate of diarrhea from as low as and up to of the cases how is this disparity in the data possible more clinical studies are required to elucidate the percentage of covid19 patients that develop intestinal symptoms and if these depend on other factors such as age gender or other comorbidities gastrointestinal symptoms are accompanied by inflammation or intestinal damage there is a loss of intestinal barrier integrity and gut microbes that can activate innate and adaptive immune cells to release proinflammatory cytokines into the circulatory system leading to systemic inflammation some intestinal signaling pathways can regulate inflammation through dendritic cells figure immunomodulation of the innate host immunity through the activation of epithelial receptors could represent a novel therapeutic target to eliminate sarscov2 in the early stage of the infection the viral load of coronavirus appeared in the feces of of the infected patients an interaction between viral shedding of sarscov2 in stool has been reported but its association with infection was yet to be determined identifying fecal coronavirus rna may also lead researchers to ask new questions is there transmission of the coronavirus found in fecal samples what these researchers found is the presence of the coronavirus in the stool but there is controversial studies about if viral load was infectious nor examples of transmission previously a study involving patients with covid19 reported that the median duration of viral shedding was days in survivors range – days cultivable sarscov2 was detected in stool or urine specimens for longer than weeks in three convalescent patients days suggesting that it may remain viable 0c contradictorily high sarscov2 titres detectable in the first week of illness with an early peak observed at symptom onset to day of disease [ ] although sarscov2 rna shedding in respiratory and stool can be prolonged the duration of a viable virus is relatively shortlived the presence of sarscov2 including live virus with infectious capacity in the feces of asymptomatic individuals implies that covid19 could be transmitted through the fecal route sarscov2 shedding in stool samples is detectable over a longer period than in nasopharyngeal swabs donors for fecal microbiota transplant for sarscov2 must be strict and validated to prevent the potential risk of transmission the results show that the elevated levels of fecal calprotectin in patients with covid19 add to the growing evidence that sarscov2 infection causes an inflammatory response in the intestine calprotectin concentrations were significantly higher in covid19 patients who had suffered from diarrhea and with more elevated serum il6 levels in the diagnosis and especially in the followup of covid19related diarrhea the calprotectin measurement could play a potential role in monitoring the disease also diarrhea may be secondary to virusinduced inflammation which in turn is due to the entry of inflammatory cells into the intestinal mucosa including neutrophils and lymphocytes and thus disruption of the gut microbiota viral sarscov2 ps were detected in feces during the second phase of covid19 accompanied by a decrease in the peak of inflammation therefore covid19related inflammatory diarrhea was associated with reduced levels of fecal sarscov2 rna however intestinal damage can manifest after respiratory symptoms as reported in a clinical case there may be a pathogenic role of sarscov2 on the gi in zheijiang china it was detected that half of the covid19 patients tested positive for rtpcr in their feces and intestinal microbial dysbiosis was also identified viral strains alive from feces were isolated indicating potential infectiousness of feces [ ] another analysis conducted from studies in china usa and singapore had also detected 0c sarscov2 rna in fecal specimens of patients at an average of this percentage is according to their clinical forecast intestinal problems or with more severe disease furthermore the fecal test for sarscov2 rna also bears a potential implication for physicians to decide the subsequent isolation strategies for those with positive fecal samples the possibility of the fecaloral route of transmission of sarscov2 should be investigated all candidates for fecal microbiota transplantation and healthy donors should be screened for the virus one could consider studies on the gut microbiome and its therapeutic role in transplanting feces in healthy donors to critically ill covid19 patients still precautions should be taken because infectious sarscov2 ps are known to have been found in covid19 positive feces the use of fecal transplants could be one of the immediate solutions in critically ill patients with a weakened immune system sarscov2 interaction with intestinal ace2 receptors sarscov2 enters cells primarily through binding of protein s to angiotensinconverting enzyme ii ace2 receptors to infected cells the central role of ace2 in the cleavage of angiotensin i to angiotensin ii a peptide involved in vascular homeostasis vasomotor tone and blood pressure regulation ace2 receptors are expressed in various human cells susceptible to viral infection including epithelial cells in the lungs small intestine and colon tubular cells of the kidney neuronal and glial cells in the brain enterocytes vascular endothelial cells smooth muscle cells and cardiomyocytes a very recent study has reported on the expression pattern of ace2 across more than different cell types corresponding to all major human tissues and ans based on rigorous immunohistochemical analysis sarscov2 also uses the receptors for transmembrane protease serine tmrpss2 an enzyme that is expressed in the small intestinal epithelial cells to entry to the infected cells figure the sarscov2 activity could cause ace2 modifications in the gut that increase susceptibility to 0c intestinal inflammation and diarrhea a high coexpression of ace2 and tmprss2 was detected in enterocytes and the esophagus and lungs the coexpression of ace2 and tmprss2 transcripts was highest in the small intestine with expressed in enterocytes and in colon cells as demonstrated by a singlecell rna sequencing study in gi ace2 is not only playing an essential role in intestinal inflammation but also have a significant impact on the composition of the intestinal microbiota ace2 ko mice have been shown to have decreased expression of antimicrobial peptides and exhibited altered intestinal microbial composition which is restored by the administration of tryptophan likewise it is known that there is a connection of transport of ace2 amino acid with microbial ecology in the gut during sarscov2 infection in another recent study using gnotobiotic germfree rats was observed a decrease in ace2 expression in the colon that contributed to the pathology during covid19 there are numerous studies that have shown that by regulating the reninangiotensin system ras which includes ace2 one can modulate systemic inflammation angiotensin ii receptor blockers are widely used compounds that are therapeutically effective in cardiovascular disorders renal disease metabolic syndrome and diabetes under normal healthy conditions homeostasis of rasace2 occurs while a perturbation to this system is observed in cardiovascular disease hypertension and diabetes mellitus a reduction of ace2 is associated with hypertension diabetes and cardiovascular problems which represent the significant comorbidities of covid19 sarscov2 may potentially further upregulate ras in cardiovascular patients and deplete ace2 downregulation of ace2 levels in tissues has been linked to viral replication efficiency and pathogenicity leading to the imbalance of positive and negative regulation of ras rasace2 imbalance in covid19 may greatly exacerbate tissue inflammation and may contribute to more adverse covid19 outcomes in patients with preexisting cardiovascular disease and other comorbidities ace2 receptors may represent a target for covid19 treatments in patients with cardiovascular risk burden susceptible to conditions that worsen asymmetries in rasace2 however suppression of 0c ace2protective roles due to ace2 depletion upon sarscov2 infection is very likely to uphold the poor outcomes observed in covid19 patients especially in those with preexisting conditions ace2 also displays nonrasrelated roles linked with neutral amino acids transport and gut homeostasis ace2 has been implicated in the intestinal epithelium regulation of gut microbiota composition and function in summary ace2 imbalance is likely a key player for the poor outcomes in the covid19 patients with preexisting comorbidities and addresses a possible link for gut microbiota dysbiosis in this interplay the effects of covid19 on the gut microbiome primary inflammatory stimuli trigger the release of microbial products and cytokines which can cause microbial dysbiosis that can induce an inflammatory environment releasing intestinal cytokines into the circulatory system increasing the systemic inflammation of covid19 taken together a combined inflammation can potentially initiate an immune reaction that can create even more harm than the virus itself it is necessary to know the host cytokine pathways and microbiota interactions with cytokine responses in sarscov2 infection to develop novel treatment approaches it is essential to investigate how intestinal bacteria interact in response to sarscov2 infection among covid19 patients an increase of a blood proteomic risk score prs was associated with a risk of becoming a clinically severe infection a recent study has shown more than proteins in bloodrelated to the severity of covid19 these proteins included immune factors that are elevated during systemic inflammation such as creactive protein crp correlations between prs and the intestinal microbiome might be associated with the severity of covid19 this new analysis would allow us to predict if the patient would develop more severe symptoms in the next days or weeks after the infection ruminococcus gnavus was identified in covid19 patients and positively correlated with inflammatory markers while 0c clostridia was negatively correlated another small study of patients hospitalized in hong kong has served to establish a gut microbiome profile in association with covid19 severity and changes in fecal shedding of sarscov2 through the application of indepth shotgun metagenomics analysis the authors investigated longitudinal changes of the gut microbiome in covid19 the abundance of coprobacillus clostridium ramosum and clostridium hathewayi correlated with covid19 severity and it was observed an inverse correlation between the abundance of faecalibacterium prausnitzii an antiinflammatory bacterium during the hospitalization time were detected in covid19 patients bacteroides dorei bacteroides thetaiotaomicron bacteroides massiliensis and bacteroides ovatus which downregulate the expression of ace2 in the murine gut correlating inversely with sarscov2 load in fecal samples this study opens the doors to possible interventions for gut microbiota in hospitalized patients to reduce the severity of covid19 another study of just patients showed that the gut bacteria was associated with fecal sarscov2 load the gut microbiome remained substantially different in hospitalized patients from that of healthy controls a total of bacterial species were identified to be significantly associated with the fecal viral load of sarscov2 across all fecal samples this does not indicate that the gut microbiome is affected for long periods after recovery and future therapeutic interventions would be necessary sarscov2 was detected in feces of hospitalized patients with covid19 and the viral transcriptional activity was analyzed to determine the infectivity range associated with the gut microbiome fecal samples with a signature of high sarscov2 infectivity had higher abundances of bacterial species collinsella aerofaciens collinsella tanakaei streptococcus infantis and manella manii in contrast fecal samples with a signature of lowto no sarscov2 infectivity had higher abundances of scfas producing bacteria parabacteroides merdae bacteroides stercoris alistipes onderdonkii and lachnospiraceae bacterium however live sarscov2 was not isolated from the feces covid19 patients another study 0c of covid19 patients showed a significantly reduced bacterial diversity and a higher relative abundance they compared these results with h1n1 patients who showed lower bacteria diversity compared with covid19 patients specific microbial signatures were identified in covid19 patients which could help identify biomarkers that differentiate them from influenza a h1n1 patients when there is a coinfection despite this the phases of the disease were not established in this study furthermore in another study with covid19 patients was observed a reduced alpha diversity in microbes the metatranscriptional analysis revealed that there were differentially genes associated with immune pathways and cytokine signaling related to the diagnosis and severity of covid19 such as interferongamma signaling despite reporting interesting patterns the studies mentioned above are not without limitations the bioinformatics analyses employed in these studies does not guarantee specieslevel nor strainlevel bacterial identification furthermore no longitudinal analysis has been performed with the same patients to determine if these bacteria change after recovery from covid19 more studies that prospectively include infected but asymptomatic subjects positive for sarscov2 but with different degrees of symptoms would be necessary to establish a correlation with microbial markers or their products monitoring early in the disease during early onset of covid19 and over the longterm will help to delineate the role of changes in the microbiome will be critically important to elucidating this connection further another major limitation is the small number of patients and the absence of information about microbial changes in the context of covid19 that define broad groups of the population stratified by geography ethnicity gender andor age to date there are no metabolomics and metaproteomic analysis studies that are needed to explore the products of these bacteria and their function by identifying microbial metabolites associated with covid19 we can understand what components these bacteria produce during covid19 that help us understand the influence of the communication pathways of the microbiotagutperiphery axis relevant to the association between sarscov2 and 0c hyperinflammation moreover more studies investigating the correlation between sarscov2 and gut inflammation are necessary including histopathology and molecular diagnostic assessments comorbidities as risk factors for covid19 associated with loss of microbial diversity sarscov2 can infect people of all ages but older adults and people with preexisting medical conditions appear to be more vulnerable to becoming seriously ill with the virus there are many hypotheses as to why this occurs still one of the factors could be the loss of microbial diversity associated with aging and with it higher susceptibility to inflammation comorbidities play an essential role in determining the risk of severe complications and death after covid19 infection covid19 comorbidities and risk factors include asthma hypertension smoking male gender or alzheimer's disease or dementia changes in gut microbiota have been previously linked to all of these comorbidities and this deregulation could also be associated with changes in the immune system and the susceptibility to suffer more severe consequences of covid19 and genderrelated differences in vulnerability to complications of covid19 our microbiome changes as we age which favors less diversity and a more significant inflammatory state covid19 appears to be more dangerous in older people men and with comorbidities the gut microbiota evolves during human life the infant microbiota shows reduced diversity and will remain unstable for the first few years of experience until it becomes an adultlike microbiota the gut microbiota within an individual is considered stable throughout adult life in the elderly gut microbiota diversity decreases and dysbiosis increases and is associated with cognitive deficits depression and inflammatory markers a change that is found repeatedly in the microbiota of the elderly is the decrease in the ratio of firmicutes to bacteroidetes [ ] decreased bacterial diversity as well as lower levels in specific bacterial groups have also been observed in very elderly [ ] at the genus and species 0c level the findings vary significantly between studies although bacteroides clostridium and lactobacillus appear recurrently altered in elderly individuals tragically a high rate of covid19 fatalities is associated with groups of people over years old the causes may be the inability to overcome the infection the weakness of the immune system and the reduced microbiome diversity causing the coronavirus to strongly attack this group of the population causing a higher mortality rate another recent study has found significant associations between dietary patterns and measures of gut microbial composition in older men the group of the population with the highest mortality rate from covid19 obesity is also associated with changes in the intestinal flora [ ] and is another risk factor in the severity of covid19 therefore another comorbidity in adults and children in the united states at least of patients who die from covid19 have obesity which is similar to the reported rates of cardiovascular disease in the same highrisk group it is necessary to study the relationship between obesity and the severity of the covid19 disease adipose tissue can serve as a reservoir for the spread of sarscov2 virus clearance and systemic immune activation adipocytes in obese patients express higher levels of ace2 and a reduction or elimination of already inflamed adipose tissue can reduce systemic viral spread viral entry and prolongation in obese individuals there is marked dysregulation of myeloid and lymphoid responses within adipose tissue associated with dysregulation of cytokine profiles obese patients also have heightened levels of proinflammatory adipokines leukotrienes chemerin among others which may exacerbate their risk for cytokine storm syndrome and death the alteration of the immune system causes changes in the intestinal flora and it remains to be seen whether the coronavirus also induces changes in the bacteria that modulate many of these functions of the intestinal brainmicrobiota axis the bacterial intestinal flora plays a critical role in the regulation of neurological functions such as depression or anxiety [ ] surely the results of these studies allow us to know 0c what the role of intestinal flora is in covid19 and its relationship with neurological problems at long term furthermore diabetes is another disease associated with increased severity of symptoms and complications of covid19 and this can be attributed to systemic inflammation and gutmetabolite dysfunction individuals suffering from cardiovascular disease who become infected with sarscov2 are at increased risk of developing a worse prognosis of covid19 and also develop cardiovascular complications including myocardial infarction arrhythmias stroke or heart feature or myocardial suppression cardiovascular disease is accompanied by an imbalance of gut microbiota and a decreased microbiome diversity hypertension is likely to be influenced by diet lifestyle factors and microbiome notably an increase in shortchain fatty acids scfa was previously associated with decreased blood pressure and improved arterial compliance changes in the gut microbiota composition through diet to deal with covid19 the impact of dietary patterns on susceptibility to and severity of infection with the sarscov2 virus has been largely ignored to date the commensal microbiome forms a dynamic environment that can be altered and cause dysbiosis from virus infection but can be positively modulated by diet components and probiotic treatments several studies show than an optimal immune response depends on proper diet and nutrition to control sarscov2 infection [ ] in general malnutrition can compromise the immune response therefore affecting the vulnerability of the response to covid19 consideration of the dietary and nutritional components the factors during viral infection can serve to strengthen the immune system for the prevention of infections and a meaningful and balanced basis for an immune response is an adequate and balanced diet intake of a sufficient amount of protein is crucial for the production of antibodies also a low level of vitamin a or zinc has been associated with an increased risk of infection branchedchain amino acids can maintain the hairy morphology of 0c the intestines and increase intestinal immunoglobulin levels thereby improving the intestinal barrier therefore highquality proteins are an essential component of an antiinflammatory diet nutritional dietary components known to exert antiinflammatory and antioxidant properties include omega3 fatty acids with high antiinflammatory and antioxidant capacity including vitamin c vitamin e and phytochemicals such as carotenoids and polyphenols that are widely present in plantbased foods undoubtedly omega3 fatty acids appear to have the most potent antiinflammatory capability several of these components can interact with cellular signaling components related to antiinflammatory and antioxidant effects an Answer:

Colon_Cancer

it is hard to comprehend how much of their lives poor people spend waiting in the emergency room dealing with indifferent bureaucracies that are supposed to address basic needs in the laundromat where people must scramble for available machines and keep a close eye on their laundry waiting adds yet more pressure and pain on feet that may already be swollen ¦ many poor people spend years of their lives on waiting lists for a public housing unit a section voucher a bed in rehab a hearing in landlordtenant court they wait for erratic buses food at a food pantry or a bed in a shelter their names fill long lists of people in dire need williams p waiting is a pervasive aspect of poverty just as poor people are most likely to wait for laundry machines housing food so too they are most likely to wait for their healthcare and this can have especially grave consequences andaya 2018b oostrom the link between the timing of treatment and health outcomes is well documented in health disparities research it has become axiomatic that timely adherence to and receipt of medications screenings and followup health services is vital for decreasing inequities in health outcomes bickham and lim dickman as a result much public health intervention work aims to identify and rectify the sources of protracted times to diagnosis andor treatment diamant koopmanschap similarly healthcare anizations are increasingly attentive to time elements identifying time as a critical quality metric shorter wait times particularly for appointments or between diagnosis and the initiation of treatment reflect greater efficiency and improved patient satisfaction and outcomes bleustein michael nevertheless anizationlevel effort to reduce wait times is unevenly distributed bureaucratic interventions disproportionately advantage wealthier and privately insured patients the task of waiting corresponding author email address jeanhunlethwustledu jm hunleth 101016jsocscimed2020113296 received in revised form august accepted august socialsciencemedicine2642020113296availableonline19august2020027795362020publishedbyelsevierltd 0caa lee in and for healthcare”in waiting rooms for future appointments procedures and treatments”is performed overwhelmingly by people who are poor kennedy oostrom in this we focus on what we are calling active waiting a concept that we suggest more accurately describes the lived experience of gaps in healthcare action than does the more frequently used word delay when contemporary health disparities researchers examine patient behavior and action as it relates to the timing of treatment they often focus on patients™ delaying diagnostic tests or treatments while understanding why a person might delay a diagnostic procedure or treatment regimen can identify barriers eg long wait times on phones or for followup procedures a lack of transportation or money that discourage individuals from taking more immediate actions diamant thinking in terms of delays can also place excessive responsibility and blame on individuals for nonaction and nonadherence hunleth œwhy did she delay scheduling her followup appointment is not the same as œwhy did she wait to schedule her appointment and though waiting can lead to assumptions about passivity or nonaction social scientists have demonstrated that waiting is not a passive state auyero mulcahy we derive our own focus on active waiting as opposed to delay from narrative interviews we carried out with people who have chronic illnesses and who have difficulties affording their medications studies of chronic illness in the us offer important insights into waiting people living with illness and in poverty wait a lot during the many appointments needed to manage their conditions ie foron health service and treatment providers and for disability insurance and paychecks ie foron bureaucratic actors of key importance active waiting unlike delay remains true to the linguistic preferences of the individuals with whom we have worked participants in the study we describe below spoke of their experiences of temporality in healthcare as of waiting and not as of delaying further the concept of active waiting allows us and others working in and studying healthcare to move beyond analyses that either locate responsibility for wait times solely with the individuals ng the waiting or perpetuate overlydeterministic views of systemic barriers that erase experience and social action to frame our conceptualization of active waiting we bring together two scholarships one on the political economy of healthcare which includes the elucidation of bureaucratic time and the other on waiting as a feeling and an experience which gains meaning within power relations and through social interactions bureaucratic time and the agency of waiting patients who then comply with bureaucratic state demands auyero waiting may be imposed but these researchers show that it always entails a response understanding the experience of waiting in the us requires recognizing that the actions people take while waiting are interpreted using neoliberal ideologies that privilege among other things individual responsibility mulligan rylkobauer and farmer this focus on individual responsibility can lead to the blaming and shaming of people who time healthcare inappropriately or do not act appropriately within others™ expected timeframes holmes scholars have shown that this privileging of individual responsibility reinforces racialized and classed hierarchies of deservingness horton notions of individual responsibility and thus of individual failure place pressure on patients to do and in ng they avoid or mitigate the likelihood of blame and shame while they wait as we have previously shown the focus on individual responsibility can lead people to blame themselves and their loved ones for waiting too long to seek care hunleth sociologist andreas g¨ottlich suggests that waiting is an interaction that depends on œthe mutual interpretations of the actors”those who wait and who are waited upon”as well as others who behold the scenes of waiting g¨ottlich p that is people wait in relationship to and with others researchers who center the interpretive acts and emotions of ˜waiters™ in healthcare settings such as waiting rooms offer several insights into what we refer to as active waiting in a study of interactions in a waiting room strathman and hay suggested that œwhen patients are told that they cannot get an appointment within the desired”even physician recommended”timeframe it is like being told that their health and by extension they as persons are less important than bureaucratic schedules strathmann and hay p that is waiting can add to a sense of being devalued or neglected andaya sj¨oling and it can negatively influence peoples™ perceptions of their quality of care and confidence in their care provider bleustein given the above work we understand active waiting to be the experiences and responses that people devise to navigate shorten or otherwise survive waiting and also to anticipate and craft possible futures within the relationality and power dynamics of bureaucratic time resisting wait times by seeking information and other resources is part of active waiting as is delaying and complying active waiting is composed of such discrete actions that people might take as patients in one place or time but is irreducible to those actions and must be situated in how people manage multiple wait times at various scales further we include not only action but also how waiting on bureaucratic time feels carr teucher and casson have used the phrase œlived wait time to communicate the fact that feelings of time differ by circumstances researchers have shown that chronic illness lends itself to particular feelings of time and of life including those of time as suspended eg feelings that life is œon hold brown mulcahy sj¨oling we expand this by focusing on how chronic illness feels while waiting on powerful others to make life or death decisions while wait time is lived people also live during their wait times daily lives are not easily paused sj¨oling this means that the circumstances of individuals™ days and lives often change while they wait on healthcare less has been said about the waiting that goes on outside of clinical settings yet is ultimately tied to health in fact waiting is an implicit and normalized mechanism of bureaucratic systems in what follows we consider how these various aspects of time and waiting combine for people experiencing illness and financial strain we use active waiting”conceptualized as an action orientation relationship and feeling that is irreducible to a singular time and place and which shapes how individuals approach their health in particular times and places we do so in order to best examine how various aspects of time and waiting interweave in the lives of people who are chronically ill and poor though healthcare bureaucracy in the us requires most people to wait for care how long people wait how waiting is structured and the consequences of waiting vary people who do not have access to insurance or other resources must endure state and federal evaluations of their lowincome statuses abilities to work severities of illness citizenship and more to qualify for assistance tickamyer as funding for social programs like medicaid food stamps and disability are rolled back and work requirements become stricter demonstrating qualifications to obtain support is increasingly difficult and time consuming whittle while social welfare programs offer access to health services they often require long waits in waiting or exam rooms at facilities that accept medicaid or offer services on a sliding fee scale becker oostrom researchers have identified how people deal with the bureaucratic schedules that shape the course of their treatment mulcahy described how some people with cancer resisted the assumptions that they must be ˜patient™ patients by seeking information and resources to shorten their waiting periods still other researchers have shown that long waittimes for healthcare personnel in clinics and waiting rooms can lead to other forms of action waiting can push individuals out of the healthcare system or lead them to delay care becker and it can induce feelings of pervasive uncertainty and powerlessness among socialsciencemedicine26420201132962 0cthis research comes from a mixed methods study investigating cost related issues that affect adherence to medications recruitment occurred in and amidst political uncertainty about changes to healthcare legislation on the national stage a new and controversial presidential administration was beginning its term this administration had goals to repeal the affordable care act aca and introduce block grant funding to medicaid which remained a possibility throughout our data collection period for people with chronic illnesses the aca was significant it mandated that preexisting conditions be covered by insurance plans and that insurers offer at least a minimum prescription drug benefit while also creating platforms for uninsured individuals to purchase plans additionally national conversations about changes to prescription drug pricing were prominent in news coverage most of our narrative interviews were conducted in st louis missouri though a few occurred in east st louis illinois missouri did not expand their medicaid program with the rollout of the aca illinois did st louis is a metropolitan area with significant racial and economic health inequities to address the health needs of those without insurance in light of not expanding medicaid the gateway to better health program was created which provided basic coverage to people living below the poverty line this program was set to end in december during our study but its extension was later approved beyond that date many of the people who participated in this study were using or had used this program recruitment interviews our research team recruited people between the ages of and who had one or more chronic illnesses for which they were prescribed medication and who identified as having difficulties affording their prescriptions the study had a mixed methods design participants in the survey n were recruited from newspaper ads federally qualified health centers and a multispecialty clinic a subsample of the survey participants n also participated in a narrative interview focused on financial strain while we have presented the mixed methods results elsewhere this focuses solely on the narrative interviews we initially aimed to recruit interview participants stratified evenly based on prescription insurance coverage age and gender however this was adjusted based on the availability of participants the final number of interviews was table the interview guide was based on the mcgill illness narrative interview mini groleau we revised the guide and refer to it as the modified financial and illness narrative interview it is structured similarly to the mini to elicit different types of narratives but with an added focus on financial history two local nonprofit community anizations were selected as interview locations due to their proximity to and rapport with the participant population nonclinical environments available private interview spaces and anizational relationships with study team members trained research team members conducted interviews and a note taker was present in most cases the interviewer obtained informed consent and interviewees were compensated with a gift card interviews were audiorecorded and lasted about “ minutes immediately after the interview the interviewer and note taker wrote detailed field notes using a structured template that included their observations about body language any conversations not recorded a summary of the financial and illness narrative and reflective memos analysis interviews were professionally transcribed verbatim deidentified and checked for accuracy before being uploaded with their respective fieldnotes to nvivo we describe the codebook development below which was designed with the mixed methods study in mind during that codebook development the team identified the repetition of comments about waiting across the interviews the team developed a codebook using deductive and inductive codes deductive codes were based on questions of interest from the quantitative survey these focused primarily on medication cost coping access to basic needs and related concepts the team identified inductive codes while conducting interviews and refined and added to these codes while reviewing transcripts during the analysis and throughout the coding process we took note of a crosscutting theme in the narratives when asked to describe what it was like to deal with chronic illness participants said that chronic illness was about waiting we coded all discussions of waiting in the interviews defined as any mention of waits or delays and in the fieldnotes while waiting is a broad concept and was used to reference different processes we chose to include in our analysis all aspects of waiting described by participants rather than focus on just one eg waiting for health insurance coverage we did so to acknowledge that people may experience waiting that is interrelated and different from the temporal distinctions made by many healthcare practitioners four team members coded the interviews using the final codebook all transcripts were coded by at least two team members and discrepancies were reconciled through consensus between the two coders al memoed on the waiting code throughout this process focusing on issues such as what kinds of things people waited for how this influenced their decisions and emotions the outcomes of their waiting on their health and healthcare and how waiting shaped their aspirations for the future the team including coders interviewers and investigators compared how waiting was discussed within and across the transcripts and narrative types elicited by the guide as well as the intersection of waiting with other codes interpretations and exemplar quotes were evaluated by multiple team members to guard against selective use and bias in interpretation of the data and to encourage reflexivity all study activities were approved by washington university™s institutional review board results aa lee methods setting table interview participant characteristics raceethnicity nonhispanic black or african american nonhispanic white other gender female male had continuous health coverage in the past year had a gap in coverage in the past year age mean range health coverage n we anize our results to make clear the meanings and experiences of active waiting in the first section we outline the breadth of waiting that shapes how people wait and answers the question œwaiting for what the second section addresses the question œwaiting for whom and describes how participants ascribed meaning to waiting according to their social and economic positions and in relation to those in power what some participants referred to as the œwaiting game finally and building on the previous sections we focus on waiting as an active process that people manage through a variety of tactics in ng so we detail the ways in which people actively wait living their lives within structures of power that make them wait socialsciencemedicine26420201132963 0caa lee waiting for what big and small waits because we cover multiple forms of waiting we wish to be specific about what participants referred to when they talked about waiting the list was long and it included waiting in waiting rooms for transportation on the phone for surgery for paychecks and welfare checks for housing and for approval for disability and insurance benefits the things participants waited for varied according to their own unique health and social circumstances these waits were often predictable and included the spaces in which people wait eg waiting rooms the bureaucracies that enforce waiting eg disability claims the conditions that create waiting eg strained social safety net and the financial demands that when unmet lengthen wait times eg a ride when one does not have transportation a paycheck to afford a prescription the variation in chronic illnesses and in life circumstances of participants in the study however meant that they discussed the structures of waiting in different ways and put emphasis on different aspects within the predictability and variability there was an overarching theme of waiting for one or a succession of big things to happen what we term ˜big waits™ big waits were for things that had a hopedfor endpoint ones that might change an individual™s health situation for example many participants talked about waiting for disability assistance to come through or for health insurance coverage to start or for stable housing and they often centered their discussions of waiting around this primary event the endpoints could be a decision made by a social service agency or could be an anticipated and desired life change one woman told us that her big wait for insurance coverage was almost over because she would soon be eligible for medicare which multiple others also mentioned that they were awaiting œthe good news for me is“ a year from this august “ i turn but um i always feel like it™s all pending there is nothing concrete in any of the healthcare while participants talked about big waits in terms of their endpoints they also talked about how such endpoints led to other big waits take kate for example kate is a black woman in her early fifties who previously worked in home healthcare she has osteoarthritis graves™ disease hepatitis c and depression after both her mother and husband died a few years ago she struggled to afford housing and continue working through her arthritis pain when we interviewed kate she had been homeless for several months and was recently accepted off a waiting list for transitional housing kate had several big waits that came in succession and were contingent upon one another she waited for housing assistance she waited for medical release to work after sustaining injuries in a car accident she waited to consult with a doctor about surgeries she waited on a disability hearing scheduled months away though she had been diagnosed with hepatitis c her doctor would not begin treatment until her disability application was approved because of the high costs of the medication which would be covered if she were on disability kate worried that all of these big waits would have longterm repercussions on her health until the disability [benefits are approved] or something [else comes through] and september [the date of her disability hearing] comes they ain™t going to even give me the medicine ¦ we will just wait and see so i am just in limbo until until my hearing ¦ i know i ain™t going to die right away but i do want to get the medication kate found herself waiting a lot on a lot the interdependence of her waiting meant that one wait exacerbated other waits while taking care of her sick family members she put off her own health needs and bill payments leading to her eventual housing instability her lack of a consistent mailing address delayed her ability to submit a disability application even after she found more stable housing and was able to see a doctor the disability application delays led to postponed medical treatments without getting her health and injuries under control she could not work and get out of transitional housing for kate each big wait led to another big wait with worsening and compounding repercussions on her health and wellbeing other participants described having waited years for their disability applications to be approved or often denied in the meantime they incurred greater debt hired lawyers relied on family for support postponed treatments and endured these big waits “ waiting for disability benefits and for other significant changes in their financial or familial situations “ also created a number of smallerscale daytoday situations in which study participants waited one uninsured woman described waiting to fill her prescription and waiting to see her doctors while she searched for a full time job to afford both œyou wait to the extreme before you do anything about whatever the problem is because you don™t want to go to the hospital it costs too much many participants missed appointments or went without medications while waiting for new insurance benefits or payday or financial assistance paperwork to go through holding off on care was just one way to wait actively available to those whose actions were constrained by chronic illness and financial strain while big waits figured strongly into participants™ imaginations of their futures small waits were not insignificant allen™s diabetes had damaged his kidneys so severely prior to getting medicaid coverage that he needed dialysis he described the cycle of waiting while on dialysis allen “ whose big wait had been first getting medicaid coverage and then once covered getting on a kidney transplant list “ did not have a car or a steady way to get to his weekly dialysis appointments a few times a week he waited to be picked up by a medicaidcovered transportation company he had little control over when they would arrive and their arrival was often sporadic when they arrived early he had to wait at the clinic when they were late he either had to pay for his own cab or push back his dialysis appointment to later in the day and wait even more optimistic allen expressed gratitude that he was able to use medicaid and live with his brother while waiting for a kidney transplant his own big wait within multiple small waits while enduring the small waits he imagined a better future after the transplant describing to us what he would write in a book about his experiences œkidney transplant got him back to a better life now he™s back in the work force he hasn™t looked back to the sickness yet ¦ that™s my story the daytoday waiting was frustrating but the promise of this story along with having family able and willing to assist him an unmarried man without children shaped how he waited and his feelings while waiting big and small waits contribute to the dynamic aspect of active waiting where social ties and financial resources regulate how one is able to navigate and survive waiting waiting was drawn out for those without the economic resources to avoid institutions such as slidingscale clinics welfare agencies and financial assistance programs throughout the interviews participants described waiting as chronic but they also held onto a sense that once a waiting period passed then their future goals of health and financial stability would be that much closer for kate waiting was incremental and each waiting period required different tactics the big waits participants described both promised and suspended the future people grappled with the present while anticipating what might happen when and if the waiting ended some people imagined a time when they could afford healthcare holding off certain appointments until that time while others imagined a future œin the work force and attended appointments diligently even in the face of smaller or shorterterm waits such small waits too structured participants™ responses in how they waited based on what they were waiting for waiting for whom the œwaiting game many participants identified feeling like the systems in place to assist chronically ill and financially strained people were œplaying games with their lives by making them wait they were made to wait by social service programs appointment schedules hospital and clinic waiting rooms insurance company call centers the waiting punctuated their socialsciencemedicine26420201132964 0caa lee daytoday lives and left them guessing about the reasons behind their extensive chronic waiting they guessed at answering œwhy meœ”why they had to wait they also guessed at a course of action to shorten or otherwise deal with waiting the œwaiting game they described was one in which the odds were stacked against them the rules were opaque and endurance was necessary to win some study participants searched for ways to get around the game or to play it to their advantage how they played the waiting game “ which we recognize as active waiting “ depended on both their interpretations of the meaning of waiting and on the resources to which they had access in our interviews some people brought up suspicions about being forced to wait for appointments and in waiting rooms due to their economic status age race and other discriminatory factors one participant talked about how once in the examining room she felt as if her time waiting was not reciprocated by providers œthey do not want to spend a lot of time with you you know theythey™ve got to make a living too so they move on some speculated that doctors reserved time for people of a higher economic status these patientprovider dynamics that waiting exemplified left people feeling slighted by the medical system œah i sat there in that waiting room for hours and and they still wasn™t ready to see me ¦ no one had the respect ah even the courtesy to come out and say ˜well the doctor is running a little bit behind™ people felt that their lives were undervalued and unimportant when their time was not acknowledged or respected that bureaucratic systems seemed to withhold resources by imposing waiting suggested to some a denial of their belonging and deservingness as patients and as humans worthy of care experiences of medical racism and sexism frequently underpinned participants™ feelings that their time was being wasted rachel a black woman told us that she had been working and paying taxes since she was a kid and still the welfare system did not support her when she needed it recognized that high blood pressure was œblack peoples™ number one killer and explained that her waiting also stressed her body contributing to her anger these indignities played out in the clinic adding to the daily stresses she said she already felt as a black woman and grandmother living near the ferguson neighborhood where an officer murdered michael brown in and was not indicted for that murder participants also talked about waiting as a game that required endurance not just because of a lengthy office wait or the months or years it could take to get a disability application approved or denied but because of the opacity of the process they questioned application processes wondering if the wait to hear back about welfare and assistance services was a test of endurance œit is just a waiting game [for disability] until they get tired tired of you being in front of them and they decide to give it to you that is what they hope for is [you] giving up on it but if you keep with it sooner or later you will get it while a number of participants expressed optimism for a better future once a big wait was over waiting out bureaucratic processes wore down that optimism œi don™t believe i can keep going through this too much longer erica said she was exhausted from the strain of balancing utility payments with medical costs while waiting for her new insurance plan to begin and while coping with illness unexplained waiting on bureaucratic systems whether for an application to be approved or for insurance to kick in or to be seen by a busy doctor in a slidingscale clinic felt like an unfair game designed to keep them from receiving necessary medical care by framing waiting as a game the participants explained that there were rules that shaped the length and types of waiting and that they did not write them the rules were far from transparent and enforced differently based on race and social status as sherry made evident in her frustration with being made to wait waiting is not a passive state that happens without mental and physical consequences when people responded with patience and endurance through the game just like when they responded with anger and confrontation their waiting was active put differently participants described fashioning tactics in part based on their perceptions of the intentions of those for whom they waited they kept turning me down for disability and they kept saying i was too young to have problems i had you know but my body was breaking down and the doctors would never [help with the application] which was really so unfair and now that™s the part that really makes me feel a little sad like there is so much “ i hate to say racism “ but just unfair treatment because i™m a woman because of my age and then because of my race and so we™re talking like years [voice breaking] ¦ it made me be really poor rachel™s doctors held the power to demonstrate that she qualified for disability but as she recounted they were unwilling to fill out the documentation she needed to apply for disability she felt that the us had enough resources to

cancer7523

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: it is hard to comprehend how much of their lives poor people spend waiting in the emergency room dealing with indifferent bureaucracies that are supposed to address basic needs in the laundromat where people must scramble for available machines and keep a close eye on their laundry waiting adds yet more pressure and pain on feet that may already be swollen ¦ many poor people spend years of their lives on waiting lists for a public housing unit a section voucher a bed in rehab a hearing in landlordtenant court they wait for erratic buses food at a food pantry or a bed in a shelter their names fill long lists of people in dire need williams p waiting is a pervasive aspect of poverty just as poor people are most likely to wait for laundry machines housing food so too they are most likely to wait for their healthcare and this can have especially grave consequences andaya 2018b oostrom the link between the timing of treatment and health outcomes is well documented in health disparities research it has become axiomatic that timely adherence to and receipt of medications screenings and followup health services is vital for decreasing inequities in health outcomes bickham and lim dickman as a result much public health intervention work aims to identify and rectify the sources of protracted times to diagnosis andor treatment diamant koopmanschap similarly healthcare anizations are increasingly attentive to time elements identifying time as a critical quality metric shorter wait times particularly for appointments or between diagnosis and the initiation of treatment reflect greater efficiency and improved patient satisfaction and outcomes bleustein michael nevertheless anizationlevel effort to reduce wait times is unevenly distributed bureaucratic interventions disproportionately advantage wealthier and privately insured patients the task of waiting corresponding author email address jeanhunlethwustledu jm hunleth 101016jsocscimed2020113296 received in revised form august accepted august socialsciencemedicine2642020113296availableonline19august2020027795362020publishedbyelsevierltd 0caa lee in and for healthcare”in waiting rooms for future appointments procedures and treatments”is performed overwhelmingly by people who are poor kennedy oostrom in this we focus on what we are calling active waiting a concept that we suggest more accurately describes the lived experience of gaps in healthcare action than does the more frequently used word delay when contemporary health disparities researchers examine patient behavior and action as it relates to the timing of treatment they often focus on patients™ delaying diagnostic tests or treatments while understanding why a person might delay a diagnostic procedure or treatment regimen can identify barriers eg long wait times on phones or for followup procedures a lack of transportation or money that discourage individuals from taking more immediate actions diamant thinking in terms of delays can also place excessive responsibility and blame on individuals for nonaction and nonadherence hunleth œwhy did she delay scheduling her followup appointment is not the same as œwhy did she wait to schedule her appointment and though waiting can lead to assumptions about passivity or nonaction social scientists have demonstrated that waiting is not a passive state auyero mulcahy we derive our own focus on active waiting as opposed to delay from narrative interviews we carried out with people who have chronic illnesses and who have difficulties affording their medications studies of chronic illness in the us offer important insights into waiting people living with illness and in poverty wait a lot during the many appointments needed to manage their conditions ie foron health service and treatment providers and for disability insurance and paychecks ie foron bureaucratic actors of key importance active waiting unlike delay remains true to the linguistic preferences of the individuals with whom we have worked participants in the study we describe below spoke of their experiences of temporality in healthcare as of waiting and not as of delaying further the concept of active waiting allows us and others working in and studying healthcare to move beyond analyses that either locate responsibility for wait times solely with the individuals ng the waiting or perpetuate overlydeterministic views of systemic barriers that erase experience and social action to frame our conceptualization of active waiting we bring together two scholarships one on the political economy of healthcare which includes the elucidation of bureaucratic time and the other on waiting as a feeling and an experience which gains meaning within power relations and through social interactions bureaucratic time and the agency of waiting patients who then comply with bureaucratic state demands auyero waiting may be imposed but these researchers show that it always entails a response understanding the experience of waiting in the us requires recognizing that the actions people take while waiting are interpreted using neoliberal ideologies that privilege among other things individual responsibility mulligan rylkobauer and farmer this focus on individual responsibility can lead to the blaming and shaming of people who time healthcare inappropriately or do not act appropriately within others™ expected timeframes holmes scholars have shown that this privileging of individual responsibility reinforces racialized and classed hierarchies of deservingness horton notions of individual responsibility and thus of individual failure place pressure on patients to do and in ng they avoid or mitigate the likelihood of blame and shame while they wait as we have previously shown the focus on individual responsibility can lead people to blame themselves and their loved ones for waiting too long to seek care hunleth sociologist andreas g¨ottlich suggests that waiting is an interaction that depends on œthe mutual interpretations of the actors”those who wait and who are waited upon”as well as others who behold the scenes of waiting g¨ottlich p that is people wait in relationship to and with others researchers who center the interpretive acts and emotions of ˜waiters™ in healthcare settings such as waiting rooms offer several insights into what we refer to as active waiting in a study of interactions in a waiting room strathman and hay suggested that œwhen patients are told that they cannot get an appointment within the desired”even physician recommended”timeframe it is like being told that their health and by extension they as persons are less important than bureaucratic schedules strathmann and hay p that is waiting can add to a sense of being devalued or neglected andaya sj¨oling and it can negatively influence peoples™ perceptions of their quality of care and confidence in their care provider bleustein given the above work we understand active waiting to be the experiences and responses that people devise to navigate shorten or otherwise survive waiting and also to anticipate and craft possible futures within the relationality and power dynamics of bureaucratic time resisting wait times by seeking information and other resources is part of active waiting as is delaying and complying active waiting is composed of such discrete actions that people might take as patients in one place or time but is irreducible to those actions and must be situated in how people manage multiple wait times at various scales further we include not only action but also how waiting on bureaucratic time feels carr teucher and casson have used the phrase œlived wait time to communicate the fact that feelings of time differ by circumstances researchers have shown that chronic illness lends itself to particular feelings of time and of life including those of time as suspended eg feelings that life is œon hold brown mulcahy sj¨oling we expand this by focusing on how chronic illness feels while waiting on powerful others to make life or death decisions while wait time is lived people also live during their wait times daily lives are not easily paused sj¨oling this means that the circumstances of individuals™ days and lives often change while they wait on healthcare less has been said about the waiting that goes on outside of clinical settings yet is ultimately tied to health in fact waiting is an implicit and normalized mechanism of bureaucratic systems in what follows we consider how these various aspects of time and waiting combine for people experiencing illness and financial strain we use active waiting”conceptualized as an action orientation relationship and feeling that is irreducible to a singular time and place and which shapes how individuals approach their health in particular times and places we do so in order to best examine how various aspects of time and waiting interweave in the lives of people who are chronically ill and poor though healthcare bureaucracy in the us requires most people to wait for care how long people wait how waiting is structured and the consequences of waiting vary people who do not have access to insurance or other resources must endure state and federal evaluations of their lowincome statuses abilities to work severities of illness citizenship and more to qualify for assistance tickamyer as funding for social programs like medicaid food stamps and disability are rolled back and work requirements become stricter demonstrating qualifications to obtain support is increasingly difficult and time consuming whittle while social welfare programs offer access to health services they often require long waits in waiting or exam rooms at facilities that accept medicaid or offer services on a sliding fee scale becker oostrom researchers have identified how people deal with the bureaucratic schedules that shape the course of their treatment mulcahy described how some people with cancer resisted the assumptions that they must be ˜patient™ patients by seeking information and resources to shorten their waiting periods still other researchers have shown that long waittimes for healthcare personnel in clinics and waiting rooms can lead to other forms of action waiting can push individuals out of the healthcare system or lead them to delay care becker and it can induce feelings of pervasive uncertainty and powerlessness among socialsciencemedicine26420201132962 0cthis research comes from a mixed methods study investigating cost related issues that affect adherence to medications recruitment occurred in and amidst political uncertainty about changes to healthcare legislation on the national stage a new and controversial presidential administration was beginning its term this administration had goals to repeal the affordable care act aca and introduce block grant funding to medicaid which remained a possibility throughout our data collection period for people with chronic illnesses the aca was significant it mandated that preexisting conditions be covered by insurance plans and that insurers offer at least a minimum prescription drug benefit while also creating platforms for uninsured individuals to purchase plans additionally national conversations about changes to prescription drug pricing were prominent in news coverage most of our narrative interviews were conducted in st louis missouri though a few occurred in east st louis illinois missouri did not expand their medicaid program with the rollout of the aca illinois did st louis is a metropolitan area with significant racial and economic health inequities to address the health needs of those without insurance in light of not expanding medicaid the gateway to better health program was created which provided basic coverage to people living below the poverty line this program was set to end in december during our study but its extension was later approved beyond that date many of the people who participated in this study were using or had used this program recruitment interviews our research team recruited people between the ages of and who had one or more chronic illnesses for which they were prescribed medication and who identified as having difficulties affording their prescriptions the study had a mixed methods design participants in the survey n were recruited from newspaper ads federally qualified health centers and a multispecialty clinic a subsample of the survey participants n also participated in a narrative interview focused on financial strain while we have presented the mixed methods results elsewhere this focuses solely on the narrative interviews we initially aimed to recruit interview participants stratified evenly based on prescription insurance coverage age and gender however this was adjusted based on the availability of participants the final number of interviews was table the interview guide was based on the mcgill illness narrative interview mini groleau we revised the guide and refer to it as the modified financial and illness narrative interview it is structured similarly to the mini to elicit different types of narratives but with an added focus on financial history two local nonprofit community anizations were selected as interview locations due to their proximity to and rapport with the participant population nonclinical environments available private interview spaces and anizational relationships with study team members trained research team members conducted interviews and a note taker was present in most cases the interviewer obtained informed consent and interviewees were compensated with a gift card interviews were audiorecorded and lasted about “ minutes immediately after the interview the interviewer and note taker wrote detailed field notes using a structured template that included their observations about body language any conversations not recorded a summary of the financial and illness narrative and reflective memos analysis interviews were professionally transcribed verbatim deidentified and checked for accuracy before being uploaded with their respective fieldnotes to nvivo we describe the codebook development below which was designed with the mixed methods study in mind during that codebook development the team identified the repetition of comments about waiting across the interviews the team developed a codebook using deductive and inductive codes deductive codes were based on questions of interest from the quantitative survey these focused primarily on medication cost coping access to basic needs and related concepts the team identified inductive codes while conducting interviews and refined and added to these codes while reviewing transcripts during the analysis and throughout the coding process we took note of a crosscutting theme in the narratives when asked to describe what it was like to deal with chronic illness participants said that chronic illness was about waiting we coded all discussions of waiting in the interviews defined as any mention of waits or delays and in the fieldnotes while waiting is a broad concept and was used to reference different processes we chose to include in our analysis all aspects of waiting described by participants rather than focus on just one eg waiting for health insurance coverage we did so to acknowledge that people may experience waiting that is interrelated and different from the temporal distinctions made by many healthcare practitioners four team members coded the interviews using the final codebook all transcripts were coded by at least two team members and discrepancies were reconciled through consensus between the two coders al memoed on the waiting code throughout this process focusing on issues such as what kinds of things people waited for how this influenced their decisions and emotions the outcomes of their waiting on their health and healthcare and how waiting shaped their aspirations for the future the team including coders interviewers and investigators compared how waiting was discussed within and across the transcripts and narrative types elicited by the guide as well as the intersection of waiting with other codes interpretations and exemplar quotes were evaluated by multiple team members to guard against selective use and bias in interpretation of the data and to encourage reflexivity all study activities were approved by washington university™s institutional review board results aa lee methods setting table interview participant characteristics raceethnicity nonhispanic black or african american nonhispanic white other gender female male had continuous health coverage in the past year had a gap in coverage in the past year age mean range health coverage n we anize our results to make clear the meanings and experiences of active waiting in the first section we outline the breadth of waiting that shapes how people wait and answers the question œwaiting for what the second section addresses the question œwaiting for whom and describes how participants ascribed meaning to waiting according to their social and economic positions and in relation to those in power what some participants referred to as the œwaiting game finally and building on the previous sections we focus on waiting as an active process that people manage through a variety of tactics in ng so we detail the ways in which people actively wait living their lives within structures of power that make them wait socialsciencemedicine26420201132963 0caa lee waiting for what big and small waits because we cover multiple forms of waiting we wish to be specific about what participants referred to when they talked about waiting the list was long and it included waiting in waiting rooms for transportation on the phone for surgery for paychecks and welfare checks for housing and for approval for disability and insurance benefits the things participants waited for varied according to their own unique health and social circumstances these waits were often predictable and included the spaces in which people wait eg waiting rooms the bureaucracies that enforce waiting eg disability claims the conditions that create waiting eg strained social safety net and the financial demands that when unmet lengthen wait times eg a ride when one does not have transportation a paycheck to afford a prescription the variation in chronic illnesses and in life circumstances of participants in the study however meant that they discussed the structures of waiting in different ways and put emphasis on different aspects within the predictability and variability there was an overarching theme of waiting for one or a succession of big things to happen what we term ˜big waits™ big waits were for things that had a hopedfor endpoint ones that might change an individual™s health situation for example many participants talked about waiting for disability assistance to come through or for health insurance coverage to start or for stable housing and they often centered their discussions of waiting around this primary event the endpoints could be a decision made by a social service agency or could be an anticipated and desired life change one woman told us that her big wait for insurance coverage was almost over because she would soon be eligible for medicare which multiple others also mentioned that they were awaiting œthe good news for me is“ a year from this august “ i turn but um i always feel like it™s all pending there is nothing concrete in any of the healthcare while participants talked about big waits in terms of their endpoints they also talked about how such endpoints led to other big waits take kate for example kate is a black woman in her early fifties who previously worked in home healthcare she has osteoarthritis graves™ disease hepatitis c and depression after both her mother and husband died a few years ago she struggled to afford housing and continue working through her arthritis pain when we interviewed kate she had been homeless for several months and was recently accepted off a waiting list for transitional housing kate had several big waits that came in succession and were contingent upon one another she waited for housing assistance she waited for medical release to work after sustaining injuries in a car accident she waited to consult with a doctor about surgeries she waited on a disability hearing scheduled months away though she had been diagnosed with hepatitis c her doctor would not begin treatment until her disability application was approved because of the high costs of the medication which would be covered if she were on disability kate worried that all of these big waits would have longterm repercussions on her health until the disability [benefits are approved] or something [else comes through] and september [the date of her disability hearing] comes they ain™t going to even give me the medicine ¦ we will just wait and see so i am just in limbo until until my hearing ¦ i know i ain™t going to die right away but i do want to get the medication kate found herself waiting a lot on a lot the interdependence of her waiting meant that one wait exacerbated other waits while taking care of her sick family members she put off her own health needs and bill payments leading to her eventual housing instability her lack of a consistent mailing address delayed her ability to submit a disability application even after she found more stable housing and was able to see a doctor the disability application delays led to postponed medical treatments without getting her health and injuries under control she could not work and get out of transitional housing for kate each big wait led to another big wait with worsening and compounding repercussions on her health and wellbeing other participants described having waited years for their disability applications to be approved or often denied in the meantime they incurred greater debt hired lawyers relied on family for support postponed treatments and endured these big waits “ waiting for disability benefits and for other significant changes in their financial or familial situations “ also created a number of smallerscale daytoday situations in which study participants waited one uninsured woman described waiting to fill her prescription and waiting to see her doctors while she searched for a full time job to afford both œyou wait to the extreme before you do anything about whatever the problem is because you don™t want to go to the hospital it costs too much many participants missed appointments or went without medications while waiting for new insurance benefits or payday or financial assistance paperwork to go through holding off on care was just one way to wait actively available to those whose actions were constrained by chronic illness and financial strain while big waits figured strongly into participants™ imaginations of their futures small waits were not insignificant allen™s diabetes had damaged his kidneys so severely prior to getting medicaid coverage that he needed dialysis he described the cycle of waiting while on dialysis allen “ whose big wait had been first getting medicaid coverage and then once covered getting on a kidney transplant list “ did not have a car or a steady way to get to his weekly dialysis appointments a few times a week he waited to be picked up by a medicaidcovered transportation company he had little control over when they would arrive and their arrival was often sporadic when they arrived early he had to wait at the clinic when they were late he either had to pay for his own cab or push back his dialysis appointment to later in the day and wait even more optimistic allen expressed gratitude that he was able to use medicaid and live with his brother while waiting for a kidney transplant his own big wait within multiple small waits while enduring the small waits he imagined a better future after the transplant describing to us what he would write in a book about his experiences œkidney transplant got him back to a better life now he™s back in the work force he hasn™t looked back to the sickness yet ¦ that™s my story the daytoday waiting was frustrating but the promise of this story along with having family able and willing to assist him an unmarried man without children shaped how he waited and his feelings while waiting big and small waits contribute to the dynamic aspect of active waiting where social ties and financial resources regulate how one is able to navigate and survive waiting waiting was drawn out for those without the economic resources to avoid institutions such as slidingscale clinics welfare agencies and financial assistance programs throughout the interviews participants described waiting as chronic but they also held onto a sense that once a waiting period passed then their future goals of health and financial stability would be that much closer for kate waiting was incremental and each waiting period required different tactics the big waits participants described both promised and suspended the future people grappled with the present while anticipating what might happen when and if the waiting ended some people imagined a time when they could afford healthcare holding off certain appointments until that time while others imagined a future œin the work force and attended appointments diligently even in the face of smaller or shorterterm waits such small waits too structured participants™ responses in how they waited based on what they were waiting for waiting for whom the œwaiting game many participants identified feeling like the systems in place to assist chronically ill and financially strained people were œplaying games with their lives by making them wait they were made to wait by social service programs appointment schedules hospital and clinic waiting rooms insurance company call centers the waiting punctuated their socialsciencemedicine26420201132964 0caa lee daytoday lives and left them guessing about the reasons behind their extensive chronic waiting they guessed at answering œwhy meœ”why they had to wait they also guessed at a course of action to shorten or otherwise deal with waiting the œwaiting game they described was one in which the odds were stacked against them the rules were opaque and endurance was necessary to win some study participants searched for ways to get around the game or to play it to their advantage how they played the waiting game “ which we recognize as active waiting “ depended on both their interpretations of the meaning of waiting and on the resources to which they had access in our interviews some people brought up suspicions about being forced to wait for appointments and in waiting rooms due to their economic status age race and other discriminatory factors one participant talked about how once in the examining room she felt as if her time waiting was not reciprocated by providers œthey do not want to spend a lot of time with you you know theythey™ve got to make a living too so they move on some speculated that doctors reserved time for people of a higher economic status these patientprovider dynamics that waiting exemplified left people feeling slighted by the medical system œah i sat there in that waiting room for hours and and they still wasn™t ready to see me ¦ no one had the respect ah even the courtesy to come out and say ˜well the doctor is running a little bit behind™ people felt that their lives were undervalued and unimportant when their time was not acknowledged or respected that bureaucratic systems seemed to withhold resources by imposing waiting suggested to some a denial of their belonging and deservingness as patients and as humans worthy of care experiences of medical racism and sexism frequently underpinned participants™ feelings that their time was being wasted rachel a black woman told us that she had been working and paying taxes since she was a kid and still the welfare system did not support her when she needed it recognized that high blood pressure was œblack peoples™ number one killer and explained that her waiting also stressed her body contributing to her anger these indignities played out in the clinic adding to the daily stresses she said she already felt as a black woman and grandmother living near the ferguson neighborhood where an officer murdered michael brown in and was not indicted for that murder participants also talked about waiting as a game that required endurance not just because of a lengthy office wait or the months or years it could take to get a disability application approved or denied but because of the opacity of the process they questioned application processes wondering if the wait to hear back about welfare and assistance services was a test of endurance œit is just a waiting game [for disability] until they get tired tired of you being in front of them and they decide to give it to you that is what they hope for is [you] giving up on it but if you keep with it sooner or later you will get it while a number of participants expressed optimism for a better future once a big wait was over waiting out bureaucratic processes wore down that optimism œi don™t believe i can keep going through this too much longer erica said she was exhausted from the strain of balancing utility payments with medical costs while waiting for her new insurance plan to begin and while coping with illness unexplained waiting on bureaucratic systems whether for an application to be approved or for insurance to kick in or to be seen by a busy doctor in a slidingscale clinic felt like an unfair game designed to keep them from receiving necessary medical care by framing waiting as a game the participants explained that there were rules that shaped the length and types of waiting and that they did not write them the rules were far from transparent and enforced differently based on race and social status as sherry made evident in her frustration with being made to wait waiting is not a passive state that happens without mental and physical consequences when people responded with patience and endurance through the game just like when they responded with anger and confrontation their waiting was active put differently participants described fashioning tactics in part based on their perceptions of the intentions of those for whom they waited they kept turning me down for disability and they kept saying i was too young to have problems i had you know but my body was breaking down and the doctors would never [help with the application] which was really so unfair and now that™s the part that really makes me feel a little sad like there is so much “ i hate to say racism “ but just unfair treatment because i™m a woman because of my age and then because of my race and so we™re talking like years [voice breaking] ¦ it made me be really poor rachel™s doctors held the power to demonstrate that she qualified for disability but as she recounted they were unwilling to fill out the documentation she needed to apply for disability she felt that the us had enough resources to Answer:

Colon_Cancer

" pharmacology and toxicology laboratory csirinstitute of himalayan bioresource technology preproof 0c preproofinfection f0b7 systemic oxidative stress and inflammation are significant outcomes of sarscov2 highlights f0b7 activated gsk3 following sarscov2 infection provoke the oxidative stress and inflammation in the host f0b7 gsk3 phosphorylates nucleocapsid protein of sarscov2 and helps in disease progression f0b7 inhibition of gsk3 can be a suitable target in curbing of covid19 pandemic 0cwith the host defence mechanism by the help of gsk3 protein the virally infected cells show the coronavirus disease covid19 outbreak caused by severe acute respiratory syndrome coronavirus sarscov2 had turned out to be highly pathogenic and transmittable researchers throughout the globe are still struggling to understand this strain's aggressiveness in search of putative therapies for its control crosstalk between oxidative stress and systemic inflammation seems to support the progression of the infection glycogen synthase kinase3 gsk3 is a conserved serinethreonine kinase that mainly participates in cell proliferation development stress and inflammation in humans nucleocapsid protein of sarscov2 is an important structural protein responsible for viral replication and interferes activated gsk3 protein that degrades the nuclear factor erythroid 2related factor nrf2 protein resulting in excessive oxidative stress activated gsk3 also modulates crebdna activity phosphorylates nfκb and degrades catenin thus provokes systemic inflammation preproofinteraction between these two pathophysiological events oxidative stress and inflammation enhance mucous secretion coagulation cascade and hypoxia which ultimately leads to multiple ans failure resulting in the death of the infected patient the present review aims to highlight the pathogenic role of gsk3 in viral replication initiation of oxidative stress and inflammation during sarscov2 infection the review also summarizes the potential gsk3 pathway modulators as putative therapeutic interventions in combating the covid19 keywords covid19 gsk3 nfκb nucleocapsid protein oxidative stress sarscovpandemic list of abbreviations ace2 angiotensinconverting enzyme ad alzheimer™s disease adp adenosine diphosphate aiibb3 glycoprotein iibiiia ards acute respiratory distress syndrome 0care antioxidant response elements asc apoptosisassociated specklike protein containing a card atp adenosine triphosphate balf bronchoalveolar lavage bzip basic leucine zipper cats “ catalase cbp creb binding protein covid19 coronavirus disease creb camp response elementbinding protein cul3 cullin gpx glutathione peroxidase gsh intracellular glutathione gsk3 glycogen synthase kinase3 damp death associated molecular pattern gcsf granulocyte colony stimulating factor hcv hepatitis c virus hdac3 histone deacetylase ho1 heme oxygenase1 ifnÎ interferongamma preproofnfκb nuclear factorκb nlrp3 nodlike receptors protein mcp1 monocyte chemoattractant peptide mip1α macrophage inflammatory protein 1α myd88 myeloid differentiation primary response nadph nicotinamide adenine dinucleotide phosphate hydrogen ikk ikb kinase il6 interleukin iraks interleukin il 1rassociated kinase iκb inhibitor of kappa b keap1 kelchlike ech associated protein licl lithium chloride nlrp3 nucleotidebinding domain nodlike receptor protein nox nadph oxidase nprotein nucleocapsid protein nrf2 nuclear factor erythroid 2related factor 0cntd nterminal domain o superoxide anion o2 oxygen molecule oxpls oxidized phospholipids pamp pathogen associated molecular pattern par proteaseactivated receptors pd parkinson™s disease pedv porcine epidemic diarrhea virus ros reactive oxygen species sarscov2 severe acute respiratory syndrome coronavirus tak1 transforming growth factor tgfactivated kinase tf tissue factor tirap tirdomaincontaining adaptor protein sgmrna sub genomic messenger rna sods superoxide dismutase ppr pattern recognition receptor psgl pselectin glycoprotein ligand1 rigi retinoic acidinducible gene i preproofvwf von willebrand factor xo xanthine oxidase xor xanthine oxidoreductase tlr3 toll like receptor3 tnf tumor necrosis factor tnfr tumor necrosis factor receptor tnfα tumour necrosis factoralpha traf6 tumour necrosis factor receptor associated factor trs transcription regulating sequence introduction in late december wuhan china got attention worldwide after getting several patients diagnosed with pneumonia following a viral infection on 11th february the pathogenic strain of the virus was taxonomically designated as severe respiratory syndrome coronavirus sarscov2 by the international committee on taxonomy of viruses ictv the 0cassociated diseased condition was termed covid19 by the world health anization who the who announced sarscov2 virus infection a pandemic as it infected nearly million persons and engulfed more than worldwide sarscov2 is a member of coronaviruses consisting of kb singlestranded positivesense rna as genetic material it shows genetic similarity between another human coronavirus ie sarscov while similarity with bat coronavirus ratg1 and shares a high similarity index with pangolin coronavirus respiratory droplets are the primary source of viral transmission either through nasopharyngeal or oral route dry cough and high fever are the sarscov virusassociated respiratory disease replication within the host cell in disease progression the present review provides an inthe infected cells however in the case of sarscov2 infection aggressive inflammation significant symptoms observed in patients within days following viral infection the disease pathophysiology of covid19 also shows a close resemblance with previous reported and oxidative stress help in viral replication and damage the airway epithelium cell that results in acute respiratory distress syndrome ards which makes the condition worst glycogen synthase kinase3 gsk3 is a serinethreonine evolutionary conserved central molecule that the majority of respiratory viral infections are associated with the recruitment of immune cells the release of proinflammatory cytokines oxidative stress and finally phagocytosis of mainly participates in cell proliferation migration development apoptosis and immune regulation acquired and innate activation of gsk3 is associated with suppression of host immunity and inhibition of antioxidant response it is also supporting viral genome preproofdepth knowledge of oxidative stress inflammation and viral replication related to gsk3 during sarscov2 infection further the review highlights the gsk3 pathway modulators' gsk3 is a versatile serinethreonine kinase that regulates glycogen metabolism it consists of two isoforms gsk3α and gsk3 encoded by two separate genes both the isoforms share sequence similarity between kinase domains despite they never compensate for each other's' loss of function gsk3 has two prime functional domains a substratebinding domain which acquires substrates to gsk3 while the other kinase domain is responsible for phosphorylation of the substrate the nterminal region of gsk3 contains atp binding domain whereas the cterminal region consists of a large conserved activation loop responsible for the enzyme's full activation activation of gsk3 depends on the siteputative role as therapeutic interventions in combating the covid19 pandemic gsk3 structure 0cspecific phosphorylation that is controlled by various kinases gsk3 prefers prephosphorylate substrate by recognizing consequence sequences stxxxphosphost on substrate gsk3 is also involved in wntcatenin and sonic hedgehog cell signalling pathways mediating in cell proliferation differentiation maturation and cell adhesion transcription factors cjun creb stat3 cebpα nfat myc nfκb and p53 are the major substrate of gsk3 that can manipulate the expression of several other genes impaired activity of gsk3 has recognized in several clinical conditions such as metabolic disorders cancers alzheimer's disease ad parkinson's disease pd bipolar disorders and various other neurodegenerative diseases sarscov2 infection and inflammation covid19 patients' systemic cytokine profile shows a close resemblance with cytokine release syndrome characterized by macrophage activation an elevated level of cytokines like tumour necrosis factoralpha tnfα interleukin6 il6 and interferongamma ifnÎ further elevated levels of these cytokines trigger ards characterized by a low level of oxygen in the severity of symptoms and death in sarscov2 infected patients depends on the viral infection and is greatly affected by the aggressive behaviour of the host immune system blood and difficulty in breathing leading to the death of the infected patients previous data on sarscov demonstrated that the virus predominately affects the endothelium cells of preproofin counterdefence the virus encodes numerous immunesuppressive proteins that help employs the same host receptor angiotensinconverting enzyme ace2 for infection like sarscov indicating that both the viruses target the same set of cells for infection the as an antagonist of interferon signalling interruptions in interferon signalling happened at various stages preventing the recognition of viral rna through pattern recognition receptor expression of the ace2 receptor is reduced in the lungs following sarscov infection disrupting the reninangiotensin system that affects fluidelectrolyte balance blood pressure it to evade from host immune response and helps in replication similarly to counter such problem sarscov2 evolves with numerous structural and nonstructural proteins that act the airway alveoli vascular system and macrophages in the pulmonary an sarscov2 increases the vascular permeability and inflammation in the airway ppr inhibiting the synthesis of type i interferon protein via interrupting the tolllike receptor1 tlr1 and retinoic acidinducible gene i rigi signalling disturbing stat signalling and initiating the host mrna degradation and interrupting host translation machinery fig1 0cat the time of replication cytopathic viruses including sarscov2 show a massive death and injury of the infected epithelial and endothelial cells triggering the excessive release of cytokines and chemokines in addition to this inflammationinduced cell deathpyroptosis also observed in sarscov2 patients that further provoke the systemic inflammatory response pyroptosis signalling proceeds via nodlike receptors protein nlrp3 present on the cell membrane activate caspase1 through asc apoptosisassociated specklike protein containing a caspase recruitment domain adaptor protein activated caspase1 further triggers the synthesis of proinflammatory cytokines such as il1 and il6 fig1 these cytokines further attract the other immune cells mostly tlymphocytes and monocytes at the site of infection bronchoalveolar lavage balf fluid from the sever lymphocyte and immune cells' requirement at the site of infection in most of the patients these recruited cells clear the infection recedes the inflammatory response and leads to recovery however some patients show cytokine storms because of an imbalance in the population of monocytederived fcn1 macrophage in addition to these responses sever cases of sarscov2 infection also disclose a significant expansion in the population of proinflammatory monocytes cd14 and cd16 in the peripheral blood as compared to mild covid19 patients showed ccl2 and ccl7 chemokines which require the recruitment of ccr2 monocytes further balf analysis also revealed a highly inflammatory around of sarscov2 infected patients show lymphopeniainfiltration of preproofsevere hospitalized covid19 patients' blood plasma exhibits a higher level of alleviation in the t cell population which is more noticeable in severe cases the level of helper t cell cd4 cytotoxic t cell cd8 and regulatory t cell were below the average level in severe cases of covid19 as compared to mild cases cd8 t cells directly attack and kill the virusinfected cells while cd4 participates in the production of cytokines to recruit other immune cells at the same time regulatory t cell maintains the normal immune homeostasis along with inhibition of proliferation the proinflammatory activity of maximum immune cascade that further inflames the lungs sarscov2 infected patients also show cases lymphocytes natural killer cells and bcells fig1 granulocyte colonystimulating factor gcsf il2 il6 il10 monocyte chemoattractant peptide mcp1 macrophage inflammatory protein 1α mip1α and tnfα the blood plasma of the infected patients shows a significantly higher level of il6 in severe cases compared to mild or nonsevere cases which further contributes to macrophage activation syndrome pulmonary infiltrationbased assessment in ards patients also revealed that a 0cmore significant portion of lung injury is associated with a higher level of il6 in peripheral blood all of this evidences suggest that sarscov2 infection is responsible for dysregulation of the host immune system with the abnormal synthesis of cytokines chemokines and a decrease in the level of lymphocytes that ultimately leads to cytokine storm responsible of multian failure role of nuclear factorκb in disease progression nuclear factorκb nfκb is the leading player that responds immediately following the a pathogenic stimulus provoked by a bacteria or a virus invasion exposure of mitogen proinflammatory cytokines growth factors and stress activates ikb kinase ikk which relb and crel are grouped in firstclass characterized by the presence of transactivation domain while nfkb1 p50 and nfkb2 p52 belongs to the second group that is devoid of transcriptionalmodulation activity so both the classes of proteins need to be heterodimerized with each other to perform their functions under normal physiological conditions rela and p50 the heterodimer's predominant form is inactivated in the cytoplasm by ikb protein pathogen's invasion by promoting inflammation controlling cell proliferation and survival nfκb is a heterodimeric transcription factor that belongs to the rel protein family there are 05rel proteins present in mammalian cells that further divided into two classes rela p65 preproofmembranelike tolllike receptor tlr pathogen associated molecular pattern pamp and death associated molecular pattern damp are inflammatory stimulating molecules suggested that the nucleocapsid protein of sarscov directly interacts with nfκb translocate it to the nucleus and finally upregulates il6 gene expression ample of shreds of evidence is there that shows sarscov directly or indirectly activates nfκb protein excessive cytokine release especially il6 plays a crucial role in sarscov2 infection and further progression of pathogenic conditions nfκb is a transcription factor that controls the expression of proinflammatory genes responsible for the cytokine storm a study following infection nfκb also activated by receptors present on the cell surface further phosphorylates and degrades ikb protein via ubiquitination process released by virusinfected cells which act as ligands for tlr subsequently activating nfκb protein via myd88dependent pathway oxidative stress is another important factor responsible for cytokine storm generation via crosstalk between nuclear factor erythroid related factor nrf2 and nfκb pathway nfκb suggested as a negative regulator of nrf2 driven genes either by recruiting histone deacetylase hdac3 which promote local histone hypoacetylation or deprive the cbp creb binding protein fig1 0c sarscov2 infection and oxidative stress oxygen is a crucial molecule in the aerobic system to maintain normal life processes under normal cellular conditions the oxygen molecule utilized to generate chemical energy in the form of atp in a very tight and controlled manner the oxygen molecule combustion generates a small number of reactive oxygen species ros which utilized for usual cell signalling cascades ros are oxygen molecules with an unpaired electron that behaves as free radicals and reactive metabolites several ros forms were discovered so far such as peroxidase oxygenfree radicals nitrogen oxide and singlet oxygen molecules generally ros associated cellular damage is processed via sophisticated antioxidant machinery involving both enzymatic catalase cats superoxide dismutase sods and glutathione peroxidase gpx and nonenzymatic glutathione and nicotinamide adenine dinucleotide phosphate mitochondrial dna get degraded under the influence of oxidative stress subsequently hydrogen [nadph] mechanisms in normal physiological conditions the antioxidant systems can work simultaneously to combat the exceeded levels of ros however in a pathological state ros overwhelmed the antioxidant mechanism and generated œoxidative stress in cells all the crucial cellular components such as proteins lipids nuclear and the available literature of clinical and preclinical experiments proposed that oxidative preproofensures the clearance of the virus but due to imbalanced host immune system they also start to release excessive cytokines that further aggravate to cytokine storm the recruited phagocytic cell participates in ros generation along with inflammatory response nicotinamide adenine dinucleotide phosphate oxidases nadph oxidase and xanthine cov2 infection activates the host airway epithelium and alveolar macrophage further releasing cytokines to attract another immune cell from the blood neutrophils and monocyte that further differentiate into macrophage at the site of injury recruitment of these cells burst is another prompting factor for mortality following sarscov infection sarstriggering the process of cell death oxidase xo are the two wellknown enzymes responsible for oxidative stress in respiratory viral infections nadph oxidase nox is an evolutionary conserved membranebounded enzyme complex that catalyzes the molecular oxygen into superoxide human™s nadph oxidase family consists of members nox15 duox1 and duox2 its cterminal region comprises nadph binding site flavin adenine dinucleotide binding domain while the nterminal region consists of transmembrane α helical domains with four conserved hemebinding sites nox2 is predominantly expressed in the recruited 0cphagocytes neutrophils and macrophages at the viral infection site and contributes to oxidative stress a study reported that alveolar macrophage depended oxidative stress is responsible for acute lung injury progression following h5n1 viral infection in mice mostly via oxidized phospholipid and superoxide however the same pathological events reduced following the suppression of p47phox a regulatory subunit of nox2 in a study influenza a virusinfected nox2y mice showed reduced oxidative stress improved alveoli epithelium condition less production of superoxide and reduced airway inflammation compared to wild type mice fig inflammation xor is converted into xo by oxidation of cysteine amino acid or calciumin superoxide synthesis via nox2 enzyme complex xanthine oxidase xo is another dependent proteolysis xo shows more affinity toward molecular oxygen resulting in the transfer of a univalent and divalent electron to oxygen that further generates superoxide and ros generating enzyme that participates in oxidative stress following respiratory viral infection in the mammalian system this enzyme is existing in interchangeable form between hydrogen peroxide respectively fig2 in vitro rhino virus™s infection in primary bronchial and a549 respiratory epithelial cell lines decreased the intracellular glutathione xo to xanthine oxidoreductase xor xor is predominantly distributed in healthy tissues and reduces nad to nadh by utilizing electron form substrate while during similarly ex vivo influenza a virusinfected alveolar macrophage exhibited an increase preproofdecreased superoxide generation thus revealed that xo also participates in oxidative stress during infection in vivo analysis also revealed that xo is the main contributor to and serum analysis however allopurinol and chemical modified superoxide dismutase decreased the oxidative stress and mortality rate this evidences revealed that xo also superoxide synthesis during a respiratory viral infection mouse infected with influenza viral showed a higher mortality rate which found to be associated with xo and superoxide in balf gsh level leading to oxidative stress via enhanced superoxide production serine protease inhibitor or xo inhibitor oxypurinol treatment enhanced the intracellular levels of gsh and participates in the viral associated disease progression via oxidative stress a part of these activated phagocyte releases prooxidant mediators such as tnf and il1 which further enhances the oxidative stress in host cells during viral infection tnf binds with the complex ii of the mitochondrial respiratory chain hampering oxidative phosphorylation via restricting electrons transport as a result the electron transport chain becomes leakier and lastly it enhances superoxide production tnf also helps in detachment of nfκb protein from ikb complex resulting in suppression of antioxidant gene expression via binding to their 0c1keap1 binding domain keap1 is a cystine rich and cytoplasmic protein whose npromoter region following translocation from the cytoplasm to the nucleus fig during stress condition neutrophils also release lactoferrin along with lysosomal protein under the influence of il1 which further binds to iron and start to accumulate in the reticuloendothelial system when an ironbinding threshold reached superoxide ions combine with free iron to generate hydroxyl radicals via fenton reaction and enhances oxidative stress nrf2 a key regulator of antioxidant genes nrf2 is the main transcription factor that plays an important role to overcome oxidative stress it is a basic leucine zipper bzip protein that belongs to the cap ˜n™ collar family of transcription factors nrf2 consist of highly conserved functional domain termed as neh nrf2ech homologies “ neh1 is a leucine zipper domain through which nrf2 interact with other transcription factors whereas neh2 is the kelchlike ech associated protein however during stress conditions nrf2 detached from the keap1 protein translocate to the terminal domain binds with cul3dependent e3 ubiquitin ligase complex while cterminal domain binds with nrf2 protein under normal physiological conditions keap1 protein nucleus heterodimerize with small musculoaponeurotic fibrosarcoma mafs proteins and finally initiate or supress the transcription of genes that consists of electrophile response elements ere or antioxidant response elements are in their promoters nrf2 regulates preproofbecause of its highly vascular nature and indirect contact with environmental oxidant which had already proven in numerous of respiratory disease it was found that lungspecific nrf2 conditional knockout rodents showed pulmonary protective behaviour in respiratory disorders more than genes expression belonging to oxidative stress inflammation autophagy metabolism and excretion the pulmonary system is more exposed to oxidative stress ubiquitinates the nrf2 resulting in its proteasomal degradation infection systemic oxidative stress and inflammation linked thrombus formation in sarscovabnormal coagulation a higher level of ddimers and low platelet count are the signs of poor prognosis and significant reasons for multiple an failure and death in severe cases of covid19 microthrombus had reported in the lungs the heart the kidneys and the brain of covid19 patients cytokine storm induces aberrant coagulation by expressing the tissue factor tf pathway tf is a member of cytokine receptor 0csuperfamily and type i integral membrane glycoprotein which is highly abundant in the vasculature subendothelium especially in the brain lungs gut skin as well as in the monocytes in response to proinflammatory cytokines especially il6 the expression of tf is upregulated in the monocytes and the perivascular cells resulting in tf exposure to circulation the exposed portion of tf forms a complex with circulating factor vii thus enhance its catalytic activity that further activates downstream circulating factors such as ix and x activated factor x participates in the transformation of prothrombin into thrombin that finally leads to the formation of blood clots by conversion of fibrinogen into fibrin fig main consequences of the cytokine storm that also provokes thrombin production via proteasediphosphate adp thromboxane a2 translocate cell adhesion molecule pselectin and cd40 ligand on the surface of platelet along with activation of the integrin aiibb3 receptor the released thromboxane a2 and adp trigger activation of neighbouring platelets via activated receptors par signalling pathway par is a unique gprotein coupled cell surface receptor that carries its ligand and remains inactive until unmasked by proteolytic cleavage by the tffactorviia complex thrombin mediated par activated platelets undergo a morphological transformation release platelet activators such as serotonin adenosine thromboxane receptor and p2y12 respectively activated aiibb3 on platelets' surface binds with von willebrand factor vwf and fibrinogen that contributes to platelet aggregation platelet activation different proinflammatory events and fibrin clot formation are the preproofalong with cytokine storm oxidized phospholipids oxpls also participate in the recognition receptors in an experimental model of acute lung injury oxpls triggered cytokine storm release via tlr4triftraf6nfκb pathway il6 further promoted tf platelets during viral infection adherent leukocyteplatelets interaction provides positive feedback to amplify the overall inflammatory response and procoagulation events these activated endothelium cells following par signalling also exposes cell adhesion molecules eselectin pselectin icam1 and vcam1 and expresses monocyte chemo coagulation cascade via the tlr4 receptors present on the monocytes and endothelium cells attractant proteini that facilitates recruitment adhesion and migration of leukocytes and events are prothrombotic which further contributes to blood clot formation fig oxpls concerned as pams patterns that are recognized by numerous conserved patternexpression on monocytes and activated the endothelium cell to express monocyte adherent protein for their requirement which finally participated in inflammatory events thrombotic complications can be reduced in preexisting metabolic and cardiovascular disorders in covid19 patients by interfering with the oxpls activated monocyte or 0cwhich consists of domains including the nterminal domain ntddomain cterminal domain ctddomain and linker region lkrdomain nterminal domain enriched with endothelial cell additionally during inflammation the natural anticoagulant pathways such as tf pathway inhibitors or antithrombin are nearly diminished subsequently facilitating coagulation cascade gsk3 and sarscov2 infection the virus has to undergo many complex processes that are tightly regulated to infect a host cell it begins with viral genomic rna entry into the host cytosol transcription and finally budding off as viral progeny these viral progenies are similar to their parent in morphology and function that consists of structural proteins spike s protein envelop e protein matrix m protein and nucleocapsid n protein the n protein of severe acute respiratory syndrome coronavirus is the most abundant protein existing in an infected host cell among all be responsible for nuclear localization signal the cterminal domain of the n protein is also responsible for protein dimerization both the domains of n protein ie domain and protein sequencing also revealed that n protein is highly conserved among the species preproofinterestingly to perform such activity nprotein should recognize the viral genomic nucleocapsid protection of viral genome timely replication and proper transmission is the capsid's primary function nprotein also inhibits host cell proliferation and cytokines by rna associate with it and finally oligomerize by selfassociation to form capsid or terminal domain mapped between and amino acids is enriched with lysin thought to arginine motif responsible for the multimerization of the nprotein and predicted as a hot spot region for phosphorylation in brief nprotein divided into three main domains that play diverse functions during different stages of the virus life cycle nprotein is a type of capsid protein whose primary function is to pack the virus's genomic rna into the protective positive charge amino acids which is responsible for binding with viral rna whereas cother proteins covering domain are linked to each other through linker region domain that consists of serineinteracting with elongation factors 1α resulting in a halt of translation mechanism moreover the nprotein of sarscov also hijacks the host innate immune system for the progress of new viral progeny and associated disease development posttranslational phosphorylation of the virus nprotein is essential for their activity which results in an increased affinity of nprotein toward virus rna rather than nonviral rna gsk3 found 0cto be an essential kinase responsible for the phosphorylation of nprotein on the serine residue in linkerregion fig sarscov infected veroe6 cells showed an reduction in viral titer and cytopathic effects following the treatment of gsk3 inhibitor kenpaullone and lithium chloride thus suggested phosphorylation by this kinase be strongly linked with the viral replication several subgenomic mrnas synthesized due to discontinued transcription mechanisms during the coronavirus replication which encodes major structural proteins transcription regulating sequence trs responsible for the discontinuous transcription process exists in front of each gene body trs and after the leader sequence leader trs templateswitching events happen via base pairing between the body trs and leader trs to synthesize the discontinuous minusstranded rnas discontinuous nested plusstrand this discontinuous transcription mechanism tightly controlled for the successful compilation participate in discontinues to a continuous process of virus replication moreover of the virus life cycle among all the structural proteins nprotein tightly regulates the discontinued transcription mechanism as the synthesis of subgenomic mrna is reduced subgenomic mrna transcribed from the previously generated minusstranded rnas phosphorylation of nprotein at the serinearginine motif also inhibits the tra

cancer7524

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " pharmacology and toxicology laboratory csirinstitute of himalayan bioresource technology preproof 0c preproofinfection f0b7 systemic oxidative stress and inflammation are significant outcomes of sarscov2 highlights f0b7 activated gsk3 following sarscov2 infection provoke the oxidative stress and inflammation in the host f0b7 gsk3 phosphorylates nucleocapsid protein of sarscov2 and helps in disease progression f0b7 inhibition of gsk3 can be a suitable target in curbing of covid19 pandemic 0cwith the host defence mechanism by the help of gsk3 protein the virally infected cells show the coronavirus disease covid19 outbreak caused by severe acute respiratory syndrome coronavirus sarscov2 had turned out to be highly pathogenic and transmittable researchers throughout the globe are still struggling to understand this strain's aggressiveness in search of putative therapies for its control crosstalk between oxidative stress and systemic inflammation seems to support the progression of the infection glycogen synthase kinase3 gsk3 is a conserved serinethreonine kinase that mainly participates in cell proliferation development stress and inflammation in humans nucleocapsid protein of sarscov2 is an important structural protein responsible for viral replication and interferes activated gsk3 protein that degrades the nuclear factor erythroid 2related factor nrf2 protein resulting in excessive oxidative stress activated gsk3 also modulates crebdna activity phosphorylates nfκb and degrades catenin thus provokes systemic inflammation preproofinteraction between these two pathophysiological events oxidative stress and inflammation enhance mucous secretion coagulation cascade and hypoxia which ultimately leads to multiple ans failure resulting in the death of the infected patient the present review aims to highlight the pathogenic role of gsk3 in viral replication initiation of oxidative stress and inflammation during sarscov2 infection the review also summarizes the potential gsk3 pathway modulators as putative therapeutic interventions in combating the covid19 keywords covid19 gsk3 nfκb nucleocapsid protein oxidative stress sarscovpandemic list of abbreviations ace2 angiotensinconverting enzyme ad alzheimer™s disease adp adenosine diphosphate aiibb3 glycoprotein iibiiia ards acute respiratory distress syndrome 0care antioxidant response elements asc apoptosisassociated specklike protein containing a card atp adenosine triphosphate balf bronchoalveolar lavage bzip basic leucine zipper cats “ catalase cbp creb binding protein covid19 coronavirus disease creb camp response elementbinding protein cul3 cullin gpx glutathione peroxidase gsh intracellular glutathione gsk3 glycogen synthase kinase3 damp death associated molecular pattern gcsf granulocyte colony stimulating factor hcv hepatitis c virus hdac3 histone deacetylase ho1 heme oxygenase1 ifnÎ interferongamma preproofnfκb nuclear factorκb nlrp3 nodlike receptors protein mcp1 monocyte chemoattractant peptide mip1α macrophage inflammatory protein 1α myd88 myeloid differentiation primary response nadph nicotinamide adenine dinucleotide phosphate hydrogen ikk ikb kinase il6 interleukin iraks interleukin il 1rassociated kinase iκb inhibitor of kappa b keap1 kelchlike ech associated protein licl lithium chloride nlrp3 nucleotidebinding domain nodlike receptor protein nox nadph oxidase nprotein nucleocapsid protein nrf2 nuclear factor erythroid 2related factor 0cntd nterminal domain o superoxide anion o2 oxygen molecule oxpls oxidized phospholipids pamp pathogen associated molecular pattern par proteaseactivated receptors pd parkinson™s disease pedv porcine epidemic diarrhea virus ros reactive oxygen species sarscov2 severe acute respiratory syndrome coronavirus tak1 transforming growth factor tgfactivated kinase tf tissue factor tirap tirdomaincontaining adaptor protein sgmrna sub genomic messenger rna sods superoxide dismutase ppr pattern recognition receptor psgl pselectin glycoprotein ligand1 rigi retinoic acidinducible gene i preproofvwf von willebrand factor xo xanthine oxidase xor xanthine oxidoreductase tlr3 toll like receptor3 tnf tumor necrosis factor tnfr tumor necrosis factor receptor tnfα tumour necrosis factoralpha traf6 tumour necrosis factor receptor associated factor trs transcription regulating sequence introduction in late december wuhan china got attention worldwide after getting several patients diagnosed with pneumonia following a viral infection on 11th february the pathogenic strain of the virus was taxonomically designated as severe respiratory syndrome coronavirus sarscov2 by the international committee on taxonomy of viruses ictv the 0cassociated diseased condition was termed covid19 by the world health anization who the who announced sarscov2 virus infection a pandemic as it infected nearly million persons and engulfed more than worldwide sarscov2 is a member of coronaviruses consisting of kb singlestranded positivesense rna as genetic material it shows genetic similarity between another human coronavirus ie sarscov while similarity with bat coronavirus ratg1 and shares a high similarity index with pangolin coronavirus respiratory droplets are the primary source of viral transmission either through nasopharyngeal or oral route dry cough and high fever are the sarscov virusassociated respiratory disease replication within the host cell in disease progression the present review provides an inthe infected cells however in the case of sarscov2 infection aggressive inflammation significant symptoms observed in patients within days following viral infection the disease pathophysiology of covid19 also shows a close resemblance with previous reported and oxidative stress help in viral replication and damage the airway epithelium cell that results in acute respiratory distress syndrome ards which makes the condition worst glycogen synthase kinase3 gsk3 is a serinethreonine evolutionary conserved central molecule that the majority of respiratory viral infections are associated with the recruitment of immune cells the release of proinflammatory cytokines oxidative stress and finally phagocytosis of mainly participates in cell proliferation migration development apoptosis and immune regulation acquired and innate activation of gsk3 is associated with suppression of host immunity and inhibition of antioxidant response it is also supporting viral genome preproofdepth knowledge of oxidative stress inflammation and viral replication related to gsk3 during sarscov2 infection further the review highlights the gsk3 pathway modulators' gsk3 is a versatile serinethreonine kinase that regulates glycogen metabolism it consists of two isoforms gsk3α and gsk3 encoded by two separate genes both the isoforms share sequence similarity between kinase domains despite they never compensate for each other's' loss of function gsk3 has two prime functional domains a substratebinding domain which acquires substrates to gsk3 while the other kinase domain is responsible for phosphorylation of the substrate the nterminal region of gsk3 contains atp binding domain whereas the cterminal region consists of a large conserved activation loop responsible for the enzyme's full activation activation of gsk3 depends on the siteputative role as therapeutic interventions in combating the covid19 pandemic gsk3 structure 0cspecific phosphorylation that is controlled by various kinases gsk3 prefers prephosphorylate substrate by recognizing consequence sequences stxxxphosphost on substrate gsk3 is also involved in wntcatenin and sonic hedgehog cell signalling pathways mediating in cell proliferation differentiation maturation and cell adhesion transcription factors cjun creb stat3 cebpα nfat myc nfκb and p53 are the major substrate of gsk3 that can manipulate the expression of several other genes impaired activity of gsk3 has recognized in several clinical conditions such as metabolic disorders cancers alzheimer's disease ad parkinson's disease pd bipolar disorders and various other neurodegenerative diseases sarscov2 infection and inflammation covid19 patients' systemic cytokine profile shows a close resemblance with cytokine release syndrome characterized by macrophage activation an elevated level of cytokines like tumour necrosis factoralpha tnfα interleukin6 il6 and interferongamma ifnÎ further elevated levels of these cytokines trigger ards characterized by a low level of oxygen in the severity of symptoms and death in sarscov2 infected patients depends on the viral infection and is greatly affected by the aggressive behaviour of the host immune system blood and difficulty in breathing leading to the death of the infected patients previous data on sarscov demonstrated that the virus predominately affects the endothelium cells of preproofin counterdefence the virus encodes numerous immunesuppressive proteins that help employs the same host receptor angiotensinconverting enzyme ace2 for infection like sarscov indicating that both the viruses target the same set of cells for infection the as an antagonist of interferon signalling interruptions in interferon signalling happened at various stages preventing the recognition of viral rna through pattern recognition receptor expression of the ace2 receptor is reduced in the lungs following sarscov infection disrupting the reninangiotensin system that affects fluidelectrolyte balance blood pressure it to evade from host immune response and helps in replication similarly to counter such problem sarscov2 evolves with numerous structural and nonstructural proteins that act the airway alveoli vascular system and macrophages in the pulmonary an sarscov2 increases the vascular permeability and inflammation in the airway ppr inhibiting the synthesis of type i interferon protein via interrupting the tolllike receptor1 tlr1 and retinoic acidinducible gene i rigi signalling disturbing stat signalling and initiating the host mrna degradation and interrupting host translation machinery fig1 0cat the time of replication cytopathic viruses including sarscov2 show a massive death and injury of the infected epithelial and endothelial cells triggering the excessive release of cytokines and chemokines in addition to this inflammationinduced cell deathpyroptosis also observed in sarscov2 patients that further provoke the systemic inflammatory response pyroptosis signalling proceeds via nodlike receptors protein nlrp3 present on the cell membrane activate caspase1 through asc apoptosisassociated specklike protein containing a caspase recruitment domain adaptor protein activated caspase1 further triggers the synthesis of proinflammatory cytokines such as il1 and il6 fig1 these cytokines further attract the other immune cells mostly tlymphocytes and monocytes at the site of infection bronchoalveolar lavage balf fluid from the sever lymphocyte and immune cells' requirement at the site of infection in most of the patients these recruited cells clear the infection recedes the inflammatory response and leads to recovery however some patients show cytokine storms because of an imbalance in the population of monocytederived fcn1 macrophage in addition to these responses sever cases of sarscov2 infection also disclose a significant expansion in the population of proinflammatory monocytes cd14 and cd16 in the peripheral blood as compared to mild covid19 patients showed ccl2 and ccl7 chemokines which require the recruitment of ccr2 monocytes further balf analysis also revealed a highly inflammatory around of sarscov2 infected patients show lymphopeniainfiltration of preproofsevere hospitalized covid19 patients' blood plasma exhibits a higher level of alleviation in the t cell population which is more noticeable in severe cases the level of helper t cell cd4 cytotoxic t cell cd8 and regulatory t cell were below the average level in severe cases of covid19 as compared to mild cases cd8 t cells directly attack and kill the virusinfected cells while cd4 participates in the production of cytokines to recruit other immune cells at the same time regulatory t cell maintains the normal immune homeostasis along with inhibition of proliferation the proinflammatory activity of maximum immune cascade that further inflames the lungs sarscov2 infected patients also show cases lymphocytes natural killer cells and bcells fig1 granulocyte colonystimulating factor gcsf il2 il6 il10 monocyte chemoattractant peptide mcp1 macrophage inflammatory protein 1α mip1α and tnfα the blood plasma of the infected patients shows a significantly higher level of il6 in severe cases compared to mild or nonsevere cases which further contributes to macrophage activation syndrome pulmonary infiltrationbased assessment in ards patients also revealed that a 0cmore significant portion of lung injury is associated with a higher level of il6 in peripheral blood all of this evidences suggest that sarscov2 infection is responsible for dysregulation of the host immune system with the abnormal synthesis of cytokines chemokines and a decrease in the level of lymphocytes that ultimately leads to cytokine storm responsible of multian failure role of nuclear factorκb in disease progression nuclear factorκb nfκb is the leading player that responds immediately following the a pathogenic stimulus provoked by a bacteria or a virus invasion exposure of mitogen proinflammatory cytokines growth factors and stress activates ikb kinase ikk which relb and crel are grouped in firstclass characterized by the presence of transactivation domain while nfkb1 p50 and nfkb2 p52 belongs to the second group that is devoid of transcriptionalmodulation activity so both the classes of proteins need to be heterodimerized with each other to perform their functions under normal physiological conditions rela and p50 the heterodimer's predominant form is inactivated in the cytoplasm by ikb protein pathogen's invasion by promoting inflammation controlling cell proliferation and survival nfκb is a heterodimeric transcription factor that belongs to the rel protein family there are 05rel proteins present in mammalian cells that further divided into two classes rela p65 preproofmembranelike tolllike receptor tlr pathogen associated molecular pattern pamp and death associated molecular pattern damp are inflammatory stimulating molecules suggested that the nucleocapsid protein of sarscov directly interacts with nfκb translocate it to the nucleus and finally upregulates il6 gene expression ample of shreds of evidence is there that shows sarscov directly or indirectly activates nfκb protein excessive cytokine release especially il6 plays a crucial role in sarscov2 infection and further progression of pathogenic conditions nfκb is a transcription factor that controls the expression of proinflammatory genes responsible for the cytokine storm a study following infection nfκb also activated by receptors present on the cell surface further phosphorylates and degrades ikb protein via ubiquitination process released by virusinfected cells which act as ligands for tlr subsequently activating nfκb protein via myd88dependent pathway oxidative stress is another important factor responsible for cytokine storm generation via crosstalk between nuclear factor erythroid related factor nrf2 and nfκb pathway nfκb suggested as a negative regulator of nrf2 driven genes either by recruiting histone deacetylase hdac3 which promote local histone hypoacetylation or deprive the cbp creb binding protein fig1 0c sarscov2 infection and oxidative stress oxygen is a crucial molecule in the aerobic system to maintain normal life processes under normal cellular conditions the oxygen molecule utilized to generate chemical energy in the form of atp in a very tight and controlled manner the oxygen molecule combustion generates a small number of reactive oxygen species ros which utilized for usual cell signalling cascades ros are oxygen molecules with an unpaired electron that behaves as free radicals and reactive metabolites several ros forms were discovered so far such as peroxidase oxygenfree radicals nitrogen oxide and singlet oxygen molecules generally ros associated cellular damage is processed via sophisticated antioxidant machinery involving both enzymatic catalase cats superoxide dismutase sods and glutathione peroxidase gpx and nonenzymatic glutathione and nicotinamide adenine dinucleotide phosphate mitochondrial dna get degraded under the influence of oxidative stress subsequently hydrogen [nadph] mechanisms in normal physiological conditions the antioxidant systems can work simultaneously to combat the exceeded levels of ros however in a pathological state ros overwhelmed the antioxidant mechanism and generated œoxidative stress in cells all the crucial cellular components such as proteins lipids nuclear and the available literature of clinical and preclinical experiments proposed that oxidative preproofensures the clearance of the virus but due to imbalanced host immune system they also start to release excessive cytokines that further aggravate to cytokine storm the recruited phagocytic cell participates in ros generation along with inflammatory response nicotinamide adenine dinucleotide phosphate oxidases nadph oxidase and xanthine cov2 infection activates the host airway epithelium and alveolar macrophage further releasing cytokines to attract another immune cell from the blood neutrophils and monocyte that further differentiate into macrophage at the site of injury recruitment of these cells burst is another prompting factor for mortality following sarscov infection sarstriggering the process of cell death oxidase xo are the two wellknown enzymes responsible for oxidative stress in respiratory viral infections nadph oxidase nox is an evolutionary conserved membranebounded enzyme complex that catalyzes the molecular oxygen into superoxide human™s nadph oxidase family consists of members nox15 duox1 and duox2 its cterminal region comprises nadph binding site flavin adenine dinucleotide binding domain while the nterminal region consists of transmembrane α helical domains with four conserved hemebinding sites nox2 is predominantly expressed in the recruited 0cphagocytes neutrophils and macrophages at the viral infection site and contributes to oxidative stress a study reported that alveolar macrophage depended oxidative stress is responsible for acute lung injury progression following h5n1 viral infection in mice mostly via oxidized phospholipid and superoxide however the same pathological events reduced following the suppression of p47phox a regulatory subunit of nox2 in a study influenza a virusinfected nox2y mice showed reduced oxidative stress improved alveoli epithelium condition less production of superoxide and reduced airway inflammation compared to wild type mice fig inflammation xor is converted into xo by oxidation of cysteine amino acid or calciumin superoxide synthesis via nox2 enzyme complex xanthine oxidase xo is another dependent proteolysis xo shows more affinity toward molecular oxygen resulting in the transfer of a univalent and divalent electron to oxygen that further generates superoxide and ros generating enzyme that participates in oxidative stress following respiratory viral infection in the mammalian system this enzyme is existing in interchangeable form between hydrogen peroxide respectively fig2 in vitro rhino virus™s infection in primary bronchial and a549 respiratory epithelial cell lines decreased the intracellular glutathione xo to xanthine oxidoreductase xor xor is predominantly distributed in healthy tissues and reduces nad to nadh by utilizing electron form substrate while during similarly ex vivo influenza a virusinfected alveolar macrophage exhibited an increase preproofdecreased superoxide generation thus revealed that xo also participates in oxidative stress during infection in vivo analysis also revealed that xo is the main contributor to and serum analysis however allopurinol and chemical modified superoxide dismutase decreased the oxidative stress and mortality rate this evidences revealed that xo also superoxide synthesis during a respiratory viral infection mouse infected with influenza viral showed a higher mortality rate which found to be associated with xo and superoxide in balf gsh level leading to oxidative stress via enhanced superoxide production serine protease inhibitor or xo inhibitor oxypurinol treatment enhanced the intracellular levels of gsh and participates in the viral associated disease progression via oxidative stress a part of these activated phagocyte releases prooxidant mediators such as tnf and il1 which further enhances the oxidative stress in host cells during viral infection tnf binds with the complex ii of the mitochondrial respiratory chain hampering oxidative phosphorylation via restricting electrons transport as a result the electron transport chain becomes leakier and lastly it enhances superoxide production tnf also helps in detachment of nfκb protein from ikb complex resulting in suppression of antioxidant gene expression via binding to their 0c1keap1 binding domain keap1 is a cystine rich and cytoplasmic protein whose npromoter region following translocation from the cytoplasm to the nucleus fig during stress condition neutrophils also release lactoferrin along with lysosomal protein under the influence of il1 which further binds to iron and start to accumulate in the reticuloendothelial system when an ironbinding threshold reached superoxide ions combine with free iron to generate hydroxyl radicals via fenton reaction and enhances oxidative stress nrf2 a key regulator of antioxidant genes nrf2 is the main transcription factor that plays an important role to overcome oxidative stress it is a basic leucine zipper bzip protein that belongs to the cap ˜n™ collar family of transcription factors nrf2 consist of highly conserved functional domain termed as neh nrf2ech homologies “ neh1 is a leucine zipper domain through which nrf2 interact with other transcription factors whereas neh2 is the kelchlike ech associated protein however during stress conditions nrf2 detached from the keap1 protein translocate to the terminal domain binds with cul3dependent e3 ubiquitin ligase complex while cterminal domain binds with nrf2 protein under normal physiological conditions keap1 protein nucleus heterodimerize with small musculoaponeurotic fibrosarcoma mafs proteins and finally initiate or supress the transcription of genes that consists of electrophile response elements ere or antioxidant response elements are in their promoters nrf2 regulates preproofbecause of its highly vascular nature and indirect contact with environmental oxidant which had already proven in numerous of respiratory disease it was found that lungspecific nrf2 conditional knockout rodents showed pulmonary protective behaviour in respiratory disorders more than genes expression belonging to oxidative stress inflammation autophagy metabolism and excretion the pulmonary system is more exposed to oxidative stress ubiquitinates the nrf2 resulting in its proteasomal degradation infection systemic oxidative stress and inflammation linked thrombus formation in sarscovabnormal coagulation a higher level of ddimers and low platelet count are the signs of poor prognosis and significant reasons for multiple an failure and death in severe cases of covid19 microthrombus had reported in the lungs the heart the kidneys and the brain of covid19 patients cytokine storm induces aberrant coagulation by expressing the tissue factor tf pathway tf is a member of cytokine receptor 0csuperfamily and type i integral membrane glycoprotein which is highly abundant in the vasculature subendothelium especially in the brain lungs gut skin as well as in the monocytes in response to proinflammatory cytokines especially il6 the expression of tf is upregulated in the monocytes and the perivascular cells resulting in tf exposure to circulation the exposed portion of tf forms a complex with circulating factor vii thus enhance its catalytic activity that further activates downstream circulating factors such as ix and x activated factor x participates in the transformation of prothrombin into thrombin that finally leads to the formation of blood clots by conversion of fibrinogen into fibrin fig main consequences of the cytokine storm that also provokes thrombin production via proteasediphosphate adp thromboxane a2 translocate cell adhesion molecule pselectin and cd40 ligand on the surface of platelet along with activation of the integrin aiibb3 receptor the released thromboxane a2 and adp trigger activation of neighbouring platelets via activated receptors par signalling pathway par is a unique gprotein coupled cell surface receptor that carries its ligand and remains inactive until unmasked by proteolytic cleavage by the tffactorviia complex thrombin mediated par activated platelets undergo a morphological transformation release platelet activators such as serotonin adenosine thromboxane receptor and p2y12 respectively activated aiibb3 on platelets' surface binds with von willebrand factor vwf and fibrinogen that contributes to platelet aggregation platelet activation different proinflammatory events and fibrin clot formation are the preproofalong with cytokine storm oxidized phospholipids oxpls also participate in the recognition receptors in an experimental model of acute lung injury oxpls triggered cytokine storm release via tlr4triftraf6nfκb pathway il6 further promoted tf platelets during viral infection adherent leukocyteplatelets interaction provides positive feedback to amplify the overall inflammatory response and procoagulation events these activated endothelium cells following par signalling also exposes cell adhesion molecules eselectin pselectin icam1 and vcam1 and expresses monocyte chemo coagulation cascade via the tlr4 receptors present on the monocytes and endothelium cells attractant proteini that facilitates recruitment adhesion and migration of leukocytes and events are prothrombotic which further contributes to blood clot formation fig oxpls concerned as pams patterns that are recognized by numerous conserved patternexpression on monocytes and activated the endothelium cell to express monocyte adherent protein for their requirement which finally participated in inflammatory events thrombotic complications can be reduced in preexisting metabolic and cardiovascular disorders in covid19 patients by interfering with the oxpls activated monocyte or 0cwhich consists of domains including the nterminal domain ntddomain cterminal domain ctddomain and linker region lkrdomain nterminal domain enriched with endothelial cell additionally during inflammation the natural anticoagulant pathways such as tf pathway inhibitors or antithrombin are nearly diminished subsequently facilitating coagulation cascade gsk3 and sarscov2 infection the virus has to undergo many complex processes that are tightly regulated to infect a host cell it begins with viral genomic rna entry into the host cytosol transcription and finally budding off as viral progeny these viral progenies are similar to their parent in morphology and function that consists of structural proteins spike s protein envelop e protein matrix m protein and nucleocapsid n protein the n protein of severe acute respiratory syndrome coronavirus is the most abundant protein existing in an infected host cell among all be responsible for nuclear localization signal the cterminal domain of the n protein is also responsible for protein dimerization both the domains of n protein ie domain and protein sequencing also revealed that n protein is highly conserved among the species preproofinterestingly to perform such activity nprotein should recognize the viral genomic nucleocapsid protection of viral genome timely replication and proper transmission is the capsid's primary function nprotein also inhibits host cell proliferation and cytokines by rna associate with it and finally oligomerize by selfassociation to form capsid or terminal domain mapped between and amino acids is enriched with lysin thought to arginine motif responsible for the multimerization of the nprotein and predicted as a hot spot region for phosphorylation in brief nprotein divided into three main domains that play diverse functions during different stages of the virus life cycle nprotein is a type of capsid protein whose primary function is to pack the virus's genomic rna into the protective positive charge amino acids which is responsible for binding with viral rna whereas cother proteins covering domain are linked to each other through linker region domain that consists of serineinteracting with elongation factors 1α resulting in a halt of translation mechanism moreover the nprotein of sarscov also hijacks the host innate immune system for the progress of new viral progeny and associated disease development posttranslational phosphorylation of the virus nprotein is essential for their activity which results in an increased affinity of nprotein toward virus rna rather than nonviral rna gsk3 found 0cto be an essential kinase responsible for the phosphorylation of nprotein on the serine residue in linkerregion fig sarscov infected veroe6 cells showed an reduction in viral titer and cytopathic effects following the treatment of gsk3 inhibitor kenpaullone and lithium chloride thus suggested phosphorylation by this kinase be strongly linked with the viral replication several subgenomic mrnas synthesized due to discontinued transcription mechanisms during the coronavirus replication which encodes major structural proteins transcription regulating sequence trs responsible for the discontinuous transcription process exists in front of each gene body trs and after the leader sequence leader trs templateswitching events happen via base pairing between the body trs and leader trs to synthesize the discontinuous minusstranded rnas discontinuous nested plusstrand this discontinuous transcription mechanism tightly controlled for the successful compilation participate in discontinues to a continuous process of virus replication moreover of the virus life cycle among all the structural proteins nprotein tightly regulates the discontinued transcription mechanism as the synthesis of subgenomic mrna is reduced subgenomic mrna transcribed from the previously generated minusstranded rnas phosphorylation of nprotein at the serinearginine motif also inhibits the tra Answer:

Colon_Cancer

"‚ammatory bowel disease ibd is a long life disease with remission and relapse periods ibd arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ‚ammation andintestinal ulcers in addition ibd has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [“] ulcerative colitisuc and crohn's diseases cd are known as two main forms of ibdaccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized in this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way for example 5asa which is acommon drug in the treatment of ibd is less eï¬ective on maintainingremission in cd patients on the other hand antibiotic therapy is notrecommended for the treatment uc but it can be eï¬ective on cd patients diï¬erential diagnosis is a serious challenge because cdand uc have significant similarities in terms of their clinical endoscopic and histological features however there are some diï¬erencesbetween uc and cd which are summarized in table1 in addition tointestinal complications uc and cd also have significant extraintestinal manifestations for example it was shown that uc is significantly associated with primary sclerosing cholangitis and cd is alsoassociated with cholelithiasis especially in cases that the ileum is involved furthermore cd can cause fistulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections both cd and uc can cause several disorderssuch as arthritis erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of ibd the latest statistics showed that the globalŽ corresponding author at department of clinical biochemistry and laboratory medicine faculty of medicine tabriz university of medical sciences daneshgahstreet po box tabriz iranemail address vagharimtbzmedacir m vagharitabari101016jcca202008025received july received in revised form august accepted august available online august elsevier bv all rights reserved 0cf khakikhatibi table1clinical endoscopic and histological features of cd and ucclinical featuresfeaturesrectal bleedingabdominal painfevermucus defectionintestinal obstructionperineal diseasepostoperative recurrenceasca positiveanca positiveendoscopic featurescdoccasionallyfrequentlyfrequentlyoccasionallyyesyesyesfrequentlynot commonucfrequentlyoccasionallynot commonfrequentlynonononot commonfrequentlyfeaturescduclocationmucosal involvementdepth of ulcerationfistulacobblestone appearanceaphthous ulcerationmucosal friabilityhistological featuresfeaturesgranulomascrypt abscessespatchinessany part of gi tractdiscontinuousdeepyesyesfrequentlynot commoncdfrequentlynot commonfrequentlycolon and rectumcontinuoussuperficialnonooccasionallyfrequentlyucrarefrequentlynot commonprevalence of ibd currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem according to areport published in ibd has the highest prevalence rate ineurope and its prevalence in the newly industrialized countries of asiaafrica and south america also appears to be increased over the pastthree decades unfortunately the peak of the disease is at the young age of“ years old therefore in addition to the suï¬ering from ‚icts on the patients it also has many negative eï¬ects on societymoreover many financial burdens are annually imposed on countriesfor controlling and treating this chronic disease the invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage ibd which are unpleasant for patients as well as having thehigh associated costs now the gold standard method for diagnosingibd and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures therefore in recent years many studies have been conducted tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofibd a high proportion of these studies have investigated the efficacy offecal calprotectin in diagnosing and monitoring patients althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring ibd patients so in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of ibd the role of fecal calprotectin in diagnosis and management ofibdthe efficacy of fecal calprotectin as an laboratory marker in various areas of ibd diagnosis and management have been studied including ibd diï¬erentiation from irritable bowel syndrome ibs evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andclinica chimica acta “response to treatment in following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting findings in all ofthe abovementioned areas calprotectin a clinically valuable proteincalprotectin is an antimicrobial protein mainly secreted by neutrophils this protein competes with bacteria over zinc thus kills thebacteria however this is not the only contribution that it has to antimicrobial activity moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsserum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection at theearly stages of ‚ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reflecting disease activity in ‚ammation of thejoints in addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker in neonatal sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspecificity of that have been reported for serum calprotectin indiagnosis of neonatal sepsis it has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the first of onset areassociated with a poor prognosis at the first three months serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low crp levels so they appear to be more efficient at reflecting disease activity some studies have also investigated the efficacy of serum calprotectin in the diagnosis of cancers correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation alsoregarding the efficacy of serum and saliva calprotectin for the diagnosisof ibd impressive results have been reported a study onpatients with ibd both uc and cd have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in ibd diagnosis compared to crp and albumin this studyalso indicated that the combination of serum calprotectin with crp oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with cd however no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with cd and uc as well as a slight correlation betweenserum calprotectin level and crp that was observed only in patientswith uc another study showed that the serum level of calprotectin was significantly higher in patients with cd compared to healthyindividuals in addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease the efficacy of salivary calprotectin in the diagnosisof ibd has also been studied which showed that salivary calprotectinsignificantly increased in patients with ibd compared to healthy individuals in this study auc values for unstimulated saliva and stimulated saliva to distinguish ibd patients from healthy individualswere reported to be and respectively however thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of ibd that is discussed in the following 0cf khakikhatibi clinica chimica acta “ laboratory measurement and reference intervalfecal calprotectin is a stable protein that remains stable for “ daysat room temperature this property is an excellent advantage for alaboratory marker also it seems that keeping the specimen at refrigerated temperature °c can increase the stability of fecal calprotectin however evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature on the other hand it is not also recommended tokeep samples in the refrigerator for more than days it seemsthat fecal calprotectin remains stable up to one year at ˆ’ °c measurement of fecal calprotectin can be done both qualitatively andquantitatively accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette however in the qualitative one only positive or negative results are reported and despite of sensitivity test specificity in theevaluation of disease activity was reported to be only it seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom ibd patients rapidly however some studies have shown that it isnot accurate enough in this case as well nevertheless asignificant concordance has been reported between home test resultsibdoc and fecal calprotectin laboratory measurement results whenquantum blue calprotectin elisa kit was used notably the agreements between results were and depending on the selectedcutoï¬s several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin these tests reportpositive results ranged from to µgg there are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin these kits are usually designed in terms of the elisamethod and some have a measurement range between and µgg moreover the chemiluminescence immunoassays cliamethod can also detect values between and µgg fluoro enzyme immunoassays feia and particle enhanced turbidimetric immunoassays petia can also be used for the measurement of fecalcalprotectin in this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals among healthy adults there is asignificant agreement on µgg as an upper limit one study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric ibd table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies according to these reports age can aï¬ect fecal calprotectinlevels fecal calprotectin and ibd diagnosisonly a small percentage of patients complaining of abdominal painand diarrhea have ibd in many cases ibs as a functional gastrointestinal disorder is known as the cause of such clinical symptomspatients with ibs have normal colonoscopy results while ibd patientsindicate abnormal colonoscopy results and have intestinal ulcersunfortunately the significant prevalence of ibs and the overlap between clinical symptoms and ibd can increase the colonoscopy ratetherefore a noninvasive diagnostic marker can be very helpful in thisregard notably the first evidence of the efficacy of fecal calprotectin inthe diagnosis of ibd was obtained in the 1990s røseth in proposed a method for measuring calprotectin in stool specimens one of the first and most interesting studies regarding fecal calprotectinutility in ibd diagnosis was the study by røseth published in in this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls this study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals subsequent studies somehow confirmed and complemented the findings of this study in another study published in auc values of ci “ were reported for fecal calprotectin in thediagnosis of colorectal ‚ammation moreover in a study onchildren with ibd it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with esr levels in astudy published in kolho reported auc values of ci “ for fecal calprotectin in the diagnosis of pediatric ibd in a study on patients with crohn disease a sensitivity of and a specificity of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease the results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ‚ammatory biomarkers such as crp andesr in the diagnosis of ibd diamanti reported a sensitivity of and a specificity of for fecal calprotectin at a cutoï¬ of μgg in ibd diagnosis in our recent study a sensitivityof and a specificity of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of ibd however oursample size was and the majority of patients were in the active phaseof the disease in another study conducted on patients with ulcerative colitis asensitivity of and a specificity of at cutoï¬ of μgg havebeen reported in this regard in one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withibd from patients without ibd patients with diseases other than ibdpatients with ibs and healthy persons with sensitivity and specificity caviglia in their study reported a sensitivity of and a specificity of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between ibs and ibd howeversome studies have reported significantly lower values accordingly in astudy on patients with ulcerative colitis kalantari reported asensitivity of and a specificity of at a cutoï¬ of μgg besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with crohn's disease asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting ce findings anddiagnosis of crohn's disease in another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity oftable reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesagesmedian levels of fecal calprotectin range µggnumber of subjectsused kitup to monthchildren “ yearschildren “ yearsadultsover years “ “ “ “ “bühlmann elisabühlmann elisacalpro® calprotectin elisa test alpphicalphicalreference 0cf khakikhatibi were reported for this biomarker at μgg in the diagnosis ofsmall bowel ‚ammation in crohn's disease given these findings it seems that fecal calprotectin has no ideal sensitivity and specificity for the diagnosis of ibd where the small intestine is involvedbesides there are some preanalytical limitations which are explainedin the next sections therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy howeverin a metaanalysis performed to evaluate the efficacy of fecal calprotectin and some other ‚ammatory markers to diï¬erentiate betweenibd and ibs the probability of ibd was less than at fecal calprotectin values lower than µgg or crp values lower than mgdl therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of ibd in patients with ibslike symptoms aswell as reducing the rate of colonoscopy moreover it should be notedthat although a systematic review has reported pooled sensitivity andspecificity above for fecal calprotectin to diï¬erentiate between ibdand ibs it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points hence performing extensive studies indiï¬erent countries on the healthy population and the ibd patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspecificity and minimum falsepositive resultstable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of ibdfrom ibs and table4 summarizes some metaanalysis results in thisregard as shown in table the most important limitation of the majority of clinical studies conducted to date is the small sample size alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between ibdand nonibd diseases fecal calprotectin and endoscopic and histologic activity evaluationundoubtedly one of the most serious challenges in the managementof ibd is evaluating the endoscopic and histologic activities of thedisease nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withibd as noted earlier several scoring systems have been devised toscore disease activity based on the findings of colonoscopy and histopathologic examinations in recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels in addition many studies have been performed inthe last decade all of which cannot be reviewed in this article the firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s in one of the first studiesroseth found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis furthermore in another study they observed that ibdpatients who were in remission clinically and had normal fecal calprotectin levels less than mgl had normal colonoscopy results these interesting findings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andclinica chimica acta “table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdsample sizepooled sensitivitypooled specificityreferences mucosal healing in ibd patients also these studies were the startingpoint of extensive studies that have been conducted up to now in astudy conducted on patients with crohn's disease sipponen investigated the sensitivity and specificity of fecal calprotectin in predicting endoscopic activity of crohn's disease correspondinglythe researchers used the crohn's disease endoscopic index of severitycdeis scoring system in their study to evaluate the endoscopic activity of crohn's disease as a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin besides the findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of crohn's disease with sensitivity and specificity in another study cdeis and mayo disease activity indexmdai were used to evaluate the endoscopic activity of crohn's diseaseand ulcerative colitis respectively according to the results of thatstudy on ibd patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to therachmilewitz clinical activity index in addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentification was obtained as some studies have also shown the superiority of fecal calprotectinover traditional ‚ammatory markers like crp besides one studyfound that fecal calprotectin was more strongly correlated with thesimple endoscopic score for crohn's disease sescd compared to thecrp and even crohn's disease activity index cdai the modifiedbaron index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis as a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to crp and clinical activity of the disease in thisregard similar results were also observed in our recent study in whichthe ulcerative colitis endoscopic index of severity uceis and sescdwere used therefore fecal calprotectin appears to be superior totraditional ‚ammatory markers in the prediction of ibd endoscopicactivity the high values of sensitivity and specificity that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients™ monitoring however severalrecent studies have reported some significantly lower values accordingly in a recent study in which mayo endoscopic score [mes] wasused to evaluate the endoscopic activity of ulcerative colitis atable summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdnumber of ibd patientsage grouplocationcut oï¬sensitivityspecificity cd and uc cd and uc cd and uc and unclassified68cd and uc cd and uc and unclassified cd and uc cd and uc uc cd ucadultsadultsadultsboth adult and pediatricpediatricadultspediatricadultsadultsboth adult and pediatrictaiwanchinaitalyspainfinlandiranitalyirandenmarkindia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggaucreferences spsrefidbib60 0cf khakikhatibi clinica chimica acta “table summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in ibd patientsage groupstudylocationusedendoscopicactivity indexcorrelationcoefficientrreferencenumberof ibdpatients cd uc uc cd ucadultsadultsadultsadultsadultsfinlandiranswitzerlandswitzerlandswitzerland modifiedcdeisuceisrachmilewitzsescd uc cdadultsadults uc cd uc cd cd uc ucadultsadultsadultsadultsadultsadultsadultsbaron scorerachmilewitzsescdgermanyusa andcanadajapanitalyitalybrazilfrancefrancesouth korea uceismattssescdmayo scoresescdcdeismayo score sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactivemes or from mes or in another study the sensitivityand specificity of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating mes ‰¤ in patients with ulcerative colitis were and respectively overall as presented in table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and ibd endoscopic activity although some of these studies reported a strong correlation some others reported a relativelyweak correlation as noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and specificity of fecal calprotectin to predict the endoscopic activity of ibd undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences however fecal calprotectin does not appear to be a very reliable marker for the predictionof ibd endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy inthis regard further studies are still needed however under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate ibd endoscopic activity can be helpfulpregnant patients with ibd have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy in one study physicianglobal assessment [pga] which is a clinical symptombased criterionwas used to evaluate ibd activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with ibd the results of this study showed a significantcorrelation between fecal calprotectin and pga levels at prepregnancyduring pregnancy and postpartum stages in another study asignificant association was reported between fecal calprotectin levelsand clinical activity of ibd in pregnant women moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a specificity between and in the assessment of ibd clinical activity at diï¬erent stages ofpregnancy a recently published systematic review has also confirmed the conclusions obtained from these studies according tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith ibd clinical activity during pregnancy therefore from the viewpoint of relatively acceptable sensitivity and specificity in predictingthe endoscopic activity of ibd fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of ibd endoscopic activity inpregnant women in addition under pandemic conditions fecal calprotectin can be very helpful following the covid19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy therefore noninvasive ibd management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before the combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringcovid19 pandemic therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for ibd endoscopic activity evaluation during pandemic fecal calprotectin appears to be associated with ibd histologic activity as well given thedifficulty in the evaluation of the histologic activity of crohn's disease some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far correspondingly thesesystems score the disease's histologic activity based on histologic observationstherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory in this regard one of these histologic scoring systems isrobert™s score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the robert™s scoring system theede also used themodified harpaz index and performed some interesting studies in thisregard in one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingauc ci95 “ sensitivity specificity andcutoï¬ mgkg in another study on patients with endoscopically inactive ulcerative colitis mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg p also despite thehigh specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg in a recent study the geboes index has been used toevaluate histologic activity in patients with clinically quiescent ulcerative colitis as a result this study reported relatively low sensitivityand specificity for fecal calprotectin in the prediction of geboesscore sensitivity specificity and cut oï¬ µgg in another recent study the nancy index has been used toevaluate the histologic activity of ulcerative colitis and a high sensitivity and a low specificity were finally reported for fecalcalprotectin at a cutoï¬ of µgg in the prediction of histologic activity however some studies have reported both high sensitivityand specificity for fecal calprotectin in the prediction of histologicalremission for example one study reported a sensitivity of aswell as a specificity of for fecal calprotectin at a cutoï¬ of µggin the prediction of gs it seems that the cause of theseconflicts should be explored in the endoscopic and clinical activity ofthe disease the inclusion and exclusion criteria of these studies andpossibly in the diï¬erent indexes used for the evaluation of the histologicactivity of the disease another notable issue is that all of these studieshave been conducted on a relatively low number of patients with ulcerative colitis so the need for a study with a large sample size is stillstrongly felt moreover a large global study may be helpful in this regard prediction of relapse and response to treatmentas mentioned previously ibd has recurrence and relief periods sopredicting response to treatment and relapse is one of the significant 0cf khakikhatibi clinica chimica acta “challenges in ibd management the first evidence of the efficacy offecal calprotectin to predict recurrence dates back to the early 2000saccordingly a study published in by tibble is one of the firststudies in this regard in this study ibd patients in clinical remissionwere followed up for one year for the assessment of recurrence afterpreparing a stool sample to measure calprotectin this study has alsoshown that fecal calprotectin levels were higher in ibd patients withrecurrent disease and it was found that fecal calprotectin had a sensitivity of and a specificity of at a cutoï¬ of mgl to predictibd recurrence in a study published in costa showedthat the sensitivity and specificity of fecal calprotectin to predict ulcerative colitis recurrence are more than that of crohn's disease asensitivity of and a specificity of versus a sensitivity of and a specificity at a cutoï¬ of μgg another studyconducted on patients with ulcerative colitis has also reported appropriate sensitivity and specificity rates for fecal calprotectin in the prediction of relapse a sensitivity of and a specificity at a cutoï¬of μgg however another study was conducted on patientswith ulcerative colitis who have been followedup for year and finally a lower sensitivity was reported this study have shown that fecalcalprotectin at cutoï¬ of μgg could predict diseas

cancer7525

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "‚ammatory bowel disease ibd is a long life disease with remission and relapse periods ibd arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ‚ammation andintestinal ulcers in addition ibd has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [“] ulcerative colitisuc and crohn's diseases cd are known as two main forms of ibdaccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized in this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way for example 5asa which is acommon drug in the treatment of ibd is less eï¬ective on maintainingremission in cd patients on the other hand antibiotic therapy is notrecommended for the treatment uc but it can be eï¬ective on cd patients diï¬erential diagnosis is a serious challenge because cdand uc have significant similarities in terms of their clinical endoscopic and histological features however there are some diï¬erencesbetween uc and cd which are summarized in table1 in addition tointestinal complications uc and cd also have significant extraintestinal manifestations for example it was shown that uc is significantly associated with primary sclerosing cholangitis and cd is alsoassociated with cholelithiasis especially in cases that the ileum is involved furthermore cd can cause fistulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections both cd and uc can cause several disorderssuch as arthritis erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of ibd the latest statistics showed that the globalŽ corresponding author at department of clinical biochemistry and laboratory medicine faculty of medicine tabriz university of medical sciences daneshgahstreet po box tabriz iranemail address vagharimtbzmedacir m vagharitabari101016jcca202008025received july received in revised form august accepted august available online august elsevier bv all rights reserved 0cf khakikhatibi table1clinical endoscopic and histological features of cd and ucclinical featuresfeaturesrectal bleedingabdominal painfevermucus defectionintestinal obstructionperineal diseasepostoperative recurrenceasca positiveanca positiveendoscopic featurescdoccasionallyfrequentlyfrequentlyoccasionallyyesyesyesfrequentlynot commonucfrequentlyoccasionallynot commonfrequentlynonononot commonfrequentlyfeaturescduclocationmucosal involvementdepth of ulcerationfistulacobblestone appearanceaphthous ulcerationmucosal friabilityhistological featuresfeaturesgranulomascrypt abscessespatchinessany part of gi tractdiscontinuousdeepyesyesfrequentlynot commoncdfrequentlynot commonfrequentlycolon and rectumcontinuoussuperficialnonooccasionallyfrequentlyucrarefrequentlynot commonprevalence of ibd currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem according to areport published in ibd has the highest prevalence rate ineurope and its prevalence in the newly industrialized countries of asiaafrica and south america also appears to be increased over the pastthree decades unfortunately the peak of the disease is at the young age of“ years old therefore in addition to the suï¬ering from ‚icts on the patients it also has many negative eï¬ects on societymoreover many financial burdens are annually imposed on countriesfor controlling and treating this chronic disease the invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage ibd which are unpleasant for patients as well as having thehigh associated costs now the gold standard method for diagnosingibd and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures therefore in recent years many studies have been conducted tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofibd a high proportion of these studies have investigated the efficacy offecal calprotectin in diagnosing and monitoring patients althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring ibd patients so in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of ibd the role of fecal calprotectin in diagnosis and management ofibdthe efficacy of fecal calprotectin as an laboratory marker in various areas of ibd diagnosis and management have been studied including ibd diï¬erentiation from irritable bowel syndrome ibs evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andclinica chimica acta “response to treatment in following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting findings in all ofthe abovementioned areas calprotectin a clinically valuable proteincalprotectin is an antimicrobial protein mainly secreted by neutrophils this protein competes with bacteria over zinc thus kills thebacteria however this is not the only contribution that it has to antimicrobial activity moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsserum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection at theearly stages of ‚ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reflecting disease activity in ‚ammation of thejoints in addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker in neonatal sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspecificity of that have been reported for serum calprotectin indiagnosis of neonatal sepsis it has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the first of onset areassociated with a poor prognosis at the first three months serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low crp levels so they appear to be more efficient at reflecting disease activity some studies have also investigated the efficacy of serum calprotectin in the diagnosis of cancers correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation alsoregarding the efficacy of serum and saliva calprotectin for the diagnosisof ibd impressive results have been reported a study onpatients with ibd both uc and cd have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in ibd diagnosis compared to crp and albumin this studyalso indicated that the combination of serum calprotectin with crp oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with cd however no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with cd and uc as well as a slight correlation betweenserum calprotectin level and crp that was observed only in patientswith uc another study showed that the serum level of calprotectin was significantly higher in patients with cd compared to healthyindividuals in addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease the efficacy of salivary calprotectin in the diagnosisof ibd has also been studied which showed that salivary calprotectinsignificantly increased in patients with ibd compared to healthy individuals in this study auc values for unstimulated saliva and stimulated saliva to distinguish ibd patients from healthy individualswere reported to be and respectively however thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of ibd that is discussed in the following 0cf khakikhatibi clinica chimica acta “ laboratory measurement and reference intervalfecal calprotectin is a stable protein that remains stable for “ daysat room temperature this property is an excellent advantage for alaboratory marker also it seems that keeping the specimen at refrigerated temperature °c can increase the stability of fecal calprotectin however evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature on the other hand it is not also recommended tokeep samples in the refrigerator for more than days it seemsthat fecal calprotectin remains stable up to one year at ˆ’ °c measurement of fecal calprotectin can be done both qualitatively andquantitatively accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette however in the qualitative one only positive or negative results are reported and despite of sensitivity test specificity in theevaluation of disease activity was reported to be only it seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom ibd patients rapidly however some studies have shown that it isnot accurate enough in this case as well nevertheless asignificant concordance has been reported between home test resultsibdoc and fecal calprotectin laboratory measurement results whenquantum blue calprotectin elisa kit was used notably the agreements between results were and depending on the selectedcutoï¬s several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin these tests reportpositive results ranged from to µgg there are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin these kits are usually designed in terms of the elisamethod and some have a measurement range between and µgg moreover the chemiluminescence immunoassays cliamethod can also detect values between and µgg fluoro enzyme immunoassays feia and particle enhanced turbidimetric immunoassays petia can also be used for the measurement of fecalcalprotectin in this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals among healthy adults there is asignificant agreement on µgg as an upper limit one study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric ibd table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies according to these reports age can aï¬ect fecal calprotectinlevels fecal calprotectin and ibd diagnosisonly a small percentage of patients complaining of abdominal painand diarrhea have ibd in many cases ibs as a functional gastrointestinal disorder is known as the cause of such clinical symptomspatients with ibs have normal colonoscopy results while ibd patientsindicate abnormal colonoscopy results and have intestinal ulcersunfortunately the significant prevalence of ibs and the overlap between clinical symptoms and ibd can increase the colonoscopy ratetherefore a noninvasive diagnostic marker can be very helpful in thisregard notably the first evidence of the efficacy of fecal calprotectin inthe diagnosis of ibd was obtained in the 1990s røseth in proposed a method for measuring calprotectin in stool specimens one of the first and most interesting studies regarding fecal calprotectinutility in ibd diagnosis was the study by røseth published in in this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls this study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals subsequent studies somehow confirmed and complemented the findings of this study in another study published in auc values of ci “ were reported for fecal calprotectin in thediagnosis of colorectal ‚ammation moreover in a study onchildren with ibd it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with esr levels in astudy published in kolho reported auc values of ci “ for fecal calprotectin in the diagnosis of pediatric ibd in a study on patients with crohn disease a sensitivity of and a specificity of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease the results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ‚ammatory biomarkers such as crp andesr in the diagnosis of ibd diamanti reported a sensitivity of and a specificity of for fecal calprotectin at a cutoï¬ of μgg in ibd diagnosis in our recent study a sensitivityof and a specificity of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of ibd however oursample size was and the majority of patients were in the active phaseof the disease in another study conducted on patients with ulcerative colitis asensitivity of and a specificity of at cutoï¬ of μgg havebeen reported in this regard in one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withibd from patients without ibd patients with diseases other than ibdpatients with ibs and healthy persons with sensitivity and specificity caviglia in their study reported a sensitivity of and a specificity of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between ibs and ibd howeversome studies have reported significantly lower values accordingly in astudy on patients with ulcerative colitis kalantari reported asensitivity of and a specificity of at a cutoï¬ of μgg besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with crohn's disease asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting ce findings anddiagnosis of crohn's disease in another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity oftable reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesagesmedian levels of fecal calprotectin range µggnumber of subjectsused kitup to monthchildren “ yearschildren “ yearsadultsover years “ “ “ “ “bühlmann elisabühlmann elisacalpro® calprotectin elisa test alpphicalphicalreference 0cf khakikhatibi were reported for this biomarker at μgg in the diagnosis ofsmall bowel ‚ammation in crohn's disease given these findings it seems that fecal calprotectin has no ideal sensitivity and specificity for the diagnosis of ibd where the small intestine is involvedbesides there are some preanalytical limitations which are explainedin the next sections therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy howeverin a metaanalysis performed to evaluate the efficacy of fecal calprotectin and some other ‚ammatory markers to diï¬erentiate betweenibd and ibs the probability of ibd was less than at fecal calprotectin values lower than µgg or crp values lower than mgdl therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of ibd in patients with ibslike symptoms aswell as reducing the rate of colonoscopy moreover it should be notedthat although a systematic review has reported pooled sensitivity andspecificity above for fecal calprotectin to diï¬erentiate between ibdand ibs it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points hence performing extensive studies indiï¬erent countries on the healthy population and the ibd patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspecificity and minimum falsepositive resultstable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of ibdfrom ibs and table4 summarizes some metaanalysis results in thisregard as shown in table the most important limitation of the majority of clinical studies conducted to date is the small sample size alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between ibdand nonibd diseases fecal calprotectin and endoscopic and histologic activity evaluationundoubtedly one of the most serious challenges in the managementof ibd is evaluating the endoscopic and histologic activities of thedisease nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withibd as noted earlier several scoring systems have been devised toscore disease activity based on the findings of colonoscopy and histopathologic examinations in recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels in addition many studies have been performed inthe last decade all of which cannot be reviewed in this article the firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s in one of the first studiesroseth found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis furthermore in another study they observed that ibdpatients who were in remission clinically and had normal fecal calprotectin levels less than mgl had normal colonoscopy results these interesting findings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andclinica chimica acta “table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdsample sizepooled sensitivitypooled specificityreferences mucosal healing in ibd patients also these studies were the startingpoint of extensive studies that have been conducted up to now in astudy conducted on patients with crohn's disease sipponen investigated the sensitivity and specificity of fecal calprotectin in predicting endoscopic activity of crohn's disease correspondinglythe researchers used the crohn's disease endoscopic index of severitycdeis scoring system in their study to evaluate the endoscopic activity of crohn's disease as a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin besides the findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of crohn's disease with sensitivity and specificity in another study cdeis and mayo disease activity indexmdai were used to evaluate the endoscopic activity of crohn's diseaseand ulcerative colitis respectively according to the results of thatstudy on ibd patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to therachmilewitz clinical activity index in addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentification was obtained as some studies have also shown the superiority of fecal calprotectinover traditional ‚ammatory markers like crp besides one studyfound that fecal calprotectin was more strongly correlated with thesimple endoscopic score for crohn's disease sescd compared to thecrp and even crohn's disease activity index cdai the modifiedbaron index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis as a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to crp and clinical activity of the disease in thisregard similar results were also observed in our recent study in whichthe ulcerative colitis endoscopic index of severity uceis and sescdwere used therefore fecal calprotectin appears to be superior totraditional ‚ammatory markers in the prediction of ibd endoscopicactivity the high values of sensitivity and specificity that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients™ monitoring however severalrecent studies have reported some significantly lower values accordingly in a recent study in which mayo endoscopic score [mes] wasused to evaluate the endoscopic activity of ulcerative colitis atable summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdnumber of ibd patientsage grouplocationcut oï¬sensitivityspecificity cd and uc cd and uc cd and uc and unclassified68cd and uc cd and uc and unclassified cd and uc cd and uc uc cd ucadultsadultsadultsboth adult and pediatricpediatricadultspediatricadultsadultsboth adult and pediatrictaiwanchinaitalyspainfinlandiranitalyirandenmarkindia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggaucreferences spsrefidbib60 0cf khakikhatibi clinica chimica acta “table summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in ibd patientsage groupstudylocationusedendoscopicactivity indexcorrelationcoefficientrreferencenumberof ibdpatients cd uc uc cd ucadultsadultsadultsadultsadultsfinlandiranswitzerlandswitzerlandswitzerland modifiedcdeisuceisrachmilewitzsescd uc cdadultsadults uc cd uc cd cd uc ucadultsadultsadultsadultsadultsadultsadultsbaron scorerachmilewitzsescdgermanyusa andcanadajapanitalyitalybrazilfrancefrancesouth korea uceismattssescdmayo scoresescdcdeismayo score sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactivemes or from mes or in another study the sensitivityand specificity of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating mes ‰¤ in patients with ulcerative colitis were and respectively overall as presented in table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and ibd endoscopic activity although some of these studies reported a strong correlation some others reported a relativelyweak correlation as noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and specificity of fecal calprotectin to predict the endoscopic activity of ibd undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences however fecal calprotectin does not appear to be a very reliable marker for the predictionof ibd endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy inthis regard further studies are still needed however under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate ibd endoscopic activity can be helpfulpregnant patients with ibd have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy in one study physicianglobal assessment [pga] which is a clinical symptombased criterionwas used to evaluate ibd activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with ibd the results of this study showed a significantcorrelation between fecal calprotectin and pga levels at prepregnancyduring pregnancy and postpartum stages in another study asignificant association was reported between fecal calprotectin levelsand clinical activity of ibd in pregnant women moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a specificity between and in the assessment of ibd clinical activity at diï¬erent stages ofpregnancy a recently published systematic review has also confirmed the conclusions obtained from these studies according tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith ibd clinical activity during pregnancy therefore from the viewpoint of relatively acceptable sensitivity and specificity in predictingthe endoscopic activity of ibd fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of ibd endoscopic activity inpregnant women in addition under pandemic conditions fecal calprotectin can be very helpful following the covid19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy therefore noninvasive ibd management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before the combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringcovid19 pandemic therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for ibd endoscopic activity evaluation during pandemic fecal calprotectin appears to be associated with ibd histologic activity as well given thedifficulty in the evaluation of the histologic activity of crohn's disease some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far correspondingly thesesystems score the disease's histologic activity based on histologic observationstherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory in this regard one of these histologic scoring systems isrobert™s score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the robert™s scoring system theede also used themodified harpaz index and performed some interesting studies in thisregard in one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingauc ci95 “ sensitivity specificity andcutoï¬ mgkg in another study on patients with endoscopically inactive ulcerative colitis mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg p also despite thehigh specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg in a recent study the geboes index has been used toevaluate histologic activity in patients with clinically quiescent ulcerative colitis as a result this study reported relatively low sensitivityand specificity for fecal calprotectin in the prediction of geboesscore sensitivity specificity and cut oï¬ µgg in another recent study the nancy index has been used toevaluate the histologic activity of ulcerative colitis and a high sensitivity and a low specificity were finally reported for fecalcalprotectin at a cutoï¬ of µgg in the prediction of histologic activity however some studies have reported both high sensitivityand specificity for fecal calprotectin in the prediction of histologicalremission for example one study reported a sensitivity of aswell as a specificity of for fecal calprotectin at a cutoï¬ of µggin the prediction of gs it seems that the cause of theseconflicts should be explored in the endoscopic and clinical activity ofthe disease the inclusion and exclusion criteria of these studies andpossibly in the diï¬erent indexes used for the evaluation of the histologicactivity of the disease another notable issue is that all of these studieshave been conducted on a relatively low number of patients with ulcerative colitis so the need for a study with a large sample size is stillstrongly felt moreover a large global study may be helpful in this regard prediction of relapse and response to treatmentas mentioned previously ibd has recurrence and relief periods sopredicting response to treatment and relapse is one of the significant 0cf khakikhatibi clinica chimica acta “challenges in ibd management the first evidence of the efficacy offecal calprotectin to predict recurrence dates back to the early 2000saccordingly a study published in by tibble is one of the firststudies in this regard in this study ibd patients in clinical remissionwere followed up for one year for the assessment of recurrence afterpreparing a stool sample to measure calprotectin this study has alsoshown that fecal calprotectin levels were higher in ibd patients withrecurrent disease and it was found that fecal calprotectin had a sensitivity of and a specificity of at a cutoï¬ of mgl to predictibd recurrence in a study published in costa showedthat the sensitivity and specificity of fecal calprotectin to predict ulcerative colitis recurrence are more than that of crohn's disease asensitivity of and a specificity of versus a sensitivity of and a specificity at a cutoï¬ of μgg another studyconducted on patients with ulcerative colitis has also reported appropriate sensitivity and specificity rates for fecal calprotectin in the prediction of relapse a sensitivity of and a specificity at a cutoï¬of μgg however another study was conducted on patientswith ulcerative colitis who have been followedup for year and finally a lower sensitivity was reported this study have shown that fecalcalprotectin at cutoï¬ of μgg could predict diseas Answer:

Colon_Cancer

to probiotics general attitudes and functions since the first observation of probiotic bacteria by elie metchnikoff there have been several studies on the immunological effects of probiotics on the host immune system according to who and fao probiotics are defined as œlive microanisms which when administered in proper amounts confer a health benefit on the host among several genera of bacteria and yeasts that identified and defined as probiotics health benefits of lactobacillus and bifidobacterium on the host have been proved and are generally consumed as a part of fermented foods like those in dietary supplements there are some reports about probiotics potential in promoting health benefits by regulating allergic reactions [“] protecting the hosts against bacterial and viral infection [“] and even reducing the tumor growth in some cancer models [“] the probioticsconferred health benefits are attributable to their effects on the immune system recognition and stimulation of immune system in the gut lumen is followed through three distinct pathways engulfment of probiotics by macrophages mfs or dendritic cells dcs present immediately below m cells specialized epithelial cells dcsdirected sampling and processing of probiotics in the gut lumen and direct stimulation of intestinal epithelial cells iecs by probiotics to secrete an array of cytokines modulating the immune functions of dcs t cells and b cells in the gutassociated lymphoid tissue galt briefly the regulatory effects of probiotics on host immune responses are followed through activation of the function of dendritic cells macrophages and t and b lymphocytes [ ] in addition probiotics have proved to modulate and regulate innate and adaptive immune responses partly through the activation of tolllike receptors tlrs as the role of the intestinal epithelium is to form a physiological barrier against pathogenic microbes and detrimental substances available in the intestinal lumen this monolayer is responsible for distinguishing between pathogens and commensal bacteria as well as regulation of intestinal immune responses it has been shown that probiotics can regulate immunomodulatory responses of intestinal epithelial cells fig one family of pattern recognition receptors prrs in the innate corresponding author email addresses a_ghaemipasteuracir ghaem_amiryahoocom a ghaemi these authors contributed equally to this work 101016jmicpath2020104452 received april received in revised form august accepted august microbialpathogenesis1482020104452availableonline18august2020088240102020elsevierltdallrightsreserved 0cm mahooti abbreviation covid19 coronavirus disease sarscov2 severe acute respiratory syndrome coronavirus who world health anization food and agriculture anization fao macrophages mfs dendritic cells dcs m cells microfold cells intestinal epithelial cells iecs galt gutassociated lymphoid tissue tlrs tolllike receptors pattern recognition receptors prrs pamps pathogenassociated molecular patterns transmembrane protein tp myd88 myeloid differentiation protein rtis respiratory tract infections human rhinovirus hrv influenza virus ifv rsv respiratory syncytial virus iav influenza a virus ifn α interferon α interferon ifn tumor necrosis factor α tnfα ifnÎ interferon gamma il1 interleukin interleukin il6 interleukin il4 il8 interleukin il10 interleukin il12 interleukin t helper type th1 th2 t helper type mips macrophage inflammatory proteins mcps monocyte chemoattractant proteins bronchoalveolar lavage bal nk cell natural killer cells epss exopolysaccharides iga immunoglobulin a igg immunoglobulin g secretory immunoglobulin a siga peyer™s patches pps tfh follicular helper t acot acylcoa thioesterase cyr61 cysteinerich angiogenic inducer early growth response egr1 fos protooncogene fos rsad2 radical sadenosyl methionine domain containing klrk1 killer cell lectin like receptor k1 ilc innate lymphoid cells mediastina lymph node mln bronchoalveolar lavage fluid balf ip10 interferoninducible protein pedv porcine epidemic diarrhea virus cfs cellfree supernatants cytopathic effect cpe dcpep dctargeting peptide coe core neutralizing epitope oas oligoadenylate synthetase interferonstimulated gene isg15 swi2snf2related crebbinding protein activator srcap protein pfu a plaqueforming unit mpiv1 murine parainfluenza virus crp creactive protein cxcl8 cxc motif chemokine ligand immune system are tolllike receptors which play a pivotal role in the linking of innate and adaptive immunity tlrs can specifically recognize pathogenassociated molecular patterns pamps and convey pathogen related molecular signals into cells by transmembrane tm protein afterward tlrmediated multistep signaling cascades are initiated leading to the activation of transcriptional pathways such as nfκb against the invader pathogens this signal transmission activates both immune system arms aimed at the pathogenic microanism through a cascade reaction which is severely dependent on signaling pathway directed by tolllike receptor tlr7 and myeloid differentiation protein myd88 interestingly it has been determined that tlr7 expression considerably reduces after influenza infection in this context wu revealed that after consumption of probiotics by neomycintreated mice the balance of intestinal flora restored and thereby tlr7 pathway upregulated this evidence presents promise for the regulatory role of probiotics in host innate and adaptive immune responses as underlying mechanisms for protection from viral infection pathology of influenza virus the most common respiratory virus infection influenza virus belonging to the orthomyxoviruses family is among viruses that cause respiratory tract infections rtis several human viruses can cause rti and due to hospitalizations medical costs sick leave and school or daycare absences viral respiratory diseases can pose a considerable social and economic burden human rhinovirus hrv enterovirus influenza virus ifv respiratory syncytial virus rsv and adenovirus are common etiological agents of acute respiratory disease influenza a virus iav initiates pulmonary inflammation and intensifies chronic lung diseases in response to the infiltration of inflammatory cells and augmentation of airway hyperresponsiveness the main target and host for iav is the bronchial epithelial cell which plays a key role in influenza pathogenesis infection occurs following “ h of influenza virus replication for the first cycle and then initial high titers of virus are shed during this period ifv infection can result in several symptoms like fever cough headache and pneumonia which may become immunologically incompetent while the induction of inflammatory cytokines by influenza infection is attributed to its systemic feature it is unlikely that the virus to be propagated outside the respiratory tract during an uncomplicated infection one of the key components of the influenza virus in pathogenesis is ha domain which is recognized by the host™s neutralizing antibodies the emerged ha is directed to the cell membrane in an infected host cell fastening to the cell membrane by means of a short transmembrane region at the cterminal and once this domain attached to terminal sialic acid residues on the cell it facilitates entry and fusion of the virus due to the acidification of host cells by proton pumps ha rearranges so that the highly conserved nterminal of ha2 is exposed this exposure leads to the fusion of viral membrane with cell membranes and thus activation of the replication complex despite all known clinical and pathogenesis descriptions of the influenza virus the mechanism through which influenza virus disease being developed has not precisely understood however it is thought that local nonimmune cells which release early cytokines are the cause of many of the clinical signs some cytokines including ifnα tnfα and il1 α and located at the site of infection are responsible microbialpathogenesis14820201044522 0cm mahooti for local inflammatory reactions as well as some systemic effects [ ] afterward il6 and many other chemotactic cytokines like the neutrophil attracting interleukin8 il8 macrophage inflammatory proteins mips and monocyte chemoattractant proteins mcps are rapidly produced fever excessive sleepiness and anorexia are attributed to the activation of ifnα tnfα il1 and il6 after influenza infection neutrophil and macrophage functions are stimulated by tnfα and il1 and both cytokines potently upregulate leukocyte adhesion molecules on the vascular endothelium therefore mediating the first indispensable step for sequestration of neutrophils and or macrophages into the respiratory tract a study by van reeth demonstrated that there is a correlation between bal fluid levels of some cytokines ifnα tnfα and il1 and virus titers neutrophil infiltration and influenza disease additionally lee showed that ifnα tnfα il1 and il6 all participate in nonspecific and specific antiviral immune responses immunomodulatory role of probiotics on influenza virus in the context of preclinical studies mm2 can significantly reduce influenzaelicited proinflammatory cytokines such as il6 and tnfα moreover a slightly elevated ifnα level in the balf indicated the impact of this probiotic on the enhancement of nk cell activity these results along with the reduction of pulmonary mrna levels of nk cell activators including proinflammatory cytokine il1 and chemokines mip2 and mcp1 suggest the modulating effect of this probiotic on influenza infection in another study continuous oral administration of lactobacillus plantarum 06cc2 led to an elevation in the production of ifnα and th1 cytokines il12 and ifnÎ and reduction in the production of tnfα and il6 cytokines in balf this probiotic could also control the number of total infiltrated cells such as macrophages and neutrophils in the balf of infected mice similarly nagai revealed that days after oral administration of the yogurt fermented with l bulgaricus oll1073r1 or its exopolysaccharides epss influenza virus infection ameliorated which attributed to the development of nk cell activity of splenocytes assessment of kimchiderived lactobacillus plantarum and leuconostoc mesenteroides has confirmed their effectiveness against lethal influenza viruses h1n1 and h7n9 by decreasing the sizes of viral plaques both in vitro and in vivo in addition it has been shown that lactococcal strains or their eps induced weight regain and also reduced viral titer in the lung of mice infected with influenza virus h1n1 starosila investigated the antiviral ability of bacillus subtilis and showed that after a single dose administration of the probiotic bacteria the survival rate of mice challenged with the ifv increased song assessed the impact of oral intake of lactobacillus rhamnosus m21 on lethally ifvinfected mice an increase in the level of ifnÎ and il12 and a decline in il4 level suggested that this probiotic can modulate some disease outcomes attributed to changes in cytokine profiles such as that happens in the lung after influenza infection in our very recent study we showed that bifidobacterium bifidum can increase the level of both th1 ifn“y and il12 and th2 il4 since the manifestation of probiotics impacts on several diseases from nonviral to viral ones [“] several studies have surveyed the probiotic roles in immune responses of influenzainfected animal models it has been fully demonstrated that upon infection with influenza many cytokines such as il12 one of the mediators of th1 immuneresponse interferon ifnÎ representative of th1 cytokine il4 and il10 th2 cytokines il1α il1 il6 and tumor necrosis factor tnfα proinflammatory cytokines and ifnα and ifn are produced in the respiratory tract [“] studies on ameliorating influenza infection as well as alleviating influenza symptoms have been trying to redress the imbalance attributed to runaway cytokines production namely cytokine storm after ifv infection kawahara demonstrated that probiotic bifidobacterium longum fig schematic presentation of possible mechanisms of probiotic immunomodulation effects in the intestine probiotics trigger immunomodulation through direct and indirect interaction with intestinal epithelial cells dendritic cells extend their dendrites between intestinal epithelial cells iecs and might directly sample and process probiotics in the gut lumen leading to activation of innate and adaptive immune responses dendritic cells present immediately below m cells engulf probiotics resulting in the maturation of dcs and may derive b cells into plasma cells additionally after the interaction of probiotics with macrophages and dendritic cells presented in lamina propria these cells are activated and induce nk cell activation which leads to ifnÎ elevation to defend against viruses upon the interaction of probiotics™ pamps with different types of tolllike receptors tlrs nuclear factorκb nfκbmediated antiviral gene expression is stimulated eventually active immune cells migrate to sites of infection through lymphatic and circulatory systems to defend against respiratory viruses microbialpathogenesis14820201044523 0canother study enterococcus faecalis1 has been proved to improve the body weight and feed conversion ratio of treated broilers and also significantly elevated the total igy serum level resulting in efficient modulation of the cecal microbiota and decrease in the mortality percentage of broilers an investigation on the possible effect of interaction between lactobacilli and chicken macrophages on eliciting antiviral responses against the aiv showed that certain probiotic species such as l acidophilus and l salivarius when administered as live bacteria either alone or in combination can induce an antiviral response in chicken macrophages in another study seo reported that live leuconostoc mesenteroides yml003 significantly restored the body weight and increased the ifnÎ levels in splenic cells of lowpathogenic aiv h9n2infected chickens examining the effectiveness of enterococcus faecium ncimb and zinc oxide in modulating the immune system of piglets in confronting with swine influenza virus siv revealed that the body weights of piglets fed with the probiotic and vaccinated with trivalent influenza vaccine significantly increased and noticeably higher h3n2specific antibodies were detected among them based on these considerations probiotics administration is effective in the secretion of high concentration of cytokines from immune cells located in the airway leading to the migration of immune cells to the lung space and thereby amelioration of influenza infection fig the probiotic effects on coronavirus infections m mahooti cytokines an increase in the level of total igg antibodies in pooled sera of treated mice and igg1 and igg2a isotypes demonstrated the efficacy of the probiotic in eliciting humoral immune responses and th1th2 responses respectively moreover it revealed that the level of inflammatory cytokines like il6 which increases upon influenza infection decreased in the probiotic group suggesting the ameliorating potential of this probiotic in influenzainfected mice based on the results of body weight changes survival rates and viral titre among treatment groups of different influenza viruses park showed that lactobacillus plantarum has antiinfluenza effects that are not virus type or straindependent revealing that regular intake of that probiotic can help to alleviate the influenza symptoms concerning the effect of longterm probiotic administration kiso orally injected lactobacillus pentosus b240 to mice for weeks and evaluated its inhibitory properties against influenza challenge assessment of different cytokineschemokines in the lungs of infected animals revealed that excluding il5 administration of that probiotic did not affect the immune system regarding cytokineschemokines secretion however ah1n1 pdm infected mice survived probably due to protecting effects of the probiotic by downregulation of acots acot1 acot2 and acot5 cyr61 egr1 and fos as well as upregulation of stfa1 and antiviral rsad2 genes in the lungs of uninfected mice in agreement with all aforementioned results harata revealed that oral administration of probiotics lactobacillus gg and l gasseri tmc035 in mice infected with a lethal dose of influenza ah1n1 pdm prompted the secretion of il12 il6 ifnÎ and iga from isolated pp cells in vitro however unlike lactobacillus gg the oral administration of l gasseri had no impact on the production of ifnÎ il6 as well as total iga in vivo proving the vital role of probiotic interaction with the component cells of galt in the protection against influenza the investigation of the effects of l casei strain shirota on aged mice showed that this probiotic can enhance not only the level of ifnÎ and tnfα but also pulmonary and spleen nk cells activity and thereby ameliorates ifv infection in another study oral administration of bifidobacterium longum bb536 could significantly reduce the loss of body weight inhibit viral proliferation in the lungs and improve the symptoms of influenzainfected mice which may be related to the decreased level of il6 belkacem observed that while administration of probiotic l paracasei induced significantly higher levels of proinflammatory cytokines in probioticfed influenza mice models this trend was reversed seven days upon influenza challenge except for il33 the number of all tissueresident or circulatory myeloid cells and b cells after the probiotic consumption and before viral infection increased and the probiotic administration generated more ifnÎproducing ilc1 mainly nk cells and th2 cells during the late phase of influenza infection additionally l paracasei peptidoglycans administration before influenza infection increased dendritic cells but did not affect other cell types and significantly reduced viral loads besides the effectiveness of oral administration of probiotics intranasal administration of lactobacillus pentosus spt84 to mice proved to induce the production of il12 and ifnÎ in mediastinal lymph node mln cells and il12 and ifnα in balf thereby improved the survival rates of mice reduced the ifv titer in balf and subsequently suppressed influenza infection in mice employing the novel sublingual route lee showed that in contrary to proinflammatory cytokines the level of il12 in the lung homogenates of mice treated with lactobacillus rhamnosus significantly increased in addition besides the increase in nk cell activities and antiinfluenza virus iga the expression of cd25 by both cd8 and cd4 lymphocytes highly increased in the lungs of mice these results recommend that compared to the traditional methods sublingual delivery is a more effective way for the administration of probiotics against seasonal and pandemic influenza regarding other animal models poorbaghi et al showed that microencapsulated lactobacillus acidophilus probiotic and its symbiotic form with inulin decreased faecal shedding of h9n2 avian influenza virus aiv in both nonvaccinated and vaccinated broiler chicks in the current outbreak of coronavirus disease covid19 reported from wuhan china has again gained global attention to taking a new measure that could work out as fast as possible against such an outbreak of viruses interestingly accumulated data obtained from clinical investigations on patients who suffered from severe covid19 in a hospital in wuhan demonstrated the presence of signs for cytokine storm especially among patients in severe stages of the disease particularly the levels of cytokines and chemokines involved in both th1 such as il1b ifnÎ and th2 il4 and il10 immune responses were promoted in studied patients moreover the levels of ip10 mcp mip1a and tnfα were in direct correlation with the severity of patients™ symptoms on the other hand it has been determined that the cytokine storm may lead to a rise in platelets and longer hospitalization of covid19 patients other studies also have revealed other aspects of virallydriven manipulation of immune responses by human coronaviruses [“] therefore addressing the cytokine storm may be the key for the treatment of patients infected with sarscov2 while some reagents such as steroids can be considered as hyperinflammation suppressors their sideeffects impede to count on them as a trustworthy medicine for covid19 alternatively addressing the urgent need for standing against the increasing rate of morbidity and mortality related to the current pandemic requires employing previously approved therapies harnessing safety profiles probiotics as a safe available treatment with the ability to modulate immune responses and manipulate cytokines production have been considered to be studied against different strains of coronavirus in some studies soon moreover a clinical survey has reported an intestine microbiota imbalance in particular a decline in the level of probiotics such as lactobacillus and bifidobacterium among some covid19 patients which may result in secondary infection in response to bacterial translocation one report has shown that the recent outbreak of porcine epidemic diarrhea virus pedv can be prevented through the use of either cell free supernatants cfs or live lactic acid bacteria lab it demonstrated that probiotics though the precise mechanism is not clear could be effective against the pandemic strain of pedv in a strainspecific manner using cpe reduction assays that further confirmed by qualitative immunofluorescence in another investigation lactobacillus casei was used as a carrier for the dctargeting peptide dcpep fused with the pedv core neutralizing epitope coe antigen this survey demonstrated that this genetically engineered lactobacillus casei oral vaccine is able to induce systemic igg and mucosal siga antibody microbialpathogenesis14820201044524 0cm mahooti fig model of the interaction of active immune cells triggered by probiotics with respiratory viruses in the lung following virus infection immune cells in the airway such as dendritic cells and macrophages secrete cytokines to defend against viruses in a probioticreceived subject the high concentration of cytokines leads to the migration of immune cells to the lung space through the gut“lung axis resulting in rapid recruitment of activated t and b cells in the lung that eventually promote upregulation of virusspecific immunoglobulins and cytokines in probioticreceived subject whereas in the absence of activated immune cells the respiratory virus can cause severe lung damage due to the lack of immediate immune responses responses in mice models there have been other articles using different types of probiotics for displaying the desired genes or antigens against pedv [“] for instance liu demonstrated that their modified lactobacillus plantarum has the property to act like a strong antiviral agent against coronavirus infection in the intestinal porcine epithelial cell line probiotic impacts on other viral respiratory infections eguchi et al demonstrated that lactobacillus gasseri sbt2055 lg2055 when administered orally to mice before infection with a human rsv could suppress the virus titre in lung tissue homogenates rsv replication and the intensity of the symptoms moreover a decrease in the expression level of proinflammatory cytokines and an increase in the mrna level of ifn ifnÎ oas1a and isg15 in the mice lung upon probiotic administration are satisfactory evidence for antiviral properties of this probiotic also swi2snf2related creb binding protein activator protein srcap introduced as a candidate for the antiviral activity of lg2055 against rsv to investigate whether probiotics can control the inflammatory pathway and modulate the coagulation system upon respiratory viral infection rhamnosus crl1505 was orally administered in rsv or ifv mice models the results elucidated that this probiotic could successfully modulate tlr3triggered immune coagulation reaction in the lung upon viral infection and prevent exacerbated respiratory injuries notably this study substantiated the vital role of probioticprovoked secretion of il10 in taming the coagulation system after the viral attack additionally in a study conducted by tomosada nasal administration of lactobacillus rhamnosus crl1505 and crl1506 has proved to modulate elevated respiratory levels of the proinflammatory mediators caused by administration of the viral pathogenassociated molecular pattern polyic moreover a nasal administration of the probiotic prior to pfu of rsv challenge improved resistance against rsv infection considering the effect of probiotics on the parainfluenza virus there is only one study evaluating the antiviral effects of oral administration of lactococcus lactis subsp lactis jcm5805 in a mouse model of murine parainfluenza virus mpiv1 infection the probiotic administration resulted in a rise in the survival rate of treated mice without any weight loss and also a decline in the lung histopathology scores compared to the nontreated group which was attributed to the incorporation of jcm5805 into cd11c immune cells in pp and thereafter activation of pp pdcs and ultimately elevation of ifns expression it is of note that although no activated local pdcs were observed at lung upregulation in ifnsinduced antiviral factors in the lung may be due to the delivery of ifns from the intestine of jcm5805fed mice into the lung studies reporting the effects of probiotic bacterias on respiratory viruses have been demonstrated in table supplementary section clinical evidence of probiotic immunomodulation in a pilot study intake of lactobacillus brevis kb290 has shown to curtail the incidence of influenza infection among schoolchildren with no adverse effects associated with consuming the probioticcontaining drinks hu demonstrated that h7n9 ifv infection led to a decrease in intestinal microbial diversity and species richness among patients they observed that although administration of c butyricum probiotic was unable to alleviate the antibioticrelated disturbances in the gut microbiome of h7n9infected patients an increase in microbiota diversity and evenness gradually appeared through continuous administration of probiotics after antibiotic cessation additionally based on the evaluation of crp levels or bacteremia and pneumonia in the patients treated with probiotics the safety of probiotic administration was approved and no inflammatory effects were observed in another study conducted by wang the impact of lactobacillus rhamnosus gg administration on nursing home residents aged and older was assessed it revealed that probiotic administration reduced the risk of influenza and other respiratory viral infections among the elderly received probiotics compared to those receiving a placebo although not statistically significant the trial provided a microbialpathogenesis14820201044525 0ctable clinical studies reporting regulatory effect of probiotic bacteria on immune responses study design an openlabel parallelgroup trial main findings the risk of infection †“ subjects schoolchildren “ years of age a retrospective study patients nursing home residents years of age and older a randomized doubleblind placebo controlled pilot trial probiotics used lactobacillus brevis kb290 kb290 days per week for weeks in the form of test drink containing × cfu clostridium butyricum three times per day at the dose of cfu tablet prior to h7n9 infection lactobacillus rhamnosus gg twice a day for months in the form of capsule containing cfu m mahooti framework to assess the effectiveness of probiotics in reducing respiratory infections among senescent individuals similarly it has been shown that there is no connection between intaking the yogurt fermented with probiotic lactobacillus delbrueckii ssp bulgaricus oll1073r1 and incidence rate of influenza in humans however the immunological study showed an increase in the level of ifnÎ in the probiotictreated group while there are some available treatments for hrv the most frequent cause of the common cold most of them have failed to be efficient in clinical trials due to their drawbacks in this regard probiotics have shown to prevent or treat common colds and upper respiratory infections several studies have revealed that the rhinovirusrelated common cold pathogenesis is associated to the innate inflammatory response to the virus therefore more attempts have done to incorporate probiotics to modulate immune responses consequently leading to balanced responses and optimal outcomes in combating viral infection in this regard an investigation on the impact of bifidobacterium animalis ssp lactis bl04 on healthy adults showed a modest modulation of innate immune host responses upon infection with hrv particularly reduction of cxcl8 response in the nasal lavage resulting in a decline in the rhinovirus replication approved by a decrease in virus shedding in the nasal secretions tapiovaara demonstrated that adults consumed juice enriched with live or heatinactivated l rhamnosus gg before intranasal inoculation of hrv showed no significant differences in nasopharyngeal hrv loads compared to the placebo group another survey has illustrated that consumption of probiotic is a good strategy to prevent viral rtis in the first year of life in a cohort of preterm infants the results showed that the probioticdriven change in microbiota leads to the induction of longlasting effects which can reduce the risk of viral rtis in agreement with that study it was demonstrated that live l rhamnosus gg might be more effective in reducing the rhinovirus infection rate than the inactivated form of the same strain respiratory syncytial virus from the paramyxoviridae family is considered as the major cause of lower respiratory tract infection in infants and children around the world and is becoming an important pathogen of the elderly although most children have experienced a first rsv infection by two years of age some cases suffering premature birth bronchopulmonary dysplasia immunodeficiency and congenital heart disease are vulnerable to symptoms worsening and hospitalization as well however the probiotic administration has proved to be effective in developing protection against virallyinduced inflammation besides there is a study demonstrating that while probiotic consumption significantly reduced the number of days with respiratory symptoms during the intervention no significant effect was neither observed on the occurrence of viruses in the nasopharynx nor on the symptoms during viral episodes among daycare children clinical studies reporting regulation of immune responses by probiotic bacterias have been presented in table conclusion bifidobacterium animalis daily for days in the

cancer7526

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: to probiotics general attitudes and functions since the first observation of probiotic bacteria by elie metchnikoff there have been several studies on the immunological effects of probiotics on the host immune system according to who and fao probiotics are defined as œlive microanisms which when administered in proper amounts confer a health benefit on the host among several genera of bacteria and yeasts that identified and defined as probiotics health benefits of lactobacillus and bifidobacterium on the host have been proved and are generally consumed as a part of fermented foods like those in dietary supplements there are some reports about probiotics potential in promoting health benefits by regulating allergic reactions [“] protecting the hosts against bacterial and viral infection [“] and even reducing the tumor growth in some cancer models [“] the probioticsconferred health benefits are attributable to their effects on the immune system recognition and stimulation of immune system in the gut lumen is followed through three distinct pathways engulfment of probiotics by macrophages mfs or dendritic cells dcs present immediately below m cells specialized epithelial cells dcsdirected sampling and processing of probiotics in the gut lumen and direct stimulation of intestinal epithelial cells iecs by probiotics to secrete an array of cytokines modulating the immune functions of dcs t cells and b cells in the gutassociated lymphoid tissue galt briefly the regulatory effects of probiotics on host immune responses are followed through activation of the function of dendritic cells macrophages and t and b lymphocytes [ ] in addition probiotics have proved to modulate and regulate innate and adaptive immune responses partly through the activation of tolllike receptors tlrs as the role of the intestinal epithelium is to form a physiological barrier against pathogenic microbes and detrimental substances available in the intestinal lumen this monolayer is responsible for distinguishing between pathogens and commensal bacteria as well as regulation of intestinal immune responses it has been shown that probiotics can regulate immunomodulatory responses of intestinal epithelial cells fig one family of pattern recognition receptors prrs in the innate corresponding author email addresses a_ghaemipasteuracir ghaem_amiryahoocom a ghaemi these authors contributed equally to this work 101016jmicpath2020104452 received april received in revised form august accepted august microbialpathogenesis1482020104452availableonline18august2020088240102020elsevierltdallrightsreserved 0cm mahooti abbreviation covid19 coronavirus disease sarscov2 severe acute respiratory syndrome coronavirus who world health anization food and agriculture anization fao macrophages mfs dendritic cells dcs m cells microfold cells intestinal epithelial cells iecs galt gutassociated lymphoid tissue tlrs tolllike receptors pattern recognition receptors prrs pamps pathogenassociated molecular patterns transmembrane protein tp myd88 myeloid differentiation protein rtis respiratory tract infections human rhinovirus hrv influenza virus ifv rsv respiratory syncytial virus iav influenza a virus ifn α interferon α interferon ifn tumor necrosis factor α tnfα ifnÎ interferon gamma il1 interleukin interleukin il6 interleukin il4 il8 interleukin il10 interleukin il12 interleukin t helper type th1 th2 t helper type mips macrophage inflammatory proteins mcps monocyte chemoattractant proteins bronchoalveolar lavage bal nk cell natural killer cells epss exopolysaccharides iga immunoglobulin a igg immunoglobulin g secretory immunoglobulin a siga peyer™s patches pps tfh follicular helper t acot acylcoa thioesterase cyr61 cysteinerich angiogenic inducer early growth response egr1 fos protooncogene fos rsad2 radical sadenosyl methionine domain containing klrk1 killer cell lectin like receptor k1 ilc innate lymphoid cells mediastina lymph node mln bronchoalveolar lavage fluid balf ip10 interferoninducible protein pedv porcine epidemic diarrhea virus cfs cellfree supernatants cytopathic effect cpe dcpep dctargeting peptide coe core neutralizing epitope oas oligoadenylate synthetase interferonstimulated gene isg15 swi2snf2related crebbinding protein activator srcap protein pfu a plaqueforming unit mpiv1 murine parainfluenza virus crp creactive protein cxcl8 cxc motif chemokine ligand immune system are tolllike receptors which play a pivotal role in the linking of innate and adaptive immunity tlrs can specifically recognize pathogenassociated molecular patterns pamps and convey pathogen related molecular signals into cells by transmembrane tm protein afterward tlrmediated multistep signaling cascades are initiated leading to the activation of transcriptional pathways such as nfκb against the invader pathogens this signal transmission activates both immune system arms aimed at the pathogenic microanism through a cascade reaction which is severely dependent on signaling pathway directed by tolllike receptor tlr7 and myeloid differentiation protein myd88 interestingly it has been determined that tlr7 expression considerably reduces after influenza infection in this context wu revealed that after consumption of probiotics by neomycintreated mice the balance of intestinal flora restored and thereby tlr7 pathway upregulated this evidence presents promise for the regulatory role of probiotics in host innate and adaptive immune responses as underlying mechanisms for protection from viral infection pathology of influenza virus the most common respiratory virus infection influenza virus belonging to the orthomyxoviruses family is among viruses that cause respiratory tract infections rtis several human viruses can cause rti and due to hospitalizations medical costs sick leave and school or daycare absences viral respiratory diseases can pose a considerable social and economic burden human rhinovirus hrv enterovirus influenza virus ifv respiratory syncytial virus rsv and adenovirus are common etiological agents of acute respiratory disease influenza a virus iav initiates pulmonary inflammation and intensifies chronic lung diseases in response to the infiltration of inflammatory cells and augmentation of airway hyperresponsiveness the main target and host for iav is the bronchial epithelial cell which plays a key role in influenza pathogenesis infection occurs following “ h of influenza virus replication for the first cycle and then initial high titers of virus are shed during this period ifv infection can result in several symptoms like fever cough headache and pneumonia which may become immunologically incompetent while the induction of inflammatory cytokines by influenza infection is attributed to its systemic feature it is unlikely that the virus to be propagated outside the respiratory tract during an uncomplicated infection one of the key components of the influenza virus in pathogenesis is ha domain which is recognized by the host™s neutralizing antibodies the emerged ha is directed to the cell membrane in an infected host cell fastening to the cell membrane by means of a short transmembrane region at the cterminal and once this domain attached to terminal sialic acid residues on the cell it facilitates entry and fusion of the virus due to the acidification of host cells by proton pumps ha rearranges so that the highly conserved nterminal of ha2 is exposed this exposure leads to the fusion of viral membrane with cell membranes and thus activation of the replication complex despite all known clinical and pathogenesis descriptions of the influenza virus the mechanism through which influenza virus disease being developed has not precisely understood however it is thought that local nonimmune cells which release early cytokines are the cause of many of the clinical signs some cytokines including ifnα tnfα and il1 α and located at the site of infection are responsible microbialpathogenesis14820201044522 0cm mahooti for local inflammatory reactions as well as some systemic effects [ ] afterward il6 and many other chemotactic cytokines like the neutrophil attracting interleukin8 il8 macrophage inflammatory proteins mips and monocyte chemoattractant proteins mcps are rapidly produced fever excessive sleepiness and anorexia are attributed to the activation of ifnα tnfα il1 and il6 after influenza infection neutrophil and macrophage functions are stimulated by tnfα and il1 and both cytokines potently upregulate leukocyte adhesion molecules on the vascular endothelium therefore mediating the first indispensable step for sequestration of neutrophils and or macrophages into the respiratory tract a study by van reeth demonstrated that there is a correlation between bal fluid levels of some cytokines ifnα tnfα and il1 and virus titers neutrophil infiltration and influenza disease additionally lee showed that ifnα tnfα il1 and il6 all participate in nonspecific and specific antiviral immune responses immunomodulatory role of probiotics on influenza virus in the context of preclinical studies mm2 can significantly reduce influenzaelicited proinflammatory cytokines such as il6 and tnfα moreover a slightly elevated ifnα level in the balf indicated the impact of this probiotic on the enhancement of nk cell activity these results along with the reduction of pulmonary mrna levels of nk cell activators including proinflammatory cytokine il1 and chemokines mip2 and mcp1 suggest the modulating effect of this probiotic on influenza infection in another study continuous oral administration of lactobacillus plantarum 06cc2 led to an elevation in the production of ifnα and th1 cytokines il12 and ifnÎ and reduction in the production of tnfα and il6 cytokines in balf this probiotic could also control the number of total infiltrated cells such as macrophages and neutrophils in the balf of infected mice similarly nagai revealed that days after oral administration of the yogurt fermented with l bulgaricus oll1073r1 or its exopolysaccharides epss influenza virus infection ameliorated which attributed to the development of nk cell activity of splenocytes assessment of kimchiderived lactobacillus plantarum and leuconostoc mesenteroides has confirmed their effectiveness against lethal influenza viruses h1n1 and h7n9 by decreasing the sizes of viral plaques both in vitro and in vivo in addition it has been shown that lactococcal strains or their eps induced weight regain and also reduced viral titer in the lung of mice infected with influenza virus h1n1 starosila investigated the antiviral ability of bacillus subtilis and showed that after a single dose administration of the probiotic bacteria the survival rate of mice challenged with the ifv increased song assessed the impact of oral intake of lactobacillus rhamnosus m21 on lethally ifvinfected mice an increase in the level of ifnÎ and il12 and a decline in il4 level suggested that this probiotic can modulate some disease outcomes attributed to changes in cytokine profiles such as that happens in the lung after influenza infection in our very recent study we showed that bifidobacterium bifidum can increase the level of both th1 ifn“y and il12 and th2 il4 since the manifestation of probiotics impacts on several diseases from nonviral to viral ones [“] several studies have surveyed the probiotic roles in immune responses of influenzainfected animal models it has been fully demonstrated that upon infection with influenza many cytokines such as il12 one of the mediators of th1 immuneresponse interferon ifnÎ representative of th1 cytokine il4 and il10 th2 cytokines il1α il1 il6 and tumor necrosis factor tnfα proinflammatory cytokines and ifnα and ifn are produced in the respiratory tract [“] studies on ameliorating influenza infection as well as alleviating influenza symptoms have been trying to redress the imbalance attributed to runaway cytokines production namely cytokine storm after ifv infection kawahara demonstrated that probiotic bifidobacterium longum fig schematic presentation of possible mechanisms of probiotic immunomodulation effects in the intestine probiotics trigger immunomodulation through direct and indirect interaction with intestinal epithelial cells dendritic cells extend their dendrites between intestinal epithelial cells iecs and might directly sample and process probiotics in the gut lumen leading to activation of innate and adaptive immune responses dendritic cells present immediately below m cells engulf probiotics resulting in the maturation of dcs and may derive b cells into plasma cells additionally after the interaction of probiotics with macrophages and dendritic cells presented in lamina propria these cells are activated and induce nk cell activation which leads to ifnÎ elevation to defend against viruses upon the interaction of probiotics™ pamps with different types of tolllike receptors tlrs nuclear factorκb nfκbmediated antiviral gene expression is stimulated eventually active immune cells migrate to sites of infection through lymphatic and circulatory systems to defend against respiratory viruses microbialpathogenesis14820201044523 0canother study enterococcus faecalis1 has been proved to improve the body weight and feed conversion ratio of treated broilers and also significantly elevated the total igy serum level resulting in efficient modulation of the cecal microbiota and decrease in the mortality percentage of broilers an investigation on the possible effect of interaction between lactobacilli and chicken macrophages on eliciting antiviral responses against the aiv showed that certain probiotic species such as l acidophilus and l salivarius when administered as live bacteria either alone or in combination can induce an antiviral response in chicken macrophages in another study seo reported that live leuconostoc mesenteroides yml003 significantly restored the body weight and increased the ifnÎ levels in splenic cells of lowpathogenic aiv h9n2infected chickens examining the effectiveness of enterococcus faecium ncimb and zinc oxide in modulating the immune system of piglets in confronting with swine influenza virus siv revealed that the body weights of piglets fed with the probiotic and vaccinated with trivalent influenza vaccine significantly increased and noticeably higher h3n2specific antibodies were detected among them based on these considerations probiotics administration is effective in the secretion of high concentration of cytokines from immune cells located in the airway leading to the migration of immune cells to the lung space and thereby amelioration of influenza infection fig the probiotic effects on coronavirus infections m mahooti cytokines an increase in the level of total igg antibodies in pooled sera of treated mice and igg1 and igg2a isotypes demonstrated the efficacy of the probiotic in eliciting humoral immune responses and th1th2 responses respectively moreover it revealed that the level of inflammatory cytokines like il6 which increases upon influenza infection decreased in the probiotic group suggesting the ameliorating potential of this probiotic in influenzainfected mice based on the results of body weight changes survival rates and viral titre among treatment groups of different influenza viruses park showed that lactobacillus plantarum has antiinfluenza effects that are not virus type or straindependent revealing that regular intake of that probiotic can help to alleviate the influenza symptoms concerning the effect of longterm probiotic administration kiso orally injected lactobacillus pentosus b240 to mice for weeks and evaluated its inhibitory properties against influenza challenge assessment of different cytokineschemokines in the lungs of infected animals revealed that excluding il5 administration of that probiotic did not affect the immune system regarding cytokineschemokines secretion however ah1n1 pdm infected mice survived probably due to protecting effects of the probiotic by downregulation of acots acot1 acot2 and acot5 cyr61 egr1 and fos as well as upregulation of stfa1 and antiviral rsad2 genes in the lungs of uninfected mice in agreement with all aforementioned results harata revealed that oral administration of probiotics lactobacillus gg and l gasseri tmc035 in mice infected with a lethal dose of influenza ah1n1 pdm prompted the secretion of il12 il6 ifnÎ and iga from isolated pp cells in vitro however unlike lactobacillus gg the oral administration of l gasseri had no impact on the production of ifnÎ il6 as well as total iga in vivo proving the vital role of probiotic interaction with the component cells of galt in the protection against influenza the investigation of the effects of l casei strain shirota on aged mice showed that this probiotic can enhance not only the level of ifnÎ and tnfα but also pulmonary and spleen nk cells activity and thereby ameliorates ifv infection in another study oral administration of bifidobacterium longum bb536 could significantly reduce the loss of body weight inhibit viral proliferation in the lungs and improve the symptoms of influenzainfected mice which may be related to the decreased level of il6 belkacem observed that while administration of probiotic l paracasei induced significantly higher levels of proinflammatory cytokines in probioticfed influenza mice models this trend was reversed seven days upon influenza challenge except for il33 the number of all tissueresident or circulatory myeloid cells and b cells after the probiotic consumption and before viral infection increased and the probiotic administration generated more ifnÎproducing ilc1 mainly nk cells and th2 cells during the late phase of influenza infection additionally l paracasei peptidoglycans administration before influenza infection increased dendritic cells but did not affect other cell types and significantly reduced viral loads besides the effectiveness of oral administration of probiotics intranasal administration of lactobacillus pentosus spt84 to mice proved to induce the production of il12 and ifnÎ in mediastinal lymph node mln cells and il12 and ifnα in balf thereby improved the survival rates of mice reduced the ifv titer in balf and subsequently suppressed influenza infection in mice employing the novel sublingual route lee showed that in contrary to proinflammatory cytokines the level of il12 in the lung homogenates of mice treated with lactobacillus rhamnosus significantly increased in addition besides the increase in nk cell activities and antiinfluenza virus iga the expression of cd25 by both cd8 and cd4 lymphocytes highly increased in the lungs of mice these results recommend that compared to the traditional methods sublingual delivery is a more effective way for the administration of probiotics against seasonal and pandemic influenza regarding other animal models poorbaghi et al showed that microencapsulated lactobacillus acidophilus probiotic and its symbiotic form with inulin decreased faecal shedding of h9n2 avian influenza virus aiv in both nonvaccinated and vaccinated broiler chicks in the current outbreak of coronavirus disease covid19 reported from wuhan china has again gained global attention to taking a new measure that could work out as fast as possible against such an outbreak of viruses interestingly accumulated data obtained from clinical investigations on patients who suffered from severe covid19 in a hospital in wuhan demonstrated the presence of signs for cytokine storm especially among patients in severe stages of the disease particularly the levels of cytokines and chemokines involved in both th1 such as il1b ifnÎ and th2 il4 and il10 immune responses were promoted in studied patients moreover the levels of ip10 mcp mip1a and tnfα were in direct correlation with the severity of patients™ symptoms on the other hand it has been determined that the cytokine storm may lead to a rise in platelets and longer hospitalization of covid19 patients other studies also have revealed other aspects of virallydriven manipulation of immune responses by human coronaviruses [“] therefore addressing the cytokine storm may be the key for the treatment of patients infected with sarscov2 while some reagents such as steroids can be considered as hyperinflammation suppressors their sideeffects impede to count on them as a trustworthy medicine for covid19 alternatively addressing the urgent need for standing against the increasing rate of morbidity and mortality related to the current pandemic requires employing previously approved therapies harnessing safety profiles probiotics as a safe available treatment with the ability to modulate immune responses and manipulate cytokines production have been considered to be studied against different strains of coronavirus in some studies soon moreover a clinical survey has reported an intestine microbiota imbalance in particular a decline in the level of probiotics such as lactobacillus and bifidobacterium among some covid19 patients which may result in secondary infection in response to bacterial translocation one report has shown that the recent outbreak of porcine epidemic diarrhea virus pedv can be prevented through the use of either cell free supernatants cfs or live lactic acid bacteria lab it demonstrated that probiotics though the precise mechanism is not clear could be effective against the pandemic strain of pedv in a strainspecific manner using cpe reduction assays that further confirmed by qualitative immunofluorescence in another investigation lactobacillus casei was used as a carrier for the dctargeting peptide dcpep fused with the pedv core neutralizing epitope coe antigen this survey demonstrated that this genetically engineered lactobacillus casei oral vaccine is able to induce systemic igg and mucosal siga antibody microbialpathogenesis14820201044524 0cm mahooti fig model of the interaction of active immune cells triggered by probiotics with respiratory viruses in the lung following virus infection immune cells in the airway such as dendritic cells and macrophages secrete cytokines to defend against viruses in a probioticreceived subject the high concentration of cytokines leads to the migration of immune cells to the lung space through the gut“lung axis resulting in rapid recruitment of activated t and b cells in the lung that eventually promote upregulation of virusspecific immunoglobulins and cytokines in probioticreceived subject whereas in the absence of activated immune cells the respiratory virus can cause severe lung damage due to the lack of immediate immune responses responses in mice models there have been other articles using different types of probiotics for displaying the desired genes or antigens against pedv [“] for instance liu demonstrated that their modified lactobacillus plantarum has the property to act like a strong antiviral agent against coronavirus infection in the intestinal porcine epithelial cell line probiotic impacts on other viral respiratory infections eguchi et al demonstrated that lactobacillus gasseri sbt2055 lg2055 when administered orally to mice before infection with a human rsv could suppress the virus titre in lung tissue homogenates rsv replication and the intensity of the symptoms moreover a decrease in the expression level of proinflammatory cytokines and an increase in the mrna level of ifn ifnÎ oas1a and isg15 in the mice lung upon probiotic administration are satisfactory evidence for antiviral properties of this probiotic also swi2snf2related creb binding protein activator protein srcap introduced as a candidate for the antiviral activity of lg2055 against rsv to investigate whether probiotics can control the inflammatory pathway and modulate the coagulation system upon respiratory viral infection rhamnosus crl1505 was orally administered in rsv or ifv mice models the results elucidated that this probiotic could successfully modulate tlr3triggered immune coagulation reaction in the lung upon viral infection and prevent exacerbated respiratory injuries notably this study substantiated the vital role of probioticprovoked secretion of il10 in taming the coagulation system after the viral attack additionally in a study conducted by tomosada nasal administration of lactobacillus rhamnosus crl1505 and crl1506 has proved to modulate elevated respiratory levels of the proinflammatory mediators caused by administration of the viral pathogenassociated molecular pattern polyic moreover a nasal administration of the probiotic prior to pfu of rsv challenge improved resistance against rsv infection considering the effect of probiotics on the parainfluenza virus there is only one study evaluating the antiviral effects of oral administration of lactococcus lactis subsp lactis jcm5805 in a mouse model of murine parainfluenza virus mpiv1 infection the probiotic administration resulted in a rise in the survival rate of treated mice without any weight loss and also a decline in the lung histopathology scores compared to the nontreated group which was attributed to the incorporation of jcm5805 into cd11c immune cells in pp and thereafter activation of pp pdcs and ultimately elevation of ifns expression it is of note that although no activated local pdcs were observed at lung upregulation in ifnsinduced antiviral factors in the lung may be due to the delivery of ifns from the intestine of jcm5805fed mice into the lung studies reporting the effects of probiotic bacterias on respiratory viruses have been demonstrated in table supplementary section clinical evidence of probiotic immunomodulation in a pilot study intake of lactobacillus brevis kb290 has shown to curtail the incidence of influenza infection among schoolchildren with no adverse effects associated with consuming the probioticcontaining drinks hu demonstrated that h7n9 ifv infection led to a decrease in intestinal microbial diversity and species richness among patients they observed that although administration of c butyricum probiotic was unable to alleviate the antibioticrelated disturbances in the gut microbiome of h7n9infected patients an increase in microbiota diversity and evenness gradually appeared through continuous administration of probiotics after antibiotic cessation additionally based on the evaluation of crp levels or bacteremia and pneumonia in the patients treated with probiotics the safety of probiotic administration was approved and no inflammatory effects were observed in another study conducted by wang the impact of lactobacillus rhamnosus gg administration on nursing home residents aged and older was assessed it revealed that probiotic administration reduced the risk of influenza and other respiratory viral infections among the elderly received probiotics compared to those receiving a placebo although not statistically significant the trial provided a microbialpathogenesis14820201044525 0ctable clinical studies reporting regulatory effect of probiotic bacteria on immune responses study design an openlabel parallelgroup trial main findings the risk of infection †“ subjects schoolchildren “ years of age a retrospective study patients nursing home residents years of age and older a randomized doubleblind placebo controlled pilot trial probiotics used lactobacillus brevis kb290 kb290 days per week for weeks in the form of test drink containing × cfu clostridium butyricum three times per day at the dose of cfu tablet prior to h7n9 infection lactobacillus rhamnosus gg twice a day for months in the form of capsule containing cfu m mahooti framework to assess the effectiveness of probiotics in reducing respiratory infections among senescent individuals similarly it has been shown that there is no connection between intaking the yogurt fermented with probiotic lactobacillus delbrueckii ssp bulgaricus oll1073r1 and incidence rate of influenza in humans however the immunological study showed an increase in the level of ifnÎ in the probiotictreated group while there are some available treatments for hrv the most frequent cause of the common cold most of them have failed to be efficient in clinical trials due to their drawbacks in this regard probiotics have shown to prevent or treat common colds and upper respiratory infections several studies have revealed that the rhinovirusrelated common cold pathogenesis is associated to the innate inflammatory response to the virus therefore more attempts have done to incorporate probiotics to modulate immune responses consequently leading to balanced responses and optimal outcomes in combating viral infection in this regard an investigation on the impact of bifidobacterium animalis ssp lactis bl04 on healthy adults showed a modest modulation of innate immune host responses upon infection with hrv particularly reduction of cxcl8 response in the nasal lavage resulting in a decline in the rhinovirus replication approved by a decrease in virus shedding in the nasal secretions tapiovaara demonstrated that adults consumed juice enriched with live or heatinactivated l rhamnosus gg before intranasal inoculation of hrv showed no significant differences in nasopharyngeal hrv loads compared to the placebo group another survey has illustrated that consumption of probiotic is a good strategy to prevent viral rtis in the first year of life in a cohort of preterm infants the results showed that the probioticdriven change in microbiota leads to the induction of longlasting effects which can reduce the risk of viral rtis in agreement with that study it was demonstrated that live l rhamnosus gg might be more effective in reducing the rhinovirus infection rate than the inactivated form of the same strain respiratory syncytial virus from the paramyxoviridae family is considered as the major cause of lower respiratory tract infection in infants and children around the world and is becoming an important pathogen of the elderly although most children have experienced a first rsv infection by two years of age some cases suffering premature birth bronchopulmonary dysplasia immunodeficiency and congenital heart disease are vulnerable to symptoms worsening and hospitalization as well however the probiotic administration has proved to be effective in developing protection against virallyinduced inflammation besides there is a study demonstrating that while probiotic consumption significantly reduced the number of days with respiratory symptoms during the intervention no significant effect was neither observed on the occurrence of viruses in the nasopharynx nor on the symptoms during viral episodes among daycare children clinical studies reporting regulation of immune responses by probiotic bacterias have been presented in table conclusion bifidobacterium animalis daily for days in the Answer:

Colon_Cancer

" preproof 0c highlights f0b7 propolis made by bees from bioactive plant resins has antiviral activity f0b7 propolis potentially can interfere with host cell invasion by sarscov2 f0b7 propolis blocks proinflammatory pak1 a kinase highly expressed in covid19 patients propolis a resinous material produced by honey bees from plant exudates has long been used in traditional herbal medicine and is widely consumed as a health aid and immune system booster the covid19 pandemic has renewed interest in propolis products worldwide fortunately various aspects of the sarscov2 infection mechanism are potential targets for propolis compounds sarscov2 entry into host cells is characterized by viral spike protein f0b7 propolis is a safe widely consumed functional food with medicinal properties f0b7 standardized propolis has consistent properties for lab and clinical research preprooffactor in advanced covid19 disease propolis has also shown promise as an aid in the treatment of various of the comorbidities that are particularly dangerous in covid19 patients including induced lung inflammation fibrosis and immune system suppression propolis components have inhibitory effects on the ace2 tmprss2 and pak1 signaling pathways in addition antiviral activity has been proven in vitro and in vivo in preclinical studies propolis promoted α this immunoregulation involves monocytes and macrophages as well as jak2stat3 nfkb and inflammasome pathways reducing the risk of cytokine storm syndrome a major mortality interaction with cellular angiotensinconverting enzyme ace2 and serine protease tmprss2 this mechanism involves pak1 overexpression which is a kinase that mediates coronavirusimmunoregulation of proinflammatory cytokines including reduction in il6 il1 beta and tnfrespiratory diseases hypertension diabetes and cancer standardized propolis products with consistent bioactive properties are now available given the current emergency caused by the covid19 pandemic and limited therapeutic options propolis is presented as a promising and 0crelevant therapeutic option that is safe easy to administrate orally and is readily available as a natural supplement and functional food keywords propolis sarscov2 covid19 antiviral antiinflammatory pak1 blocker introduction the covid19 pandemic is of grave concern due its impact on human health and on the deadly disease most require more robust data in clinical trials before they can be widely and safely used isolation and stayathome measures do not effectively protect essential workers economy it is much more deadly than influenza and other types of diseases that recently have had worldwide impact forcing countries to take unusual measures such as limiting travel closing schools businesses and other locations where many people can come into contact with each other various public healthcare strategies have been adopted in an attempt to reduce the impact of the disease but with limited effectiveness as the virus continues to spread often through asymptomatic patients unfortunately tests to determine if people are infectious or were previously infected are not widely available often are costly and frequently do not provide timely and accurate results various therapeutic alternatives have been proposed and tested however preproofœnonessential professions return to the workplace they become more at risk for infection in this scenario any options that could help ameliorate disease progression and its consequences even marginally would be useful the world needs safe alternatives to help reduce the impact of this especially health care personnel who have become infected and are dying at alarming rates economic and other necessities limit how well and how long these isolation measures can be maintained especially in poor countries and in poor communities such as slums and favelas [ ] as populations gradually try to get back to normalcy reducing social distancing and people in natural products which have historically been widely used to help avoid and alleviate diseases are among the options being considered as an adjuvant treatment for sarscov2 infection because they are generally inexpensive widely available and rarely have undesirable side effects some have proven antiviral activity an important advantage of using natural remedies is that people who have other health problems or who have mild flurelated 0c infection infection by sarscov2 the virus that causes covid19 is characterized by binding symptoms but do not have the means or courage to visit an already overcrowded medical facility could take simple and inexpensive measures to help reduce the impact of infection with sarscov2 considering the large number of deaths and other types of damage that the covid19 pandemic is causing there is an urgent need to find treatments that have been approved as safe and potentially able to inhibit the new coronavirus reduce its infectivity andor alleviate the symptoms of infection [ ] along this line propolis and its components emerge as potential candidate materials that could help to reduce the pathophysiological consequences of sarscovfactors increased pak1 levels also suppress the adaptive immune response facilitating viral replication [ ] sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory impairment and pulmonary failure immunologicalinflammatory between viral spike proteins and angiotensinconverting enzyme ace2 activation of the spike protein is mediated through proteases such as tmprss2 which play important roles in the viral infection after entry followed by endocytosis coronavirus infection causes pak1 upregulation a kinase that mediates lung inflammation lung fibrosis and other critical mortality preproofphenomena such as cytokine release syndrome have been shown to be important in the spectrum of sarscov2 infection these mechanisms are associated with an dysfunction more than the viral load per se along this line a retrospective observational study found higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα in patients with severe inflammatory diseases by affecting various metabolic cycles recently several studies have shown that propolis extract and some of its components act against several important targets in the pathophysiological context of the disease caused by sarscov2 such as reducing tmprss2 expression and reducing ace2 anchorage which would otherwise facilitate entry of covid19 compared to individuals with mild disease there is considerable evidence that propolis can reduce and alleviate the symptoms of the virus into the cell this is in addition to immunomodulation of monocytes macrophages reducing production of and eliminating il1 beta and il6 reduction of the transcription factors 0cviral replication and antiinflammatory action [ ] propolis and its properties are particularly relevant to sarscov2 infection such as immune system fortification reduced nfkb and jak2 stat3 and blocking pak1 which determine inflammatory activities and fibrosis caused by covid19 various comorbidities have been associated with severe covid19 symptoms and a greater chance of patients requiring intensive care these include hypertension and diabetes also mortality rates of covid19 patients are much higher in those with cardiovascular disease chronic respiratory disease and diabetes [ ] there is considerable evidence that these conditions could be alleviated by treatment with propolis this includes research in animal models of diabetes [ ] hypertension [ ] and cardiovascular disease [ ] propolis has properties that preproofthese plant products to make propolis which they use to protect the colony the production and use of propolis by honey bees evolved to the point that these social bees have considerably fewer immune genes than solitary insect species bees in colonies that produce more propolis are healthier and live longer and propolis consumption by the bees augments their immune [ ] as this variability can affect their medicinal properties standardized propolis products have been developed to help meet the need for a product that does not vary in the main bioactive components and is safe with minimal interaction with pharmaceutical drugs and proven efficacy in clinical trials in recent decades it has been shown to have antimicrobial including the composition of propolis varies according to the plant species available in each region propolis is a product derived from resins and plant exudates plants defend themselves from pathogens mainly by producing phytochemicals many of which have been extracted and used in medicine plant defense substances collected by bees include phenols and terpenoids phytochemical compounds that show promise for the inhibition of coronavirus in humans include quercetin myricetin and caffeic acid all components of propolis honey bees and many other species of social bees recognize these antimicrobial properties and selectively collect and process response to bacterial challenge antiviral antiinflammatory immunomodulatory antioxidant and anticancer properties propolis has historically been widely used to alleviate various diseases [ ] it also has been considered among other natural alternatives as an adjuvant treatment for sarscov2 infection 0ccompared to the propolis that the bees make from these plant materials because it is generally inexpensive widely available and rarely causes undesirable side effects some types of propolis that are highly valued for their medicinal properties such as brazilian green propolis are mainly produced by bees from materials they collect from specific plants in this case baccharis dracunculifolia after the botanical origin of the propolis has been identified extracts of the plant can be made to develop useful products such as a medicinal mouthwash however the medicinal properties of these plant extracts are often inferior can be safely consumed these propolis products and the raw material for their manufacture are extensively exported by brazilian companies especially to asian countries including japan south among natural medicine alternatives propolis has been widely studied and is already extensively consumed in many countries [ ] for example propolis products such as throat sprays and extracts are produced by hundreds of companies in brazil and are sold as a health aid in nearly every pharmacy throughout the country demonstrating on a practical basis that they preproofin fact propolis has a wide spectrum of pharmacological properties and is a dietary supplement that is commonly consumed by both healthy and sick people as a preventative precaution and for treatment it is also used in veterinary medicine due its antibacterial antifungal antiviral korea and china [ ] the importance of propolis in chinese japanese russian and korean medicine is reflected in the number of patents for propolis containing products registered by including about by china and “ each for japan russia and korea since about new propolisrelated patents were applied for in the us patent office it is a key ingredient in traditional chinese medicine japanese scientists have isolated and patented various brazilian propolis components for cancer treatment demonstrating their usefulness why propolis may be a good fit for dealing with covid19 antiparasitic hepatoprotective and immunomodulatory activities in the wake of the coronavirus outbreak south korea has seen a boon in the use of functional foods according to their ministry of food and drug safety œhealth functional foods are nutrients that have been proven to be beneficial to health in march of this year in response to the coronavirus pandemic the ministry eased regulations for propolis which is considered a 0ccould help fight against the covid19 pandemic active site of this enzyme is a relevant target for drug discovery functional food and allowed new oral formulations however despite considerable evidence that propolis can reduce and alleviate disease symptoms its acceptance as a healthpromoting supplement in human medicine has been limited in many countries such as the usa because of a relevant criticism that propolis products are not standardized and vary in their components and biological activity in part this is because propolis varies with the species of plants available in each region from which the bees collect resins to produce it [ ] however standardized propolis products have recently become available to help fill the need for a product that does not vary in the main bioactive components and effectiveness [ ] one such option a standardized brazilian propolis extract blend has been tested for safety and effectiveness in clinical trials for treating kidney disease and diabetes denture stomatitis and burn patients therefore propolis as a nutraceutical or functional food should be considered as a resource that preproofcaffeic acid phenethyl ester cape galangin chrysin and caffeic acid substances found in several different types of propolis around the world appeared as potential drugs against this viral target table specifically cape was predicted to interact with sarscov2 mpro in a potential targets in order to inactive the virus and reduce the damage that it causes the main protease of coronavirus sarscov2 mpro 3chymotrypsinlike cysteine enzyme is essential similar study therefore although it will be necessary to run in vitro assays to evaluate the potential antisarscov2 effects of propolis andor its constituents these in silico results are along this line hashem evaluated various natural compounds with an in silico approach molecular docking to try to find useful options for treating sarscov2 infection curiously for coronavirus processing of polyproteins and for its life cycle and therefore inhibition of the some propolis compounds can potentially interact with sarscov2 mpro the research community has examined the genetic code of coronavirus and the mechanisms underlying the damages caused by sarscov2 to help search for drugs andor well boding propolis can interact with ace and tmprss2 potentially blocking or reducing sarscov2 invasion of the host cell 0c sarscov2 strongly binds to angiotensinconverting enzyme ace2 using this enzyme as a receptor for invasion and replication in the host cell [ ] causing damage and increasing interpersonal transmission [ ] consequently ace inhibitors have been considered as useful drug alternatives however potential deleterious effects on users of angiotensinconverting enzyme ace inhibitors and angiotensin receptor blockers arbs have tool against potential cardiovascular events inhibition of ace2 enzyme is an important target for treatment against sarscov2 myricetin caffeic acid phenethyl ester hesperetin and pinocembrin rutin interacts with zinc fingers of the active sites of ace2 a metalloprotease that presents the same zinc finger in ace1 emerged as a concern for treatment of covid19 patients an observational study involving patients did not confirm this suspicion and therefore these classes of drugs remain an important infection [ ] güler et al prepared an alcoholic extract of propolis and identified some hydroxycinnamic acids caffeic acid pcoumaric acid tcinnamic acid and cape the flavanons rutin and myricetin and the flavones hesperidin chrysin and pinocembrin using molecular docking evaluations they found that rutin had the highest binding energy to ace2 followed by preproofvarious characteristics including inhibition of ace they found strong inhibition for most of the propolis types they studied with higher than ace inhibition the best results were found in addition to the in silico evidence osés et al evaluated several types of propolis for with the propolis components catechin and pcoumaric acid ace2 and tmprss2 transmembrane serine protease on the surface of host cells are used by sarscov2 via interaction with spike glycoproteins in order to proceed with invasion and replication vardhan sahoo studied several molecules commonly found in medicinal herbs using molecular docking procedures with relevant targets such as rnadependent rna polymerase rdrp ace2 and spike glycoproteins and compared the resulting scores with those of hydroxychloroquine limonin was the most active compound however quercetin and kaempferol also propolis compounds gave high docking scores kaempferol was studied in prostate cancer models and the expression of tmprss2 was reduced showing a potential mechanism of action for an antitumoral effect kaempferol could be an important propolis component for use against covid19 since it is involved in the inhibition of tmprss2 0c potentially interacting with ace2 rdrp and spike glycoprotein sgp besides its antiviral activity table propolis blocks pak1 potentially avoiding lung fibrosis and restoring a normal immune response among the possible targets for controlling covid19 damage the major œpathogenic contributes to their suppression pak1 inhibitors can both help combat the virus and restore a normal immune response kinase pak1 is key it is an essential component in malaria and viral infections but it is also involved in a wide variety of other diseases and disease conditions including cancer inflammation and immunosuppression when abnormally activated consequences of pak1 activation include lung fibrosis which is an aggravating factor in covid19 pak1 is activated by rac xu et al demonstrated that caffeic acid and its ester cape components of propolis can inactivate rac consequently inhibiting pak1 the inactivation of pak1 directly or upstream can potentially attenuate coronavirus pathogenesis bcells and tcells are lymphocytes that produce specific antibodies against viruses and other intruders and pak1 preproofimprove the immune response its components increase neutralizing antibody titers activate phagocytosis and increase ifnγ levels and the number of lymphocytes an increase in ifnγ levels was also detected by shimizu et al who evaluated the mechanisms involved in the cape caffeic acid phenethyl ester is a potent inhibitor of activation of nfkb in myelomonocytic cells anse et al demonstrated that propolis cape quercetin hesperidin and some other propolis flavonoids can inhibit the cytokine production of th1 and th2 type t cells while increasing tgfbeta an important antiinflammatory cytokine moreover cape can attenuate oxidative stress and inflammation through downregulation of jak2stat3 signaling propolis from europe and temperate asia usually made by bees from resins collected from poplar trees has predominantly flavonoid compounds while green propolis from baccharis dracunculifolia a propolis exclusively found in brazil has various kinds of flavonoids and prenylated phenylpropanoids such as artepellin c baccharin and drupanin these and all other types of propolis can inactivate pak1 artepillin c selectively inhibits pak1 table some studies have shown that propolis can act as an immunostimulant with the ability to effects of some types of propolis in a herpes simplex animal model 0c as well as having an immunomodulatory effect in which cape inhibits il6 phosphorylation and stat3 which are important for proinflammatory th17 development besides the antiinflammatory effect of cape and kaempferol paulino et al evaluated the antiinflammatory effect of artepellin c in rat paw edema and in cell cultures demonstrating that the activity was at least in part mediated by prostaglandin e2 and no inhibition through nfkb modulation artepillin c is an important biomarker of brazilian green propolis botanical source baccharis dracunculifolia immune modulation is desirable since coronavirus infection dysregulates the immune response in the initial phases of infection which facilitates viral replication however in later stages of covid19 the body develops an exaggerated inflammatory response which can greatly damage the lungs and other ans propolis different from typical immunosuppressants can help avoid immunosuppression during the initial phases of disease and in later stages reduce an exaggerated host inflammatory response inhibiting excess il6 il2 and jak signaling cape a propolis component is also known as an immunemodulating agent and should be considered as an alternative to help reduce an exaggerated inflammatory response in a mouse model propolis had immunomodulatory action in vivo on tolllike receptor expression and on preproofreplication and infectivity potentially decreasing lung inflammation due to antiinflammatory properties while promoting immune system fortification these are useful properties that could there is ample evidence for interference of propolis andor its components with viral propolis has been tested against various viral disease anisms initial successes have prompted research to determine the most useful components which may be modified to produce more active and specific pharmaceuticals viruses that were controlled by propolis in animal models with suggestion for control in humans include influenza [ ] herpes simplex virus type and hiv [ ] shimizu et al evaluated three different types of propolis in ethanol extracts using a murine model of herpes simplex virus type despite the chemical differences proinflammatory cytokine production help minimize the symptoms and deleterious effects of covid19 figure figure propolis as an antiviral substance 0cdue to the different plant origins of the resins the bees used to produce the propolis baccharis dracunculifolia baccharis eriodata and myrceugenia euosma all three propolis extracts not only had direct antihsv1 effects but also stimulated immunological activity against intradermal hsv1 infection in mice antiviral activity of propolis has been reported for dna and rna viruses poliovirus herpes simplex virus and adenovirus in an in vitro model cultured cells the best results were obtained against poliovirus and herpes virus with inhibition of the latter at a propolis concentration of ugml the propolis components chrysine and kaempferol caused a concentrationdependent reduction of intracellular replication of herpesvirus strains when host cell monolayers were infected and subsequently cultured in a drugcontaining medium quercetin another propolis component had the same effect but only at the highest concentrations tested ugml against various human herpes simplex virus strains with a intracellular replication reduction of approximately while it reduced the infectivity of bovine herpes virus human adenovirus human coronavirus and bovine coronavirus about the reduction was in the preproofimpairment and pulmonary failure inflammatory response to infection sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory immunologicalinflammatory phenomena such as cytokine release syndrome have been shown to be important mortality factors in sarscov2 infection higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα are found in patients with severe covid compared to those of individuals with mild disease molecular mechanisms involved in this immune process are the targets of various synthetic medicines being tested in patients including ciclesonide hydroxy chloroquine ivermectin and ketorolac which are pak1 blockers pak1 raccdc42activated kinase is overexpressed in the lung in response to sarscov2 infection and is a critical mediator of the cytokine storm that frequently results in mortality the most critical cases of covid19 which require ventilatorassisted intensive care and often result in prolonged ventilator dependency and death are a result of an exaggerated case of rotavirus antiinflammatory and immunomodulatory properties of propolis 0cin hospitalized patients fortuitously propolis components are effective pak1 blockers table there is considerable evidence that propolis can reduce and alleviate the symptoms of inflammatory diseases and has immunomodulatory properties [ ] however these properties can vary according to the plant origin of the propolis as well as the extraction processsolvent used and the inflammatory protocol cell culture animal models induction by lipopolysaccharides when the propolis extracts are tested tests with animal models have shown that propolis can reduce the levels of il6 and tnfalpha which are key proinflammatory mediators and increase the levels of the regulatory cytokine il10 kaempferol a propolis component reduces il6 tnfalpha and vegf vascular endothelial growth factor via the preproofifnγ in serum fernandes et al found that propolis exerted a positive adjuvant effect on vaccines that were developed against canine coronavirus they assayed ifnγ which is an effective way to measure the cellular response induced by a vaccine in a mouse model propolis added as an adjuvant to inactivated swine herpesvirus type vaccine stimulated increased cellular propolis is considered a safe immunostimulant and a potent vaccine adjuvant propolis has been widely tested as a vaccine adjuvant because it induces an earlier immune response and provides a longer protection period it is also included as an adjuvant ingredient in traditional chinese medicine propolis flavonoids have potential as adjuvants enhancing igg il4 and propolis has potential as a vaccine adjuvant erknfkbcmycp21 pathway table tests on macrophage cell cultures also demonstrated that propolis inhibits the production of il1 beta an important component of the inflammasome inflammatory pathway in diseases such as rheumatoid arthritis lupus and other autoimmune diseases although the mechanisms of action are not well elucidated these propolis components have potential as complementary supplements in the preventive treatment of chronic inflammatory diseases and humoral responses increasing ifnγ [ ] propolis enhanced the immune response to inactivated porcine parvovirus vaccine in guinea pigs when added to a trichomonas vaginalis protein vaccine propolis increased the igg antibody response times in mice 0ccancer is considered a relevant comorbidity factor for covid19 cancer patients have a patients cancer patients compared to the protein alone propolis was also effective as an adjuvant in the immunization of cattle with bovine herpesvirus it improved the humoral and cellular responses in mice inoculated with inactivated virus vaccines propolis as an adjuvant gave a similar immune response increasing ifnγ levels to alum and freund™s adjuvant in mice vaccinated with an hiv1 polytope vaccine candidate with less risk of undesirable side effects comorbidities and evidence of how propolis can help reduce their impact in covid19 to risk a visit to a clinic or hospital to determine if they have cancer alternative therapies could help retard cancer or reduce the impact of cancer and cancer treatment in covid19 times higher risk of progressing to severe covid19 disease than patients without comorbidities also the hospital environment during the coronavirus pandemic can interfere with or delay the treatment that cancer patients should receive patients with symptoms may choose not preproofacid cape quercetin and chrysin propolis and its components normally have little impact on normal cells displaying differential cytotoxicity in liver cancer melanoma and breast cell carcinoma cell lines [ ] propolis enhances the activity of tumor various types including bladder blood brain breast colon head and neck kidney liver pancreas prostate and skin cancers propolis could help prevent cancer progression in various parts of the world it is considered an alternative therapy for cancer treatment propolis extracts have been found to inhibit tumor cell growth both in vitro and in vivo including inhibition of angiogenesis demonstrating potential for the development of new anticancer drugs various components of propolis have been shown to inhibit cancer cell growth including cinnamic propolis has potential as a complementary therapy for cancer it has shown efficacy against necrosis factor related apoptosis inducing ligand trail in cancer cells hypertension and cardiovascular disease 0c hypertension and cardiovascular disease are considered relevant comorbidities for covid19 propolis has demonstrated antihypertensive effects in rat models [ ] in cameroon it is used in popular medicine to treat various ailments including high blood pressure propolis has been widely used as a dietary supplement for its health benefits including cardiovascular protective effects [ ] in a human trial consumption of propolis improved critical blood parameters including hdl gsh and tbars levels demonstrating that obesity a natural antiobesity agent it could contribute to a reduced risk for cardiovascular disease obesity is a major comorbidity and predictor of increased mortality in covd19 patients obesity and sarscov2 both induce an inflammatory process exacerbating sarscov2 infection in the obese propolis reduced inflammation and prevented hyperlipidemia and metabolic syndromes in highly caloric diet induced obesity in mice body weight gain visceral adipose tissue liver and serum triglycerides cholesterol and nonesterified fatty acids were all reduced in the propolis fed mice [ ] caffeic acid phenethyl ester a propolis component is preproofplatelet aggregation was reduced by propolis in tests on human blood in vitro and in other in vitro tests caffeic acid phenethyl ester cape a wellstudied bioactive propolis component inhibits collagen induced platelet activation thromboembolism thrombosis and microthrombosis microthrombosis disseminated intravascular coagulation and consequent multian failure are common in severely affected covid19 patients with associated high mortality rates anticoagulants are sometimes prescribed to such patients because they can reduce mortality tang et al an elevated level of plasminogen activator inhibitor1 pai1 is a biomarker and risk factor for thrombosis and atherosclerosis [ ] various types of evidence demonstrate that propolis can reduce platelet aggregation and other thrombosisrelated parameters propolis decreased thrombotic tendencies in mice by suppressing lipopolysaccharideinduced increases in pai1 levels [ ] propolis downregulated plateletderived growth factor and platelet endothelial cell adhesion molecules in lowdensity lipoprotein knockout mice 0c old age the elderly are more often affected by chronic inflammation characterized by systemically increased levels of proinflammatory cytokines which can contribute to development of a cytokine storm a major cause of covid19 mortality propolis has antioxidant properties which could help retard o

cancer7527

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " preproof 0c highlights f0b7 propolis made by bees from bioactive plant resins has antiviral activity f0b7 propolis potentially can interfere with host cell invasion by sarscov2 f0b7 propolis blocks proinflammatory pak1 a kinase highly expressed in covid19 patients propolis a resinous material produced by honey bees from plant exudates has long been used in traditional herbal medicine and is widely consumed as a health aid and immune system booster the covid19 pandemic has renewed interest in propolis products worldwide fortunately various aspects of the sarscov2 infection mechanism are potential targets for propolis compounds sarscov2 entry into host cells is characterized by viral spike protein f0b7 propolis is a safe widely consumed functional food with medicinal properties f0b7 standardized propolis has consistent properties for lab and clinical research preprooffactor in advanced covid19 disease propolis has also shown promise as an aid in the treatment of various of the comorbidities that are particularly dangerous in covid19 patients including induced lung inflammation fibrosis and immune system suppression propolis components have inhibitory effects on the ace2 tmprss2 and pak1 signaling pathways in addition antiviral activity has been proven in vitro and in vivo in preclinical studies propolis promoted α this immunoregulation involves monocytes and macrophages as well as jak2stat3 nfkb and inflammasome pathways reducing the risk of cytokine storm syndrome a major mortality interaction with cellular angiotensinconverting enzyme ace2 and serine protease tmprss2 this mechanism involves pak1 overexpression which is a kinase that mediates coronavirusimmunoregulation of proinflammatory cytokines including reduction in il6 il1 beta and tnfrespiratory diseases hypertension diabetes and cancer standardized propolis products with consistent bioactive properties are now available given the current emergency caused by the covid19 pandemic and limited therapeutic options propolis is presented as a promising and 0crelevant therapeutic option that is safe easy to administrate orally and is readily available as a natural supplement and functional food keywords propolis sarscov2 covid19 antiviral antiinflammatory pak1 blocker introduction the covid19 pandemic is of grave concern due its impact on human health and on the deadly disease most require more robust data in clinical trials before they can be widely and safely used isolation and stayathome measures do not effectively protect essential workers economy it is much more deadly than influenza and other types of diseases that recently have had worldwide impact forcing countries to take unusual measures such as limiting travel closing schools businesses and other locations where many people can come into contact with each other various public healthcare strategies have been adopted in an attempt to reduce the impact of the disease but with limited effectiveness as the virus continues to spread often through asymptomatic patients unfortunately tests to determine if people are infectious or were previously infected are not widely available often are costly and frequently do not provide timely and accurate results various therapeutic alternatives have been proposed and tested however preproofœnonessential professions return to the workplace they become more at risk for infection in this scenario any options that could help ameliorate disease progression and its consequences even marginally would be useful the world needs safe alternatives to help reduce the impact of this especially health care personnel who have become infected and are dying at alarming rates economic and other necessities limit how well and how long these isolation measures can be maintained especially in poor countries and in poor communities such as slums and favelas [ ] as populations gradually try to get back to normalcy reducing social distancing and people in natural products which have historically been widely used to help avoid and alleviate diseases are among the options being considered as an adjuvant treatment for sarscov2 infection because they are generally inexpensive widely available and rarely have undesirable side effects some have proven antiviral activity an important advantage of using natural remedies is that people who have other health problems or who have mild flurelated 0c infection infection by sarscov2 the virus that causes covid19 is characterized by binding symptoms but do not have the means or courage to visit an already overcrowded medical facility could take simple and inexpensive measures to help reduce the impact of infection with sarscov2 considering the large number of deaths and other types of damage that the covid19 pandemic is causing there is an urgent need to find treatments that have been approved as safe and potentially able to inhibit the new coronavirus reduce its infectivity andor alleviate the symptoms of infection [ ] along this line propolis and its components emerge as potential candidate materials that could help to reduce the pathophysiological consequences of sarscovfactors increased pak1 levels also suppress the adaptive immune response facilitating viral replication [ ] sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory impairment and pulmonary failure immunologicalinflammatory between viral spike proteins and angiotensinconverting enzyme ace2 activation of the spike protein is mediated through proteases such as tmprss2 which play important roles in the viral infection after entry followed by endocytosis coronavirus infection causes pak1 upregulation a kinase that mediates lung inflammation lung fibrosis and other critical mortality preproofphenomena such as cytokine release syndrome have been shown to be important in the spectrum of sarscov2 infection these mechanisms are associated with an dysfunction more than the viral load per se along this line a retrospective observational study found higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα in patients with severe inflammatory diseases by affecting various metabolic cycles recently several studies have shown that propolis extract and some of its components act against several important targets in the pathophysiological context of the disease caused by sarscov2 such as reducing tmprss2 expression and reducing ace2 anchorage which would otherwise facilitate entry of covid19 compared to individuals with mild disease there is considerable evidence that propolis can reduce and alleviate the symptoms of the virus into the cell this is in addition to immunomodulation of monocytes macrophages reducing production of and eliminating il1 beta and il6 reduction of the transcription factors 0cviral replication and antiinflammatory action [ ] propolis and its properties are particularly relevant to sarscov2 infection such as immune system fortification reduced nfkb and jak2 stat3 and blocking pak1 which determine inflammatory activities and fibrosis caused by covid19 various comorbidities have been associated with severe covid19 symptoms and a greater chance of patients requiring intensive care these include hypertension and diabetes also mortality rates of covid19 patients are much higher in those with cardiovascular disease chronic respiratory disease and diabetes [ ] there is considerable evidence that these conditions could be alleviated by treatment with propolis this includes research in animal models of diabetes [ ] hypertension [ ] and cardiovascular disease [ ] propolis has properties that preproofthese plant products to make propolis which they use to protect the colony the production and use of propolis by honey bees evolved to the point that these social bees have considerably fewer immune genes than solitary insect species bees in colonies that produce more propolis are healthier and live longer and propolis consumption by the bees augments their immune [ ] as this variability can affect their medicinal properties standardized propolis products have been developed to help meet the need for a product that does not vary in the main bioactive components and is safe with minimal interaction with pharmaceutical drugs and proven efficacy in clinical trials in recent decades it has been shown to have antimicrobial including the composition of propolis varies according to the plant species available in each region propolis is a product derived from resins and plant exudates plants defend themselves from pathogens mainly by producing phytochemicals many of which have been extracted and used in medicine plant defense substances collected by bees include phenols and terpenoids phytochemical compounds that show promise for the inhibition of coronavirus in humans include quercetin myricetin and caffeic acid all components of propolis honey bees and many other species of social bees recognize these antimicrobial properties and selectively collect and process response to bacterial challenge antiviral antiinflammatory immunomodulatory antioxidant and anticancer properties propolis has historically been widely used to alleviate various diseases [ ] it also has been considered among other natural alternatives as an adjuvant treatment for sarscov2 infection 0ccompared to the propolis that the bees make from these plant materials because it is generally inexpensive widely available and rarely causes undesirable side effects some types of propolis that are highly valued for their medicinal properties such as brazilian green propolis are mainly produced by bees from materials they collect from specific plants in this case baccharis dracunculifolia after the botanical origin of the propolis has been identified extracts of the plant can be made to develop useful products such as a medicinal mouthwash however the medicinal properties of these plant extracts are often inferior can be safely consumed these propolis products and the raw material for their manufacture are extensively exported by brazilian companies especially to asian countries including japan south among natural medicine alternatives propolis has been widely studied and is already extensively consumed in many countries [ ] for example propolis products such as throat sprays and extracts are produced by hundreds of companies in brazil and are sold as a health aid in nearly every pharmacy throughout the country demonstrating on a practical basis that they preproofin fact propolis has a wide spectrum of pharmacological properties and is a dietary supplement that is commonly consumed by both healthy and sick people as a preventative precaution and for treatment it is also used in veterinary medicine due its antibacterial antifungal antiviral korea and china [ ] the importance of propolis in chinese japanese russian and korean medicine is reflected in the number of patents for propolis containing products registered by including about by china and “ each for japan russia and korea since about new propolisrelated patents were applied for in the us patent office it is a key ingredient in traditional chinese medicine japanese scientists have isolated and patented various brazilian propolis components for cancer treatment demonstrating their usefulness why propolis may be a good fit for dealing with covid19 antiparasitic hepatoprotective and immunomodulatory activities in the wake of the coronavirus outbreak south korea has seen a boon in the use of functional foods according to their ministry of food and drug safety œhealth functional foods are nutrients that have been proven to be beneficial to health in march of this year in response to the coronavirus pandemic the ministry eased regulations for propolis which is considered a 0ccould help fight against the covid19 pandemic active site of this enzyme is a relevant target for drug discovery functional food and allowed new oral formulations however despite considerable evidence that propolis can reduce and alleviate disease symptoms its acceptance as a healthpromoting supplement in human medicine has been limited in many countries such as the usa because of a relevant criticism that propolis products are not standardized and vary in their components and biological activity in part this is because propolis varies with the species of plants available in each region from which the bees collect resins to produce it [ ] however standardized propolis products have recently become available to help fill the need for a product that does not vary in the main bioactive components and effectiveness [ ] one such option a standardized brazilian propolis extract blend has been tested for safety and effectiveness in clinical trials for treating kidney disease and diabetes denture stomatitis and burn patients therefore propolis as a nutraceutical or functional food should be considered as a resource that preproofcaffeic acid phenethyl ester cape galangin chrysin and caffeic acid substances found in several different types of propolis around the world appeared as potential drugs against this viral target table specifically cape was predicted to interact with sarscov2 mpro in a potential targets in order to inactive the virus and reduce the damage that it causes the main protease of coronavirus sarscov2 mpro 3chymotrypsinlike cysteine enzyme is essential similar study therefore although it will be necessary to run in vitro assays to evaluate the potential antisarscov2 effects of propolis andor its constituents these in silico results are along this line hashem evaluated various natural compounds with an in silico approach molecular docking to try to find useful options for treating sarscov2 infection curiously for coronavirus processing of polyproteins and for its life cycle and therefore inhibition of the some propolis compounds can potentially interact with sarscov2 mpro the research community has examined the genetic code of coronavirus and the mechanisms underlying the damages caused by sarscov2 to help search for drugs andor well boding propolis can interact with ace and tmprss2 potentially blocking or reducing sarscov2 invasion of the host cell 0c sarscov2 strongly binds to angiotensinconverting enzyme ace2 using this enzyme as a receptor for invasion and replication in the host cell [ ] causing damage and increasing interpersonal transmission [ ] consequently ace inhibitors have been considered as useful drug alternatives however potential deleterious effects on users of angiotensinconverting enzyme ace inhibitors and angiotensin receptor blockers arbs have tool against potential cardiovascular events inhibition of ace2 enzyme is an important target for treatment against sarscov2 myricetin caffeic acid phenethyl ester hesperetin and pinocembrin rutin interacts with zinc fingers of the active sites of ace2 a metalloprotease that presents the same zinc finger in ace1 emerged as a concern for treatment of covid19 patients an observational study involving patients did not confirm this suspicion and therefore these classes of drugs remain an important infection [ ] güler et al prepared an alcoholic extract of propolis and identified some hydroxycinnamic acids caffeic acid pcoumaric acid tcinnamic acid and cape the flavanons rutin and myricetin and the flavones hesperidin chrysin and pinocembrin using molecular docking evaluations they found that rutin had the highest binding energy to ace2 followed by preproofvarious characteristics including inhibition of ace they found strong inhibition for most of the propolis types they studied with higher than ace inhibition the best results were found in addition to the in silico evidence osés et al evaluated several types of propolis for with the propolis components catechin and pcoumaric acid ace2 and tmprss2 transmembrane serine protease on the surface of host cells are used by sarscov2 via interaction with spike glycoproteins in order to proceed with invasion and replication vardhan sahoo studied several molecules commonly found in medicinal herbs using molecular docking procedures with relevant targets such as rnadependent rna polymerase rdrp ace2 and spike glycoproteins and compared the resulting scores with those of hydroxychloroquine limonin was the most active compound however quercetin and kaempferol also propolis compounds gave high docking scores kaempferol was studied in prostate cancer models and the expression of tmprss2 was reduced showing a potential mechanism of action for an antitumoral effect kaempferol could be an important propolis component for use against covid19 since it is involved in the inhibition of tmprss2 0c potentially interacting with ace2 rdrp and spike glycoprotein sgp besides its antiviral activity table propolis blocks pak1 potentially avoiding lung fibrosis and restoring a normal immune response among the possible targets for controlling covid19 damage the major œpathogenic contributes to their suppression pak1 inhibitors can both help combat the virus and restore a normal immune response kinase pak1 is key it is an essential component in malaria and viral infections but it is also involved in a wide variety of other diseases and disease conditions including cancer inflammation and immunosuppression when abnormally activated consequences of pak1 activation include lung fibrosis which is an aggravating factor in covid19 pak1 is activated by rac xu et al demonstrated that caffeic acid and its ester cape components of propolis can inactivate rac consequently inhibiting pak1 the inactivation of pak1 directly or upstream can potentially attenuate coronavirus pathogenesis bcells and tcells are lymphocytes that produce specific antibodies against viruses and other intruders and pak1 preproofimprove the immune response its components increase neutralizing antibody titers activate phagocytosis and increase ifnγ levels and the number of lymphocytes an increase in ifnγ levels was also detected by shimizu et al who evaluated the mechanisms involved in the cape caffeic acid phenethyl ester is a potent inhibitor of activation of nfkb in myelomonocytic cells anse et al demonstrated that propolis cape quercetin hesperidin and some other propolis flavonoids can inhibit the cytokine production of th1 and th2 type t cells while increasing tgfbeta an important antiinflammatory cytokine moreover cape can attenuate oxidative stress and inflammation through downregulation of jak2stat3 signaling propolis from europe and temperate asia usually made by bees from resins collected from poplar trees has predominantly flavonoid compounds while green propolis from baccharis dracunculifolia a propolis exclusively found in brazil has various kinds of flavonoids and prenylated phenylpropanoids such as artepellin c baccharin and drupanin these and all other types of propolis can inactivate pak1 artepillin c selectively inhibits pak1 table some studies have shown that propolis can act as an immunostimulant with the ability to effects of some types of propolis in a herpes simplex animal model 0c as well as having an immunomodulatory effect in which cape inhibits il6 phosphorylation and stat3 which are important for proinflammatory th17 development besides the antiinflammatory effect of cape and kaempferol paulino et al evaluated the antiinflammatory effect of artepellin c in rat paw edema and in cell cultures demonstrating that the activity was at least in part mediated by prostaglandin e2 and no inhibition through nfkb modulation artepillin c is an important biomarker of brazilian green propolis botanical source baccharis dracunculifolia immune modulation is desirable since coronavirus infection dysregulates the immune response in the initial phases of infection which facilitates viral replication however in later stages of covid19 the body develops an exaggerated inflammatory response which can greatly damage the lungs and other ans propolis different from typical immunosuppressants can help avoid immunosuppression during the initial phases of disease and in later stages reduce an exaggerated host inflammatory response inhibiting excess il6 il2 and jak signaling cape a propolis component is also known as an immunemodulating agent and should be considered as an alternative to help reduce an exaggerated inflammatory response in a mouse model propolis had immunomodulatory action in vivo on tolllike receptor expression and on preproofreplication and infectivity potentially decreasing lung inflammation due to antiinflammatory properties while promoting immune system fortification these are useful properties that could there is ample evidence for interference of propolis andor its components with viral propolis has been tested against various viral disease anisms initial successes have prompted research to determine the most useful components which may be modified to produce more active and specific pharmaceuticals viruses that were controlled by propolis in animal models with suggestion for control in humans include influenza [ ] herpes simplex virus type and hiv [ ] shimizu et al evaluated three different types of propolis in ethanol extracts using a murine model of herpes simplex virus type despite the chemical differences proinflammatory cytokine production help minimize the symptoms and deleterious effects of covid19 figure figure propolis as an antiviral substance 0cdue to the different plant origins of the resins the bees used to produce the propolis baccharis dracunculifolia baccharis eriodata and myrceugenia euosma all three propolis extracts not only had direct antihsv1 effects but also stimulated immunological activity against intradermal hsv1 infection in mice antiviral activity of propolis has been reported for dna and rna viruses poliovirus herpes simplex virus and adenovirus in an in vitro model cultured cells the best results were obtained against poliovirus and herpes virus with inhibition of the latter at a propolis concentration of ugml the propolis components chrysine and kaempferol caused a concentrationdependent reduction of intracellular replication of herpesvirus strains when host cell monolayers were infected and subsequently cultured in a drugcontaining medium quercetin another propolis component had the same effect but only at the highest concentrations tested ugml against various human herpes simplex virus strains with a intracellular replication reduction of approximately while it reduced the infectivity of bovine herpes virus human adenovirus human coronavirus and bovine coronavirus about the reduction was in the preproofimpairment and pulmonary failure inflammatory response to infection sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory immunologicalinflammatory phenomena such as cytokine release syndrome have been shown to be important mortality factors in sarscov2 infection higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα are found in patients with severe covid compared to those of individuals with mild disease molecular mechanisms involved in this immune process are the targets of various synthetic medicines being tested in patients including ciclesonide hydroxy chloroquine ivermectin and ketorolac which are pak1 blockers pak1 raccdc42activated kinase is overexpressed in the lung in response to sarscov2 infection and is a critical mediator of the cytokine storm that frequently results in mortality the most critical cases of covid19 which require ventilatorassisted intensive care and often result in prolonged ventilator dependency and death are a result of an exaggerated case of rotavirus antiinflammatory and immunomodulatory properties of propolis 0cin hospitalized patients fortuitously propolis components are effective pak1 blockers table there is considerable evidence that propolis can reduce and alleviate the symptoms of inflammatory diseases and has immunomodulatory properties [ ] however these properties can vary according to the plant origin of the propolis as well as the extraction processsolvent used and the inflammatory protocol cell culture animal models induction by lipopolysaccharides when the propolis extracts are tested tests with animal models have shown that propolis can reduce the levels of il6 and tnfalpha which are key proinflammatory mediators and increase the levels of the regulatory cytokine il10 kaempferol a propolis component reduces il6 tnfalpha and vegf vascular endothelial growth factor via the preproofifnγ in serum fernandes et al found that propolis exerted a positive adjuvant effect on vaccines that were developed against canine coronavirus they assayed ifnγ which is an effective way to measure the cellular response induced by a vaccine in a mouse model propolis added as an adjuvant to inactivated swine herpesvirus type vaccine stimulated increased cellular propolis is considered a safe immunostimulant and a potent vaccine adjuvant propolis has been widely tested as a vaccine adjuvant because it induces an earlier immune response and provides a longer protection period it is also included as an adjuvant ingredient in traditional chinese medicine propolis flavonoids have potential as adjuvants enhancing igg il4 and propolis has potential as a vaccine adjuvant erknfkbcmycp21 pathway table tests on macrophage cell cultures also demonstrated that propolis inhibits the production of il1 beta an important component of the inflammasome inflammatory pathway in diseases such as rheumatoid arthritis lupus and other autoimmune diseases although the mechanisms of action are not well elucidated these propolis components have potential as complementary supplements in the preventive treatment of chronic inflammatory diseases and humoral responses increasing ifnγ [ ] propolis enhanced the immune response to inactivated porcine parvovirus vaccine in guinea pigs when added to a trichomonas vaginalis protein vaccine propolis increased the igg antibody response times in mice 0ccancer is considered a relevant comorbidity factor for covid19 cancer patients have a patients cancer patients compared to the protein alone propolis was also effective as an adjuvant in the immunization of cattle with bovine herpesvirus it improved the humoral and cellular responses in mice inoculated with inactivated virus vaccines propolis as an adjuvant gave a similar immune response increasing ifnγ levels to alum and freund™s adjuvant in mice vaccinated with an hiv1 polytope vaccine candidate with less risk of undesirable side effects comorbidities and evidence of how propolis can help reduce their impact in covid19 to risk a visit to a clinic or hospital to determine if they have cancer alternative therapies could help retard cancer or reduce the impact of cancer and cancer treatment in covid19 times higher risk of progressing to severe covid19 disease than patients without comorbidities also the hospital environment during the coronavirus pandemic can interfere with or delay the treatment that cancer patients should receive patients with symptoms may choose not preproofacid cape quercetin and chrysin propolis and its components normally have little impact on normal cells displaying differential cytotoxicity in liver cancer melanoma and breast cell carcinoma cell lines [ ] propolis enhances the activity of tumor various types including bladder blood brain breast colon head and neck kidney liver pancreas prostate and skin cancers propolis could help prevent cancer progression in various parts of the world it is considered an alternative therapy for cancer treatment propolis extracts have been found to inhibit tumor cell growth both in vitro and in vivo including inhibition of angiogenesis demonstrating potential for the development of new anticancer drugs various components of propolis have been shown to inhibit cancer cell growth including cinnamic propolis has potential as a complementary therapy for cancer it has shown efficacy against necrosis factor related apoptosis inducing ligand trail in cancer cells hypertension and cardiovascular disease 0c hypertension and cardiovascular disease are considered relevant comorbidities for covid19 propolis has demonstrated antihypertensive effects in rat models [ ] in cameroon it is used in popular medicine to treat various ailments including high blood pressure propolis has been widely used as a dietary supplement for its health benefits including cardiovascular protective effects [ ] in a human trial consumption of propolis improved critical blood parameters including hdl gsh and tbars levels demonstrating that obesity a natural antiobesity agent it could contribute to a reduced risk for cardiovascular disease obesity is a major comorbidity and predictor of increased mortality in covd19 patients obesity and sarscov2 both induce an inflammatory process exacerbating sarscov2 infection in the obese propolis reduced inflammation and prevented hyperlipidemia and metabolic syndromes in highly caloric diet induced obesity in mice body weight gain visceral adipose tissue liver and serum triglycerides cholesterol and nonesterified fatty acids were all reduced in the propolis fed mice [ ] caffeic acid phenethyl ester a propolis component is preproofplatelet aggregation was reduced by propolis in tests on human blood in vitro and in other in vitro tests caffeic acid phenethyl ester cape a wellstudied bioactive propolis component inhibits collagen induced platelet activation thromboembolism thrombosis and microthrombosis microthrombosis disseminated intravascular coagulation and consequent multian failure are common in severely affected covid19 patients with associated high mortality rates anticoagulants are sometimes prescribed to such patients because they can reduce mortality tang et al an elevated level of plasminogen activator inhibitor1 pai1 is a biomarker and risk factor for thrombosis and atherosclerosis [ ] various types of evidence demonstrate that propolis can reduce platelet aggregation and other thrombosisrelated parameters propolis decreased thrombotic tendencies in mice by suppressing lipopolysaccharideinduced increases in pai1 levels [ ] propolis downregulated plateletderived growth factor and platelet endothelial cell adhesion molecules in lowdensity lipoprotein knockout mice 0c old age the elderly are more often affected by chronic inflammation characterized by systemically increased levels of proinflammatory cytokines which can contribute to development of a cytokine storm a major cause of covid19 mortality propolis has antioxidant properties which could help retard o Answer:

Colon_Cancer

tetrastigma hemsleyanum diels et gilg t hemsleyanum mostly known as œsan ye qing is a kind of folk plant because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource it mainly grows in the eastern central southern and south western provinces of china such as zhejiang jiangsu guangxi fujian and yunnan provinces peng and wang t hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models xu as an edible plant the leaves of t hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body sun while the aerial parts of t hemsleyanum developed as potential new traditional chinese medicine tcm preparations guo corresponding author ningbo research institute of zhejiang university ningbo zhejiang people™s republic of china email address px4142163com x peng 101016jjep2020113247 received may received in revised form july accepted august ofethnopharmacology2642021113247availableonline12august2020037887412020elsevierbvallrightsreserved 0ct ji abbreviations t hemsleyanum tetrastigma hemsleyanum diels et gilg tcm uplcesiqtofmsms ultra high performance liquid traditional chinese medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκb 5hydroxytryptamine norepinephrine dopamine prostaglandin e2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin mic gsh mda nfκb 5ht ne da pge2 mapk mitogenactivated protein kinase lps celegans caenorhabditis elegans tnfα il1 il6 il12p40 interleukin subunit p40 stnfr1 soluble tnf receptors il10 il1 il4 inos tlr4 md2 myd88 myeloid differentiation protein jnk gpt got alp sod interleukin interleukin interleukin inducible no synthase tolllike receptor myeloid differentiation factor2 cjun nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory the root tubers of t hemsleyanum are extensively used either alone or in combination with other herbal medicines in tcm clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain sun chen and guo therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal in february t hemsleyanum was awarded as the new œeight famous kinds of tcm in zhejiang province meant that it has become a key object of industrialization development of zhejiang™s dominant large varieties of medicinal materials in covid19 broke out and has caused more than deaths in china and infection cases have been reported in more than countries hua shi xuan fei mixture approval number of zhejiang medicine z20200026000 which composed of t hemsleyanum has been approved by zhejiang provincial drug administration for clinical treatment of covid19 futhermore the modern pharmacological studies had shown that t hemsleyanum also had effects of antiinflammatory ji antioxidant hossain antivirus ding antitumor lin antipyretic yang and wang antihepatic injury ma et al immunomodulatory xu antibacterial chen hypoglycemic ru 2018ab etc numerous reports have demonstrated that the biological activities of t hemsleyanum are attributed to its many chemical components fu wang has reported isolated alkaloids from the aerial parts of t hemsleyanum wang ru extracted a novel polysaccharide tdgp3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin a secretory immunoglobulin a epithelialmesenchymal transition alt ast ha ln tbili tp ifnÎ iga siga emt mmps matrix metalloproteinase timps matrixmetallo proteinase cytc cat gshpx glutathione peroxidase tregs tgf cox2 foxp3 pdl taoc ccl4 cef hvj vsv a f s1 s2 pef cff eaf baf cytochrome c catalase regulatory t cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast hemagglutinating virus of japan vesicular stomatitis virus alkalicontaining extract of t hemsleyanum ketonecontaining extract of t hemsleyanum crude extract of t hemsleyanum crude extract of t hemsleyanum petroleum ether extractions of t hemsleyanum ethanol extract chloroform extractions of t hemsleyanum ethanol extract ethyl acetate extractions of t hemsleyanum ethanol extract nbutanol extractions of t hemsleyanum ethanol extract t hemsleyanum with a molecular weight of — da by enzymolysisultrasonic assisted extraction method ru 2019ab large amounts of flavonoids were found in leaves aerial parts and root tubers of t hemsleyanum xu 2014ab deng yu in addition t hemsleyanum also contains a variety of functional components such as anic acids hu phenolic acids liu minerals fan amino acids fu etc in recent years wild resources of t hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments in it was listed in the preferentially protected crop germplasm resources of zhejiang province based on our team™s preliminary research peng peng 2016ab li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of t hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization materials and methods the available information about the traditional uses phytochemicals and pharmacological properties of t hemsleyanum was searched via web of science google scholar pubmed science direct china national knowledge infrastructure cnki and springer search using chinese or english as the retrieval languages the keywords used include t hemsleyanum root tubers of t hemsleyanum radix tetrastigma ofethnopharmacology26420211132472 0ct ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words all references were from experimental studies and published prior to april were reviewed all chemical structures were drawn using chemdraw pro software heatclearing were botanical characteristics t hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose it is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus the optimum ph is between and the root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter fig the epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section the stem of t hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate the leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base the flowers of t hemsleyanum are small yellow green and ovate the flowering stage of t hemsleyanum ranges from april to june and the fruit phase is normally from august to november when the flower withered it will form a small green round fruit with the size of millet when it is mature the fruit will turn from green to red the berries are spherical and soft spherical traditional uses t hemsleyanum belonging to the family vitaceae was firstly recorded in ben cao gang mu ming dynasty ad the aliases of sanyeqing include shi hou zi shi bao zi shi lao shu lan shan hu lei dan zi po shi zhu tu jing wan sou jia feng san ye dui golden wire hanging gourd golden bell golden wire hanging potato etc the root tubers or whole grass of t hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of tcm such as zhi wu ming shi tu kao qing dynasty wu jiangxi herbal medicine common folk herbal medicine in zhejiang all of these ancient works described the effects of toxicityremoving t dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women national compilation team of chinese herbal medicine in the tcm culture the properties of t hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional chinese medicine and zhong hua ben cao shanghai science and technology press the channel tropism was lung heart liver and kidney meridians decocting with water or mashing for external application are the traditional possess methods of t hemsleyanum considering its extensive traditional effects many prescriptions containing t hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies excitingly it has reported that jinlian disinfection drink containing san ye qing combined with interferon can treat covid19 he jinqi tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was wei moreover zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage iii primary liver cancer jiang and gong in addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites ji chemical compounds of themsleyanum the chemical constituents of t hemsleyanum have been widely investigated sun sun zeng xu 2014ab fu fan chen ding 2015a fig the aerial part a root tuber b and raw herb c of t hemsleyanum ofethnopharmacology26420211132473 0ct ji b ding a total of one hundred and fortytwo compounds have been isolated and identified from t hemsleyanum until now the information about compound name molecular weight compound formula detection method analysis sample is summarized in table flavonoids and their glycosides modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from t hemsleyanum lin zhang table to date fiftyone flavonoids and their glycosides have been extracted and identified from t hemsleyanum in this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on c3² and c4™ on the b ring of flavonoid aglycone at present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry uplcesiqtofms has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution our team used uplcesiqtofms to identify chemical constituents from the aerial part of t hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc sun according to the report liu total flavonoids of t hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase mapk and nuclear factorκb nfκb in lung tissue moreover the flavonoids of t hemsleyanum had the activity of antilung cancer wei luteolin a flavonoid found in t hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers muhammad it is certain that t hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs polysaccharide saccharide is another important active ingredient extracted from t hemsleyanum shao polysaccharide has great potential in clinical application because of its unique pharmacological activity however due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures guo table the prescriptions and traditional uses of t hemsleyanum in china prescriptions name qingteng fengshi qufengshi yaojiu main composition jiu traditional use t hemsleyanum parabarium chunianum tsiang zanthoxylum nitidum roxb dc t hemsleyanum deeringia amaranthoides lam merr blumea aromatica wall dc t hemsleyanum deeringia amaranthoides lam merr zanthoxylum nitidum roxb dc panax notoginseng burk fh chen t hemsleyanum gypsum lonicera japonica thunb houttuynia cordata thunb ophiopogon japonicus linn f kergawl t hemsleyanum t hemsleyanum lysimachia christinae hance imperata cylindrica citrus reticulata blanco t hemsleyanum ginsenoside astragalus propinquus schischkin t hemsleyanum nepeta cataria l lonicera japonica thunb saposhnikovia divaricata trucz schischk huatuo fengtongbao capsule sanyeqing gypsum decoction sanyeqing power zhonggan mixture jinqi tablet hua shi xuan fei mixture extracted the polysaccharides from roots of t hemsleyanum rtp1 rtp2 and rtp3 were successively found by protein precipitation and purification moreover further study indicated rtp31 was high purity polysaccharide with a molecular weight of kda and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively ru 2018ab extracted a polysaccharide thp from t hemsleyanum with the average molecular weight estimated as kda the results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of in ru 2019ab successfully extracted polysaccharide thdp3 from t hemsleyanum with molecular weight of kda which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of moreover tdgp3 mainly consists of †’4αdgalap1†’ †’4dgalp1†’ and †’4αdglcp1†’ residues as backbones and dmanp1†’ †’36dmanp1†’ and αdaraf1†’residues as branches phenolic acids phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring as a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects twenty three phenolic acids no52“ table have been reported in the aerial parts of t hemsleyanum such as caffeic acid chlorogenic acid 1ogalloyldglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid there were twentyone phenolic acids in the root tuber of t hemsleyanum some of which were the same as aerial parts alkaloids alkaloids are a group of basic anic compounds containing nitrogen that exist in nature alkaloids are stored in small quantities in t hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare wang fu extracted the aerial parts of t hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and treatment of joint pain wind cold dampness arthralgia treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture treatment of infantile hyperpyretic convulsion treatment of blood avalanche leucorrhea treatment of liver cancer treatment of malignant tumor treatment of covid19 usage oral administration “ ml once times a day oral administration ml once times a day oral administration capsules once times a day references ministerial standard ministerial standard ministerial standard one dose a day decoct twice in water and take it “ times after mixing oral administration oral administration ml once times a day oral administration capsules once times a day oral administration ml once times a day xu gao jiang and gong wei zhejiang provincial drug administration ofethnopharmacology26420211132474 0ct ji detection mode analysis parts of sample reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber sun sun zeng sun sun sun zeng zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun zeng sun zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun xu 2014b sun zeng sun zeng sun zeng zeng sun sun sun sun xu 2014b sun xu 2014b sun zeng sun xu 2014b sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun fu sun sun xu 2014b fan xu 2014b fan sun continued on next page uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms table chemical constituents isolated from the different parts of t hemsleyanum name flavonoids and their glycosides quercetin quercitrin quercetin3oglucoside quercetin3orutinoside quercetin3galactoside quercetin3oxylosylglucoside quercetin3oxylosylglucose7orhamnoside orientin orientin2²²orhamnoside isoorientin isoorientin2²²orhamnoside isoorientin cid0 ²²oxyloside vitexin vitexin2²²orhamnoside vitexin2²²oglucoside vitexin2²²oarabinoside isovitexin isovitexin2²²orhamnoside isovitexin2²²oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8cxylosyl6cglucoside apigenin6cαlarabinose8cdglucose eriodictyol eriodictyolohexoside i eriodictyolohexoside ii luteolin luteolin6 8dichexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3oneohesperidin kaempferol3orhamnoside kaempferol7orhamnose3oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3ocarfuran7orhamnosyl glucoside daidzein biochanin a procyanidin dimmer procyanidin b1 procyanidin b2 procyanidin trimer phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr13cnmr ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms ofethnopharmacology26420211132475 0ct ji table continued name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1ogalloyldglucose protocatechol glucoside epigallocatechin vanillic acid1ofuran celery glucosyl ester protocatechuic acid1ofuran celery glucosyl ester methoxyphenol1ofuran glycosyloglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid scid0 trolline fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid dimeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid terpenoids and steroids sitosterol daucosterol campesterol stigmasterol 6obenzoyl daucosterol ergosterol taraxerone taraxerol αamyrine pteroside z ganoderic acid h 3epipapyriferic acid oleanic acid saponins ginsenoside rh1 detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr lcms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms gcms tcl hnmr cnmr ms gcms gcms ir hnmr eims ir hnmr ms ir hnmr ms ir hnmr ms ir eims uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms hnmr cnmr ms analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber reference sun sun sun sun sun sun zeng sun xu 2014b zeng zeng zeng zeng xu 2014b xu 2014b chen fu fu fu fu fu fu fu fu fu fu sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun chen ding sun sun guo ru ru ru ru sun sun sun ding uplcesiqtofmsms root tuber sun continued on next page ofethnopharmacology26420211132476 0ct ji table continued name ginsenoside rh2 vinaginsenoside r1 amino acid and derivatives phenylalanine pyroglutamic acid glutimic acid hexose tryptophan lglutamic acid detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part reference sun sun sun sun sun sun sun respectively a carboline by comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and scid0 trolline the chemical structures were shown in fig identified as indole anic acids and derivatives the biologically essential anic acids have been isolated and characterized from t hemsleyanum as well ten anic acids and seventeen fatty acids were identified from the aerial parts and root tuber of t hemsleyanum most of which were found in the aerial parts except stearic acid propanoic acid and dihydroxy octadecenoic acid all the anic acids and fatty acids are listed in no112“ and no95“ of table respectively terpenoids and steroids terpenoids and steroids are other kinds of secondary metabolites of t hemsleyanum thirteen of these compounds have been isolated and identified no122“ table liu yang liu isolated and identified αamyrine sitosterol ergosterol taraxerone taraxerol from the aerial part of t hemsleyanum in addition daucosterol campesterol stigmasterol 6obenzoyldaucosterol pteroside z ganoderic acid h 3epipapyriferic acid and oleanic acid were successively separated tuber roots of t hemsleyanum liu and yang from the inanic elements the mineral elements of tcm are indispensable supplements to the bioactive components which are closely related to the efficacy toxicity and side effects of tcm wu wu et al demonstrated that t hemsleyanum contained twentyseven different mineral elements namely li be na mg al k ca v cr mn fe co ni cu zn ga as se rb ag cd cs ba hg ti pb u moreover ca cu ni ba al k have higher loading values which are the characteristic elements of t hemsleyanum wang wang has indicated that the contents of fe mn zn and cu in three populations of t hemsleyanum cultivated in different environments were “ “ “ “ mg kgcid0 respectively pharmacology the ethnomedical uses of t hemsleyanum have stimulated various pharmacological studies on it the extracts and isolated compounds from t hemsleyanum showed a variety of bioactivities such as antiviral antibacterial antioxidant antipyretic analgesic hepatoprotective immunoregulatory and antitumor activity the detailed pharmacological activities of t hemsleyanum were presented in table and summarized as follows antiviral activity according to yang™s literatures yang the nitrogenous alkalicontaining extract a ketonecontaining extract f crude extract s1 and crude extract s2 of t hemsleyanum had different antiviral effect on mice and chicken embryo fibroblast cef infected with hemagglutinating virus of japan hvj influenza virus pr6 vesicular stomatitis virus vsv specifically s2 strongly inhibited the proliferation of influenza virus pr6 with at the concentration of mgml and mgml s1 has obvious antiviral effect on hvj at the concentrations of mgml and mgml both f and s1 displayed a strong suppressive effect on the plaque formation of vsv in vivo a f s1 s2 have different degrees of antiviral activity when the concentration of a was gkg the protective rate was up to and that of s1 gkg was however the author did not give the sample preparation method ding had demonstrated compounds quercetin3orutinoside kaempferol kaempferol3glucoside quercitrin quercetin kaempferol3orutinoside procyanidin dimmer and epicatechin which were isolated from t hemsleyanum were positively related to the activity of t hemsleyanum against h1n1 influenza virus the ethyl acetate extracts of t hemsleyanum have been shown to obviously restrain the secretion of hbsag and hbeag released by hbv with the ic50 values of “ mgl however the specific mechanism of action needs to be further confirmed yang and wu wang had proved that the nbutanol and ethyl acetate extraction of t hemsleyanum had antiviral activity against rsv and were superior to ribavirin with the ec50 values of mgl wang moreover the t hemsleyanum extracts had different degrees of inhibition to different hiv1 strains the ec50 values were between μgml and μgml and the

cancer7528

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: tetrastigma hemsleyanum diels et gilg t hemsleyanum mostly known as œsan ye qing is a kind of folk plant because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource it mainly grows in the eastern central southern and south western provinces of china such as zhejiang jiangsu guangxi fujian and yunnan provinces peng and wang t hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models xu as an edible plant the leaves of t hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body sun while the aerial parts of t hemsleyanum developed as potential new traditional chinese medicine tcm preparations guo corresponding author ningbo research institute of zhejiang university ningbo zhejiang people™s republic of china email address px4142163com x peng 101016jjep2020113247 received may received in revised form july accepted august ofethnopharmacology2642021113247availableonline12august2020037887412020elsevierbvallrightsreserved 0ct ji abbreviations t hemsleyanum tetrastigma hemsleyanum diels et gilg tcm uplcesiqtofmsms ultra high performance liquid traditional chinese medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκb 5hydroxytryptamine norepinephrine dopamine prostaglandin e2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin mic gsh mda nfκb 5ht ne da pge2 mapk mitogenactivated protein kinase lps celegans caenorhabditis elegans tnfα il1 il6 il12p40 interleukin subunit p40 stnfr1 soluble tnf receptors il10 il1 il4 inos tlr4 md2 myd88 myeloid differentiation protein jnk gpt got alp sod interleukin interleukin interleukin inducible no synthase tolllike receptor myeloid differentiation factor2 cjun nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory the root tubers of t hemsleyanum are extensively used either alone or in combination with other herbal medicines in tcm clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain sun chen and guo therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal in february t hemsleyanum was awarded as the new œeight famous kinds of tcm in zhejiang province meant that it has become a key object of industrialization development of zhejiang™s dominant large varieties of medicinal materials in covid19 broke out and has caused more than deaths in china and infection cases have been reported in more than countries hua shi xuan fei mixture approval number of zhejiang medicine z20200026000 which composed of t hemsleyanum has been approved by zhejiang provincial drug administration for clinical treatment of covid19 futhermore the modern pharmacological studies had shown that t hemsleyanum also had effects of antiinflammatory ji antioxidant hossain antivirus ding antitumor lin antipyretic yang and wang antihepatic injury ma et al immunomodulatory xu antibacterial chen hypoglycemic ru 2018ab etc numerous reports have demonstrated that the biological activities of t hemsleyanum are attributed to its many chemical components fu wang has reported isolated alkaloids from the aerial parts of t hemsleyanum wang ru extracted a novel polysaccharide tdgp3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin a secretory immunoglobulin a epithelialmesenchymal transition alt ast ha ln tbili tp ifnÎ iga siga emt mmps matrix metalloproteinase timps matrixmetallo proteinase cytc cat gshpx glutathione peroxidase tregs tgf cox2 foxp3 pdl taoc ccl4 cef hvj vsv a f s1 s2 pef cff eaf baf cytochrome c catalase regulatory t cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast hemagglutinating virus of japan vesicular stomatitis virus alkalicontaining extract of t hemsleyanum ketonecontaining extract of t hemsleyanum crude extract of t hemsleyanum crude extract of t hemsleyanum petroleum ether extractions of t hemsleyanum ethanol extract chloroform extractions of t hemsleyanum ethanol extract ethyl acetate extractions of t hemsleyanum ethanol extract nbutanol extractions of t hemsleyanum ethanol extract t hemsleyanum with a molecular weight of — da by enzymolysisultrasonic assisted extraction method ru 2019ab large amounts of flavonoids were found in leaves aerial parts and root tubers of t hemsleyanum xu 2014ab deng yu in addition t hemsleyanum also contains a variety of functional components such as anic acids hu phenolic acids liu minerals fan amino acids fu etc in recent years wild resources of t hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments in it was listed in the preferentially protected crop germplasm resources of zhejiang province based on our team™s preliminary research peng peng 2016ab li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of t hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization materials and methods the available information about the traditional uses phytochemicals and pharmacological properties of t hemsleyanum was searched via web of science google scholar pubmed science direct china national knowledge infrastructure cnki and springer search using chinese or english as the retrieval languages the keywords used include t hemsleyanum root tubers of t hemsleyanum radix tetrastigma ofethnopharmacology26420211132472 0ct ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words all references were from experimental studies and published prior to april were reviewed all chemical structures were drawn using chemdraw pro software heatclearing were botanical characteristics t hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose it is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus the optimum ph is between and the root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter fig the epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section the stem of t hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate the leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base the flowers of t hemsleyanum are small yellow green and ovate the flowering stage of t hemsleyanum ranges from april to june and the fruit phase is normally from august to november when the flower withered it will form a small green round fruit with the size of millet when it is mature the fruit will turn from green to red the berries are spherical and soft spherical traditional uses t hemsleyanum belonging to the family vitaceae was firstly recorded in ben cao gang mu ming dynasty ad the aliases of sanyeqing include shi hou zi shi bao zi shi lao shu lan shan hu lei dan zi po shi zhu tu jing wan sou jia feng san ye dui golden wire hanging gourd golden bell golden wire hanging potato etc the root tubers or whole grass of t hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of tcm such as zhi wu ming shi tu kao qing dynasty wu jiangxi herbal medicine common folk herbal medicine in zhejiang all of these ancient works described the effects of toxicityremoving t dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women national compilation team of chinese herbal medicine in the tcm culture the properties of t hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional chinese medicine and zhong hua ben cao shanghai science and technology press the channel tropism was lung heart liver and kidney meridians decocting with water or mashing for external application are the traditional possess methods of t hemsleyanum considering its extensive traditional effects many prescriptions containing t hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies excitingly it has reported that jinlian disinfection drink containing san ye qing combined with interferon can treat covid19 he jinqi tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was wei moreover zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage iii primary liver cancer jiang and gong in addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites ji chemical compounds of themsleyanum the chemical constituents of t hemsleyanum have been widely investigated sun sun zeng xu 2014ab fu fan chen ding 2015a fig the aerial part a root tuber b and raw herb c of t hemsleyanum ofethnopharmacology26420211132473 0ct ji b ding a total of one hundred and fortytwo compounds have been isolated and identified from t hemsleyanum until now the information about compound name molecular weight compound formula detection method analysis sample is summarized in table flavonoids and their glycosides modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from t hemsleyanum lin zhang table to date fiftyone flavonoids and their glycosides have been extracted and identified from t hemsleyanum in this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on c3² and c4™ on the b ring of flavonoid aglycone at present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry uplcesiqtofms has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution our team used uplcesiqtofms to identify chemical constituents from the aerial part of t hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc sun according to the report liu total flavonoids of t hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase mapk and nuclear factorκb nfκb in lung tissue moreover the flavonoids of t hemsleyanum had the activity of antilung cancer wei luteolin a flavonoid found in t hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers muhammad it is certain that t hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs polysaccharide saccharide is another important active ingredient extracted from t hemsleyanum shao polysaccharide has great potential in clinical application because of its unique pharmacological activity however due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures guo table the prescriptions and traditional uses of t hemsleyanum in china prescriptions name qingteng fengshi qufengshi yaojiu main composition jiu traditional use t hemsleyanum parabarium chunianum tsiang zanthoxylum nitidum roxb dc t hemsleyanum deeringia amaranthoides lam merr blumea aromatica wall dc t hemsleyanum deeringia amaranthoides lam merr zanthoxylum nitidum roxb dc panax notoginseng burk fh chen t hemsleyanum gypsum lonicera japonica thunb houttuynia cordata thunb ophiopogon japonicus linn f kergawl t hemsleyanum t hemsleyanum lysimachia christinae hance imperata cylindrica citrus reticulata blanco t hemsleyanum ginsenoside astragalus propinquus schischkin t hemsleyanum nepeta cataria l lonicera japonica thunb saposhnikovia divaricata trucz schischk huatuo fengtongbao capsule sanyeqing gypsum decoction sanyeqing power zhonggan mixture jinqi tablet hua shi xuan fei mixture extracted the polysaccharides from roots of t hemsleyanum rtp1 rtp2 and rtp3 were successively found by protein precipitation and purification moreover further study indicated rtp31 was high purity polysaccharide with a molecular weight of kda and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively ru 2018ab extracted a polysaccharide thp from t hemsleyanum with the average molecular weight estimated as kda the results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of in ru 2019ab successfully extracted polysaccharide thdp3 from t hemsleyanum with molecular weight of kda which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of moreover tdgp3 mainly consists of †’4αdgalap1†’ †’4dgalp1†’ and †’4αdglcp1†’ residues as backbones and dmanp1†’ †’36dmanp1†’ and αdaraf1†’residues as branches phenolic acids phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring as a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects twenty three phenolic acids no52“ table have been reported in the aerial parts of t hemsleyanum such as caffeic acid chlorogenic acid 1ogalloyldglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid there were twentyone phenolic acids in the root tuber of t hemsleyanum some of which were the same as aerial parts alkaloids alkaloids are a group of basic anic compounds containing nitrogen that exist in nature alkaloids are stored in small quantities in t hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare wang fu extracted the aerial parts of t hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and treatment of joint pain wind cold dampness arthralgia treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture treatment of infantile hyperpyretic convulsion treatment of blood avalanche leucorrhea treatment of liver cancer treatment of malignant tumor treatment of covid19 usage oral administration “ ml once times a day oral administration ml once times a day oral administration capsules once times a day references ministerial standard ministerial standard ministerial standard one dose a day decoct twice in water and take it “ times after mixing oral administration oral administration ml once times a day oral administration capsules once times a day oral administration ml once times a day xu gao jiang and gong wei zhejiang provincial drug administration ofethnopharmacology26420211132474 0ct ji detection mode analysis parts of sample reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber sun sun zeng sun sun sun zeng zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun zeng sun zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun xu 2014b sun zeng sun zeng sun zeng zeng sun sun sun sun xu 2014b sun xu 2014b sun zeng sun xu 2014b sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun fu sun sun xu 2014b fan xu 2014b fan sun continued on next page uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms table chemical constituents isolated from the different parts of t hemsleyanum name flavonoids and their glycosides quercetin quercitrin quercetin3oglucoside quercetin3orutinoside quercetin3galactoside quercetin3oxylosylglucoside quercetin3oxylosylglucose7orhamnoside orientin orientin2²²orhamnoside isoorientin isoorientin2²²orhamnoside isoorientin cid0 ²²oxyloside vitexin vitexin2²²orhamnoside vitexin2²²oglucoside vitexin2²²oarabinoside isovitexin isovitexin2²²orhamnoside isovitexin2²²oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8cxylosyl6cglucoside apigenin6cαlarabinose8cdglucose eriodictyol eriodictyolohexoside i eriodictyolohexoside ii luteolin luteolin6 8dichexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3oneohesperidin kaempferol3orhamnoside kaempferol7orhamnose3oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3ocarfuran7orhamnosyl glucoside daidzein biochanin a procyanidin dimmer procyanidin b1 procyanidin b2 procyanidin trimer phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr13cnmr ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms ofethnopharmacology26420211132475 0ct ji table continued name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1ogalloyldglucose protocatechol glucoside epigallocatechin vanillic acid1ofuran celery glucosyl ester protocatechuic acid1ofuran celery glucosyl ester methoxyphenol1ofuran glycosyloglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid scid0 trolline fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid dimeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid terpenoids and steroids sitosterol daucosterol campesterol stigmasterol 6obenzoyl daucosterol ergosterol taraxerone taraxerol αamyrine pteroside z ganoderic acid h 3epipapyriferic acid oleanic acid saponins ginsenoside rh1 detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr lcms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms gcms tcl hnmr cnmr ms gcms gcms ir hnmr eims ir hnmr ms ir hnmr ms ir hnmr ms ir eims uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms hnmr cnmr ms analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber reference sun sun sun sun sun sun zeng sun xu 2014b zeng zeng zeng zeng xu 2014b xu 2014b chen fu fu fu fu fu fu fu fu fu fu sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun chen ding sun sun guo ru ru ru ru sun sun sun ding uplcesiqtofmsms root tuber sun continued on next page ofethnopharmacology26420211132476 0ct ji table continued name ginsenoside rh2 vinaginsenoside r1 amino acid and derivatives phenylalanine pyroglutamic acid glutimic acid hexose tryptophan lglutamic acid detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part reference sun sun sun sun sun sun sun respectively a carboline by comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and scid0 trolline the chemical structures were shown in fig identified as indole anic acids and derivatives the biologically essential anic acids have been isolated and characterized from t hemsleyanum as well ten anic acids and seventeen fatty acids were identified from the aerial parts and root tuber of t hemsleyanum most of which were found in the aerial parts except stearic acid propanoic acid and dihydroxy octadecenoic acid all the anic acids and fatty acids are listed in no112“ and no95“ of table respectively terpenoids and steroids terpenoids and steroids are other kinds of secondary metabolites of t hemsleyanum thirteen of these compounds have been isolated and identified no122“ table liu yang liu isolated and identified αamyrine sitosterol ergosterol taraxerone taraxerol from the aerial part of t hemsleyanum in addition daucosterol campesterol stigmasterol 6obenzoyldaucosterol pteroside z ganoderic acid h 3epipapyriferic acid and oleanic acid were successively separated tuber roots of t hemsleyanum liu and yang from the inanic elements the mineral elements of tcm are indispensable supplements to the bioactive components which are closely related to the efficacy toxicity and side effects of tcm wu wu et al demonstrated that t hemsleyanum contained twentyseven different mineral elements namely li be na mg al k ca v cr mn fe co ni cu zn ga as se rb ag cd cs ba hg ti pb u moreover ca cu ni ba al k have higher loading values which are the characteristic elements of t hemsleyanum wang wang has indicated that the contents of fe mn zn and cu in three populations of t hemsleyanum cultivated in different environments were “ “ “ “ mg kgcid0 respectively pharmacology the ethnomedical uses of t hemsleyanum have stimulated various pharmacological studies on it the extracts and isolated compounds from t hemsleyanum showed a variety of bioactivities such as antiviral antibacterial antioxidant antipyretic analgesic hepatoprotective immunoregulatory and antitumor activity the detailed pharmacological activities of t hemsleyanum were presented in table and summarized as follows antiviral activity according to yang™s literatures yang the nitrogenous alkalicontaining extract a ketonecontaining extract f crude extract s1 and crude extract s2 of t hemsleyanum had different antiviral effect on mice and chicken embryo fibroblast cef infected with hemagglutinating virus of japan hvj influenza virus pr6 vesicular stomatitis virus vsv specifically s2 strongly inhibited the proliferation of influenza virus pr6 with at the concentration of mgml and mgml s1 has obvious antiviral effect on hvj at the concentrations of mgml and mgml both f and s1 displayed a strong suppressive effect on the plaque formation of vsv in vivo a f s1 s2 have different degrees of antiviral activity when the concentration of a was gkg the protective rate was up to and that of s1 gkg was however the author did not give the sample preparation method ding had demonstrated compounds quercetin3orutinoside kaempferol kaempferol3glucoside quercitrin quercetin kaempferol3orutinoside procyanidin dimmer and epicatechin which were isolated from t hemsleyanum were positively related to the activity of t hemsleyanum against h1n1 influenza virus the ethyl acetate extracts of t hemsleyanum have been shown to obviously restrain the secretion of hbsag and hbeag released by hbv with the ic50 values of “ mgl however the specific mechanism of action needs to be further confirmed yang and wu wang had proved that the nbutanol and ethyl acetate extraction of t hemsleyanum had antiviral activity against rsv and were superior to ribavirin with the ec50 values of mgl wang moreover the t hemsleyanum extracts had different degrees of inhibition to different hiv1 strains the ec50 values were between μgml and μgml and the Answer:

Colon_Cancer

neprilysin nep is a neutral endopeptidase it is also known by different functional names such as common acute lymphoblastic leukemia antigen calla the cluster of differentiation cd10 endoprotease endopeptidase and membrane metalloen dopeptidase nep is a member of m13 family of zinc peptidase in the body nep cleaves many peptides such as atrial natri uretic peptides btype natriuretic peptides angiotensins i ii ii en ix bradykinin substance p endothelin i ii amyloid dorphin neurotensin vasopressin etc [“] the progression of various pathological conditions such as kidney and heart disease obesity diabetes [ ] few malignancies such as colon can a ˆ—corresponding author email address anoopkishoremanipaledu a kishore 101016jmolstruc2020129073 elsevier bv all rights reserved cer lung cancer and melanomas [“] etc is associated with the peptidase activity of nep in the us food and drug ad ministration fda approved sacubitrilvalsartan the combination of a neprilysin inhibitor and an angiotensin receptor blocker arb respectively commonly known as angiotensin receptor neprilysin inhibitor arni for heart failure with reduced ejection fraction further in clinical trials involving sacubitrilvalsartan treatment groups performed well in the renal failure population as compared to treatment with an arb valsartan alone there fore nep has gained considerable attention in the last decade for its peptide degrading property and its inhibition has therapeutic potential in multiple diseases but the known and available nep inhibitors are limited hence drug repurposing using different in silico tools can aid in speeding up the process of drug discovery for the development of new nep inhibitors the role of nep has been extensively studied in various dis eases the study report of the paradigm trial highlighted the role 0c r sankhe e rathi and s manandhar of molecular structure of nep inhibitors in the population of heart failure with reduced ejection fraction in an invivo study of subtotal nephrec tomy the renoprotective effect of sacubitrilvalsartan was found to be stronger as compared to valsartan alone according to the result of the uk harpiii trial the combination of sacubi trilvalsartan is effective and is welltolerated in the chronic kidney disease population similarly various studies are focussed on the importance of nep on chronic kidney and cardiovascular dis eases nep inhibition in streptozotocininduced diabetic mice im proved outcomes of cardiac function for heart failure with reduced ejection fraction in diabetic nephropathy the combination of the nep inhibitor thiorphan with an angiotensin receptor blocker and an angiotensinconverting enzyme ii activator showed significant improvement in the condition by modulating components of the reninangiotensin system and natriuretic peptide system the activation of the leptinaldosteroneneprilysin axis contributes to the pathogenesis of cardiac complications in obese patients in obesity and type diabetes nep inhibition showed improve ment in insulin sensitivity and glycaemic control the inhibition re sults in modulation of several peptides with glucoregulatory prop erties such as bradykinin cholecystokinin glycogen like peptide glucosedependent insulinotropic peptide secretin and vasoactive intestinal polypeptide leading to improved glucose homeostasis and weight loss a study conducted to evaluate the effect of nep on nociception concluded that nep inhibition can be a good strategy for pain management in cancers such as colon cancer [ ] lung cancer [ ] and melanomas the increased levels of nep is correlated with neoplastic progression the peptidase ac tivity of nep and its interaction with akt focal adhesion kinase is assumed to contribute to the pathogenesis of colon cancer in aggressive melanomas cd10 nep is the biomarker for detec tion a recent report has highlighted the role of arni in enhanc ing anti‚ammatory and natriuretic peptide systems in covid patients [ ] additionally the use of arni is also recom mended for patients suffering from covid19 all these find ings highlighted the need for designing novel nep inhibitors but de novo drug development is resource intensive and time consum ing hence drug discovery by repurposing the existing drugs can be an attractive strategy with the benefit of reduced developmen tal risk especially in the case of nep inhibitors the computation repurposing is known as ˜ insilico drug re purposing™ in in the us approximately of drugs ap proved was through the drug repurposing approach the con cept of drug repurposing has been already practiced in cardio vascular disorders cancer obesity erectile dysfunction smoking cessation stress psychosis etc drug repurposing using al ready approved drugs reduces the time and money on preliminary screening toxicity studies clinical trials bulk manufacturing and formulation development on the other hand the establishment of new drug candidates requires lots of time and resources a good example is the case of allopurinol which was originally approved for cancer and is now available for the treatment of gout in this context we decided to identify a series of inhibitors for nep using insilico drug repurposing the protein structure of the extracellular domain of nep with sacubitralat the active metabo lite of sacubitril was used in the current study the inhibitor bind ing pocket in the protein structure of the extracellular domain of human nep pdb id 5jmy has already been revealed by schier ing nikolaus the inhibitor binding pocket contains the catalytically essential triad of his583 his587 and glu646 for our drug repurposing study the structures of fda approved drugs were downloaded from the zinc database based on the binding pocket of the nep inhibitor the high throughput virtual screening of existing fda approved drugs was done to find out a new se ries of nep inhibitors to the best of our knowledge this is the first study based on drug repurposing approach that is being re ported and employed for the development of nep inhibitors using receptorinhibitor complex materials and methods in the current study the maestro molecular platform version by schrodinger llc was used to perform molecular dock ing and simulation studies on an hp desktop system with linux ubuntu lts platform intel haswell graphics card 8gb ram and intel core i34160 processor protein preparation and grid generation xray crystallographic structure of the extracellular domain of human nep pdb id 5jmy was downloaded from the rcsb pro tein data bank the pdb id 5jmy has a resolution of ˚a prior to docking and simulation studies the biological unit of protein was prepared using ˜protein preparation wizard™ in schrodinger suite during the process of protein preparation the protein was subjected to import and refine review and modify and minimize processes in protein preparation wizard missing side chains and residues were filled using the prime tool the active site and cat alytically important residues were retained in the protein structure the water molecules beyond ˚a were deleted and stages were generated for hetero atoms to generate low energy state protein energy minimization was done using opls3e optimized potential for liquid stimulation force field and the prepared protein was used for molecular modelling to generate a grid around the lig and the receptor grid generation workflow was used by keeping all functional residues in the grid ligand preparation the structures of fda approved drugs from zinc database were downloaded for ligand preparation the lig prep tool was employed the lowest energy 3d structures with cor ± under the opls3e related chiralities were generated at ph force field in this process all the ligands were preprocessed which includes generation of tautomers ionization state at ph ± using epik addition of hydrogen bond charged group neu tralization and ligand geometry were optimized ligand docking all the molecular docking studies were carried out using the ligand docking tool glide gridbased ligand docking with ener getics module the glide module was used for predicting ligand protein binding modes and ranking different scoring functions are involved in glide such as highthroughput virtual screening htvs standard precision sp and extra precision xp first all the drugs were docked with htvs mode but computationally htvs docking does not use descriptor and explicit water technol ogy as used in the xp mode hence to avoid falsepositive results few drugs were reanalyzed using sp and xp modes [ ] free ligand binding energy calculation the prime module was used to determine absolute ligand binding affinities to nep using mmgbsa molecular mechanics energies generalized born and surface area continuum solvation method the mmgbsa assay of top eight xp docked drugs was performed using pose viewer file of glide xp mode the prime mmgbsa method is dependent on the vsgb solvation model that uses a variabledielectric generalized born model and water as a solvent under the opls3e force field to calculate binding energy 0cr sankhe e rathi and s manandhar of molecular structure adme analysis for the assessment of the adme profile the qikprop tool from the maestro modeling platform was used the qikprop tool helps in the prediction of the druggable property of best four hits based on adme analysis during this process various descriptors such as molecular weight cardiotoxicity qplogherg predicted octanolwater partition coefficient qplogpow permeability qp pcaco polar surface area psa human oral absorption oral absorption and lipinski rule of five were calculated induced fit docking ifdsp table docking score and prime mmgbsa score of top eight drugs sr no drug dock score xp kcalmol mmgbsa 01g bind sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 zinc000000402909 zinc000000601283 zinc000000000797 zinc000003831594 zinc000028973441 ifdsp was carried out using the inducedfit docking module from maestro molecular modelling platform based on the xp glide docking score binding energy crucial residues involved and adme analysis four zinc0 zinc0 zinc0 and zinc0 drugs were selected for ifd“sp docking in ifd based on the bfactor side chains were trimmed with receptor and van der waals scaling of and respectively and a maximum of poses were set for each ligand further prime sidechain prediction and minimization were performed in which refinement of all residues within ˚a of the ligands™ pose and side chains were performed this pro cess allows the ligand structure and conformation to accommodate nearby reorienting side chains the ligands and residues were min imized in inducedfit protein structure all the ligands were rigor ously docked and ifd score for each was calculated using the for mula ˆ— prime_energy ˆ— glide score ˆ—ifd score glide_ecoul molecular dynamics md simulation the flexibility of the receptor is restricted in gridbased dock ing systems like xp and ifd these do not mimic the actual bio logical systems where the protein and drug are solvated in wa ter hence to tackle this problem md simulation was performed based on the glide docking score free binding energy and ifd score four drugs were selected for md simulation for 20ns for md simulation three steps were performed viz system builder mini mization and md simulation the docked complex of protein and ligand were selected and the system model was made by prede fined spc solvent under orthorhombic boundary conditions next the system model was subjected to energy minimization until a gradient threshold reached kcalmol ˚a balanced at k tem perature and bar pressure via npt ensemble in the final step minimized ligandprotein complex were subjected to md simula tion bioisostere replacement for optimization of adme and biological properties of top two selected compounds zinc0 and zinc0 the bioisostere replacement of functional group was performed the bioisosteric replacement tool from maestro molecular modelling platform was employed to create bioisosteric structures of better potency and adme profile further the results of the generated bioisosteres were analysed through interaction of ligands with crucial amino acid residues xp glide docking score free binding energy and adme analysis results nep was prepared at a neutral ph of αhelical α subdomains were present in the extracellular domain both helical subdomains of nep are connected with the linker region ± two and essential catalytic triad are present in the central cavity of both subdomains in the central cavity the catalytically impor tant zinc atom is coordinated with the side chains of amino acid residues his583 his587 and glu646 [ ] in the protein the cocrystallized ligand sacubitrilat is bound to the active site of nep and showed crucial interactions with his583 his587 and glu646 residues a fourth interaction was provided by the car boxylate oxygen adjacent to the p1 methyl group of sacubitri lat to generate a receptor grid receptor grid generation workflow was used and the cubic box of specific dimensions was generated around sacubitrilat to perform molecular docking studies ligand docking around ligands from zinc database were screened with htvs docking mode of glide panel htvs docking mode utilizes a small period to a large set of drugs by reducing the final torsional refinement and comprehensive sampling but during htvs dock ing mode the number of intermediate conformational sampling is limited hence a total of drugs with dock scores less than kcalmole were filtered and reanalyzed in sp docking mode after performing sp docking around drugs were subjected to an extensive xp docking mode of glide panel xp docking mode is more accurate avoids the possibility of falsepositive results and gives an appropriate correlation between a good pose of drugs and a good dock score finally based on xp dock score and pivotal interactions eight active drugs zinc0 zinc0 zinc0 zinc0 zinc0 zinc0 were identified for further screening the docking score of cocrystalized ligand sacubitralat was found to be all the eight selected drugs showed docking scores between to given in table zinc0 zinc0 all the eight drugs showed similar interaction as sacubitri lat schiering nikolaus et al had reported that the hydropho bic interaction of sacubitrilat with phe544 was towards the shal low s1 pocket of nep protein the charge positive interac tion with arg717 and polar interaction with asn542 were found to be common in sacubitrilat and selected eight drugs even in this study all the eight drugs showed hydrophobic interactions with phe544 sacubitrilat also showed interactions with asn542 arg717 arg110 and arg102 our eight selected drugs showed in teractions with atleast two of the aforementioned residues insilico docking studies also showed that all the eight drugs showed in teraction with his711 which then formed a hydrogen bond with zinc causing the stabilization of zinc transition state this in teraction with zinc and its stabilization might result in decreased catalytic activity of nep as it is a zinc dependent endopeptidase nep degrades various peptide substrates at the amino sides of hydrophobic amino acids according to the reports the pro tein structure of nep consists of a large hydrophobic pocket con taining the side chains ala543 ile558 phe563 met579 val580 0c r sankhe e rathi and s manandhar of molecular structure his583 val692 and trp693 the cocrystalized ligand sacu bitrilat showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 the eight se lected drugs also showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 but the hydrophobic interaction with ile558 met579 and trp693 were missing in interactions of zinc0 zinc0 and zinc0 respectively sacubitrilat and the selected eight drugs showed polar pipi stacking and cation interaction with his583 the interactions with side chains of ala543 ile558 phe563 met579 val580 his583 val692 and trp693 may con tribute to inhibition of the peptidase activity of nep according to previous reports amino acid residue glu584 is important for peptidase activity and residues such as ala543 and asn542 are important for nep inhibition in the current study all eight selected drugs possess interaction with glu584 asn542 and ala543 the 2d interaction diagrams with a summary of all non bonding interactions are given in table free ligand binding energy calculation the primemmgbsa was employed to calculate the binding en ergy of the top eight drugs with selected docked poses all the 01g bind eight drugs showed stability in the docked pose with 01g bind ing energy kcalmol described in table the ing energy of cocrystallized drug sacubitrilat was found to be 9651kcalmol the cocrystalized ligand and the eight drugs were found to be stable with docked pose this finding indicates that the selected drugs may act as nep inhibitors induced fit docking ifdsp after the virtual docking studies based on the ligand interac tion and binding energy of the eight drugs four ligands showing good values were taken forward for induced fit docking ifd in virtual docking protocol the interactions occur between the bind ing site of the rigid protein and the flexible ligand but this is not the case with the actual ligandprotein interactions in the body where the target protein undergoes backbone or sidechain move ments after binding with ligands this induces alteration in binding sites of the protein also in the body the ligand binding site on the proteins conforms to the ligand shape and binding mode ifd was conducted to resolve the shortcomings of rigid docking pro tocols ifd has two main applications first it generates the most accurate active complex structure of ligand which is not possi ble in virtual molecular docking with rigid protein structure sec ond ifd avoids falsenegative results of virtual docking in virtual docking screening of the ligands was done with the single confor mation of ligands however in ifd confirmers were generated for each ligand hence ifdsp was carried for zinc0 zinc0 zinc0 and zinc0 and a maximum of conformers were generated for each ligand based on molecular docking and binding energy further the ifd score and ligand interaction were analyzed for selected drugs the ifd score and 3d ligand interactions are given in fig zinc0 showed similar nonbonding interactions as predicted in xp docking the zinc0 exhibits a new hbond interaction with his711 with similar nonbonding interactions as observed in xp docking in ligand interactions of zinc0 the new hbond interaction was observed with his711 and lost with glu584 the hydrophobic interaction with ala543 val580 met579 phe689 val692 trp693 phe563 and phe106 was also lost similarly new hydrophobic interaction was observed with ile718 and lost with ile558 and phe544 the new pipi stacking interactions were observed with trp693 and phe106 and missing with amino acid residue his583 the pipi cation interaction with arg717 was retained and lost with arg110 as predicted in xp docking zinc0 retained hbond interaction with his711 and glu584 showed new hbond inter action with trp693 and lost hbond interaction with arg717 the new pipi stacking interaction was observed with phe106 zinc0 also showed new hydrophobic interaction with phe689 and met579 and hydrophobic interaction missing with tyr545 it also showed similar hydrophobic interaction patterns with other amino acid residues as predicted in xp docking adme analysis adme properties of the four drugs were analyzed using the quikprop module the adme profile was assessed using vari ous descriptor calculations such as molecular weight qplogherg qplogpow qppcaco human oral absorption psa and lipinski rule of five given in table all the selected drugs obey the lip inski rule of five molecular dynamics md simulation molecular dynamics is used to simulate ligandprotein com plexes in presence of systems with biological relevance it includes the explicit solvent representation with the entire protein the main advantage of md stimulation is that it represents the actual conditions of the biological system it provides a highly dynamic protein structure and the ligandprotein complex is solvated with water as happens in the biological system ifd however pro vides limited flexibility which is insufficient to mimic the actual conditions of a biological system hence md simulation studies were carried out to get insights into the top four drugs in terms of binding stability and nonbonding interactions with crucial amino acid within the drugbinding pocket of nep protein in a dynamic state in md simulation the frame was captured for 20ps which results in the generation of frames for 20ns stimulation time and saved in a trajectory further rmsd root mean square devi ation for nep protein and ˜lig fit prot™ for the ligands were com puted to estimate the stability of ligandprotein complex based on molecular docking score binding energy and ifd score the md simulation was carried out for four ligand protein complexes viz zinc0 01427nep docked complex complex zinc0 01533877nep docked complex complex zinc0 0601283nep docked complex complex and zinc0 03831594nep docked complex complex for com plex rmsd values for protein and ligand were found to be ˚a and ˚a respectively the rmsd values were found to be in the acceptable range ˚a but the drift in the ligandprotein complex was observed for a period of 05ns20ns in case of complex the ligandprotein stabilization was observed from 022ns and 59ns respectively and drift was observed for 720ns in complex the rmsd values are ˚a and ˚a for protein and ligand respectively for complex the rmsd values were found to be ˚a for both the complex was initially stable but there was drift for 313ns and eventually stabilization was observed for 1320ns according to the results obtained from md simulation complex is possibly more stable than complex and similarly complex showed rmsd value of ˚a for both the protein and the ligand the com plex showed initial drift from to 13ns but eventually stabi lized from 1320ns overall better stability in protein and ligand was observed in complex and compared to complexes and the rmsd plot of selected ligandprotein complexes are given in fig further the binding pattern and nonbonding interactions were analyzed for all four complexes the binding pattern was found to be different for all four complexes in complex the signifi 0cr sankhe e rathi and s manandhar of molecular structure table 2d interaction diagrams of top eight drugs with a summary of all nonbonding interactions sr no drug 2d ligand interaction diagrams nonbonding interaction sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 hbond glu584 his711 arg717 arg102 asn542 hydrophobic met579 val580 ile558 phe689 val692 trp693 phe563 phe106 ile718 ala543 phe544 polar his583 his587 asn542 salt bridge zn806 arg102 pipi stacking trp693 his583 charged positive arg102 his711 arg717 arg110 charged negative asp650 glu646 glu584 hbond arg717 glu584 ala543 asn542 hydrophobic ile718 phe689 val692 trp693 ala543 phe544 met579 val580 phe106 ile558 phe563 polar thr721 his587 his583 asn542 salt bridge zn806 his711 arg110 pipi cation his583 charged positive his711 arg717 arg110 charged negative glu646 asp650 glu584 hbond ala543 his711 glu584 hydrophobic ile558 phe544 ala543 val580 met579 ile718 phe689 val680 trp693 phe563 phe106 polar asn542 his583 his587 salt bridge zn806 pipi stacking his583 trp693 charged positive arg717 his711 charged negative asp650 glu646 hbond glu584 his711 ala543 trp693 hydrophobic ile718 ile558 ala543 phe544 phe689 ala690 val692 trp693 met579 val580 phe563 phe106 polar thr721 his587 his583 asn542 salt bridge zn806 pipi stacking trp693 charged positive arg717 his711 arg110 charged negative asp650 glu646 glu584 zinc000000402909 hbond his711 glu584 hydrophobic ile718 ala543 phe544 phe689 val692 trp693 met579 val580 phe106 phe563 polar his587 his583 asn542 pipi stacking phe106 his583 salt bridge zn806 charged positive arg717 his711 charged negative asp650 glu646 glu584 continued on next page 0c r sankhe e rathi and s manandhar of molecular structure table continued sr no drug 2d ligand interaction diagrams nonbonding interaction zinc000000601283 zinc000000000797 hbond his711 glu584 hydrophobic phe544 ala543 trp693 val692 phe689 val580 met579 phe106 ile558 phe563 polar his587 his583 asn542 salt bridge zn806 pipi stacking his583 pipi cation arg717 arg110 charged positive arg102 arg110 his711 arg717 charged negative asp709 glu646 glu584 asp650 hbond asn542 hydrophobic ile718 val580 met579 phe689 val692 trp693 ile558 ala543 phe544 phe563 phe106 polar his587 his583 asn542 salt bridge zn806 pipi stacking his711 phe544 his583 pipi cation his711 charged positive arg717 his711 charged negative glu646 glu584 asp650 zinc000003831594 hbond glu584 his711 arg717 hydrophobic val580 ala543 phe544 tyr545 phe106 phe563 ile558 trp693 val692 polar his587 his583 asn542 salt bridge zn806 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 zinc000028973441 hbond glu584 his711 hydrophobic met579 val580 phe544 ala543 phe106 trp693 val692 phe563 ile558 polar his587 his583 asn542 salt bridge zn806 pipi stacking phe106 pipi cation arg110 his711 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 asp650 0cr sankhe e rathi and s manandhar of molecular structure fig 3d ifd ligand interactions and scores of the top four selected drugs ligand interaction of a zinc0 b zinc0 czinc0 0601283d zinc0 with different amino acid residues of nep fig rmsd plot of ligandprotein complexes rmsd plot of a zinc0 b zinc0 c zinc0 d zinc0 with the active site of nep 0c r sankhe e rathi and s manandhar of molecular structure table adme analysis of top four selected drugs using qikprop compound id molecular weight qplogp ow qplogherg qplogs qppcaco oral absorption psa rule of five sacubitrilat zinc000001533877 zinc000000001427 zinc000000601283 zinc000003831594 fig ligandprotein interaction diagram obtained after md stimulation ligand interaction of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep cant hbond interactions were observed with amino acid residues glu584 ala543 and his711 and pipi interaction with his583 and trp693 as predicted in xp docking the hydrophobic interac tions with ala543 trp693 met579 and phe689 were retained in md simulation on the other hand hydrophobic interactions with ile558 phe544 and phe563 were missing in md simulation the hydrophobic interaction with ala543 val580 ile718 val692 and phe106 was weaker affecting the stability of the ligand protein complex similarly the water bridgetype interaction with glu584 was observed in complex strong hbond interaction was shown by asn542 arg717 glu584 and ala543 additional hbond interactions were also observed with his711 and glu646 the hydrophobic interaction with ala543 ile718 phe689 trp693 met579 val580 ile558 phe106and phe563 were weakly con tributing to the stability of ligandprotein complex and the inter action was lost with the amino acid residue phe544 additional water bridge type of interaction was shown by asn542 glu646 and ala543 the pipi cation interactions were retained with his583 as predicted in xp docking in complex hbond interac tion was retained with glu584 and his711 and new hbond inter action was observed with asp709 and arg110 in md simulation complex showed weak hydrophobic interaction with ala543 phe544 val580 trp693 phe563 ile558 and phe106the hy drophobic interaction was lost with amino acid residues met579 phe689 and val692 the new pipi stacking interaction was ob served with his711 however pipi stacking interaction was missing with his583 the new pipi cation interaction was observed with arg717 and pipi cation interaction was missing with arg110 as compared to xp docking the additional water bridge type of inter action was shown by asp709 and glu584 in complex hbond interaction was retained with his711 and arg717 new hbond in teractions were found with trp693 and ala543 whereas hbond interaction was lost with glu584 complex showed strong hy drophobic interaction with trp693 and ala543 whereas weak hy drophobic interaction with val680 phe106 phe563 ile558 and val692 in contrast to xp docking similarly hydrophobic interac tion was missing with amino acid residues phe544 and tyr545 the additional water bridge type of interaction was observed with ala543 among all four complexes complexes and were found to more stable the additional hbond interactions in complexes and may contribute to the stability of the ligandprotein com plexes the ligandprotein md interaction diagrams and histograms of selected complexes are given figs and bioisostere replacement the zinc0 indomethacin a nonsteroidal anti ‚ammatory drug and zinc0 tyropanoic acid a ra diocontrast agent were found to be more stable in md simulation for 20ns the zinc0 is anti‚ammatory antipyretic 0cr sankhe e rathi and s manandhar of molecular structure fig histogram of ligandprotein complexes histogram of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep and analgesic in nature it is commonly used in rheuma toid arthritis acute shoulder pains osteoarthritis spondylitis and acute gouty arthritis zinc0 is known as sodium tyropanoate which is employed in xray diagnosis and imaging of gallstones though they exhibit good binding affinity for nep one of the major disadvantages of zinc0 is its rapid elimination from the body [ ] therefore bioisostere re placement of zinc0 and zinc0 was per formed to enhance biological activity and surpass rapid excretion bioisosteres are the molecules which are generated by replace ment an atom or a group of atoms from the parent drug with other functional groups two main advantages associated with bioisostere replacement are first it will result in generation of new bioisostere molecules with similar biological characteristics of the parent drug second bioisosteres can overcome various prob lems associated with the parent drug™s activity pharmacokinetics and toxicity during the bioisosteric replacement and bioisosteric structures of zinc0 and zinc0 respec tively were generated out of these the top two bioisosteres were identified based on the ligand interactions with the crucial amino acid residues of nep docking score the binding energy calculated employing mmgbsa and adme parameters the top two selected bioisosteres of zinc0 and zinc0 are il lustrated by fig the docking scores of the bioisosteres of zinc0 structure structure are and with binding en ergies and kcalmol respectively similarly the dock ing scores of structure and of zinc0 were found to be and with binding energies and Ï Ïkcalmol respectively table further assessment was done based on the ligand interactions with crucial amino acid residues of the protein compared to the parent drugs table structure of zinc0

cancer7529

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: neprilysin nep is a neutral endopeptidase it is also known by different functional names such as common acute lymphoblastic leukemia antigen calla the cluster of differentiation cd10 endoprotease endopeptidase and membrane metalloen dopeptidase nep is a member of m13 family of zinc peptidase in the body nep cleaves many peptides such as atrial natri uretic peptides btype natriuretic peptides angiotensins i ii ii en ix bradykinin substance p endothelin i ii amyloid dorphin neurotensin vasopressin etc [“] the progression of various pathological conditions such as kidney and heart disease obesity diabetes [ ] few malignancies such as colon can a ˆ—corresponding author email address anoopkishoremanipaledu a kishore 101016jmolstruc2020129073 elsevier bv all rights reserved cer lung cancer and melanomas [“] etc is associated with the peptidase activity of nep in the us food and drug ad ministration fda approved sacubitrilvalsartan the combination of a neprilysin inhibitor and an angiotensin receptor blocker arb respectively commonly known as angiotensin receptor neprilysin inhibitor arni for heart failure with reduced ejection fraction further in clinical trials involving sacubitrilvalsartan treatment groups performed well in the renal failure population as compared to treatment with an arb valsartan alone there fore nep has gained considerable attention in the last decade for its peptide degrading property and its inhibition has therapeutic potential in multiple diseases but the known and available nep inhibitors are limited hence drug repurposing using different in silico tools can aid in speeding up the process of drug discovery for the development of new nep inhibitors the role of nep has been extensively studied in various dis eases the study report of the paradigm trial highlighted the role 0c r sankhe e rathi and s manandhar of molecular structure of nep inhibitors in the population of heart failure with reduced ejection fraction in an invivo study of subtotal nephrec tomy the renoprotective effect of sacubitrilvalsartan was found to be stronger as compared to valsartan alone according to the result of the uk harpiii trial the combination of sacubi trilvalsartan is effective and is welltolerated in the chronic kidney disease population similarly various studies are focussed on the importance of nep on chronic kidney and cardiovascular dis eases nep inhibition in streptozotocininduced diabetic mice im proved outcomes of cardiac function for heart failure with reduced ejection fraction in diabetic nephropathy the combination of the nep inhibitor thiorphan with an angiotensin receptor blocker and an angiotensinconverting enzyme ii activator showed significant improvement in the condition by modulating components of the reninangiotensin system and natriuretic peptide system the activation of the leptinaldosteroneneprilysin axis contributes to the pathogenesis of cardiac complications in obese patients in obesity and type diabetes nep inhibition showed improve ment in insulin sensitivity and glycaemic control the inhibition re sults in modulation of several peptides with glucoregulatory prop erties such as bradykinin cholecystokinin glycogen like peptide glucosedependent insulinotropic peptide secretin and vasoactive intestinal polypeptide leading to improved glucose homeostasis and weight loss a study conducted to evaluate the effect of nep on nociception concluded that nep inhibition can be a good strategy for pain management in cancers such as colon cancer [ ] lung cancer [ ] and melanomas the increased levels of nep is correlated with neoplastic progression the peptidase ac tivity of nep and its interaction with akt focal adhesion kinase is assumed to contribute to the pathogenesis of colon cancer in aggressive melanomas cd10 nep is the biomarker for detec tion a recent report has highlighted the role of arni in enhanc ing anti‚ammatory and natriuretic peptide systems in covid patients [ ] additionally the use of arni is also recom mended for patients suffering from covid19 all these find ings highlighted the need for designing novel nep inhibitors but de novo drug development is resource intensive and time consum ing hence drug discovery by repurposing the existing drugs can be an attractive strategy with the benefit of reduced developmen tal risk especially in the case of nep inhibitors the computation repurposing is known as ˜ insilico drug re purposing™ in in the us approximately of drugs ap proved was through the drug repurposing approach the con cept of drug repurposing has been already practiced in cardio vascular disorders cancer obesity erectile dysfunction smoking cessation stress psychosis etc drug repurposing using al ready approved drugs reduces the time and money on preliminary screening toxicity studies clinical trials bulk manufacturing and formulation development on the other hand the establishment of new drug candidates requires lots of time and resources a good example is the case of allopurinol which was originally approved for cancer and is now available for the treatment of gout in this context we decided to identify a series of inhibitors for nep using insilico drug repurposing the protein structure of the extracellular domain of nep with sacubitralat the active metabo lite of sacubitril was used in the current study the inhibitor bind ing pocket in the protein structure of the extracellular domain of human nep pdb id 5jmy has already been revealed by schier ing nikolaus the inhibitor binding pocket contains the catalytically essential triad of his583 his587 and glu646 for our drug repurposing study the structures of fda approved drugs were downloaded from the zinc database based on the binding pocket of the nep inhibitor the high throughput virtual screening of existing fda approved drugs was done to find out a new se ries of nep inhibitors to the best of our knowledge this is the first study based on drug repurposing approach that is being re ported and employed for the development of nep inhibitors using receptorinhibitor complex materials and methods in the current study the maestro molecular platform version by schrodinger llc was used to perform molecular dock ing and simulation studies on an hp desktop system with linux ubuntu lts platform intel haswell graphics card 8gb ram and intel core i34160 processor protein preparation and grid generation xray crystallographic structure of the extracellular domain of human nep pdb id 5jmy was downloaded from the rcsb pro tein data bank the pdb id 5jmy has a resolution of ˚a prior to docking and simulation studies the biological unit of protein was prepared using ˜protein preparation wizard™ in schrodinger suite during the process of protein preparation the protein was subjected to import and refine review and modify and minimize processes in protein preparation wizard missing side chains and residues were filled using the prime tool the active site and cat alytically important residues were retained in the protein structure the water molecules beyond ˚a were deleted and stages were generated for hetero atoms to generate low energy state protein energy minimization was done using opls3e optimized potential for liquid stimulation force field and the prepared protein was used for molecular modelling to generate a grid around the lig and the receptor grid generation workflow was used by keeping all functional residues in the grid ligand preparation the structures of fda approved drugs from zinc database were downloaded for ligand preparation the lig prep tool was employed the lowest energy 3d structures with cor ± under the opls3e related chiralities were generated at ph force field in this process all the ligands were preprocessed which includes generation of tautomers ionization state at ph ± using epik addition of hydrogen bond charged group neu tralization and ligand geometry were optimized ligand docking all the molecular docking studies were carried out using the ligand docking tool glide gridbased ligand docking with ener getics module the glide module was used for predicting ligand protein binding modes and ranking different scoring functions are involved in glide such as highthroughput virtual screening htvs standard precision sp and extra precision xp first all the drugs were docked with htvs mode but computationally htvs docking does not use descriptor and explicit water technol ogy as used in the xp mode hence to avoid falsepositive results few drugs were reanalyzed using sp and xp modes [ ] free ligand binding energy calculation the prime module was used to determine absolute ligand binding affinities to nep using mmgbsa molecular mechanics energies generalized born and surface area continuum solvation method the mmgbsa assay of top eight xp docked drugs was performed using pose viewer file of glide xp mode the prime mmgbsa method is dependent on the vsgb solvation model that uses a variabledielectric generalized born model and water as a solvent under the opls3e force field to calculate binding energy 0cr sankhe e rathi and s manandhar of molecular structure adme analysis for the assessment of the adme profile the qikprop tool from the maestro modeling platform was used the qikprop tool helps in the prediction of the druggable property of best four hits based on adme analysis during this process various descriptors such as molecular weight cardiotoxicity qplogherg predicted octanolwater partition coefficient qplogpow permeability qp pcaco polar surface area psa human oral absorption oral absorption and lipinski rule of five were calculated induced fit docking ifdsp table docking score and prime mmgbsa score of top eight drugs sr no drug dock score xp kcalmol mmgbsa 01g bind sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 zinc000000402909 zinc000000601283 zinc000000000797 zinc000003831594 zinc000028973441 ifdsp was carried out using the inducedfit docking module from maestro molecular modelling platform based on the xp glide docking score binding energy crucial residues involved and adme analysis four zinc0 zinc0 zinc0 and zinc0 drugs were selected for ifd“sp docking in ifd based on the bfactor side chains were trimmed with receptor and van der waals scaling of and respectively and a maximum of poses were set for each ligand further prime sidechain prediction and minimization were performed in which refinement of all residues within ˚a of the ligands™ pose and side chains were performed this pro cess allows the ligand structure and conformation to accommodate nearby reorienting side chains the ligands and residues were min imized in inducedfit protein structure all the ligands were rigor ously docked and ifd score for each was calculated using the for mula ˆ— prime_energy ˆ— glide score ˆ—ifd score glide_ecoul molecular dynamics md simulation the flexibility of the receptor is restricted in gridbased dock ing systems like xp and ifd these do not mimic the actual bio logical systems where the protein and drug are solvated in wa ter hence to tackle this problem md simulation was performed based on the glide docking score free binding energy and ifd score four drugs were selected for md simulation for 20ns for md simulation three steps were performed viz system builder mini mization and md simulation the docked complex of protein and ligand were selected and the system model was made by prede fined spc solvent under orthorhombic boundary conditions next the system model was subjected to energy minimization until a gradient threshold reached kcalmol ˚a balanced at k tem perature and bar pressure via npt ensemble in the final step minimized ligandprotein complex were subjected to md simula tion bioisostere replacement for optimization of adme and biological properties of top two selected compounds zinc0 and zinc0 the bioisostere replacement of functional group was performed the bioisosteric replacement tool from maestro molecular modelling platform was employed to create bioisosteric structures of better potency and adme profile further the results of the generated bioisosteres were analysed through interaction of ligands with crucial amino acid residues xp glide docking score free binding energy and adme analysis results nep was prepared at a neutral ph of αhelical α subdomains were present in the extracellular domain both helical subdomains of nep are connected with the linker region ± two and essential catalytic triad are present in the central cavity of both subdomains in the central cavity the catalytically impor tant zinc atom is coordinated with the side chains of amino acid residues his583 his587 and glu646 [ ] in the protein the cocrystallized ligand sacubitrilat is bound to the active site of nep and showed crucial interactions with his583 his587 and glu646 residues a fourth interaction was provided by the car boxylate oxygen adjacent to the p1 methyl group of sacubitri lat to generate a receptor grid receptor grid generation workflow was used and the cubic box of specific dimensions was generated around sacubitrilat to perform molecular docking studies ligand docking around ligands from zinc database were screened with htvs docking mode of glide panel htvs docking mode utilizes a small period to a large set of drugs by reducing the final torsional refinement and comprehensive sampling but during htvs dock ing mode the number of intermediate conformational sampling is limited hence a total of drugs with dock scores less than kcalmole were filtered and reanalyzed in sp docking mode after performing sp docking around drugs were subjected to an extensive xp docking mode of glide panel xp docking mode is more accurate avoids the possibility of falsepositive results and gives an appropriate correlation between a good pose of drugs and a good dock score finally based on xp dock score and pivotal interactions eight active drugs zinc0 zinc0 zinc0 zinc0 zinc0 zinc0 were identified for further screening the docking score of cocrystalized ligand sacubitralat was found to be all the eight selected drugs showed docking scores between to given in table zinc0 zinc0 all the eight drugs showed similar interaction as sacubitri lat schiering nikolaus et al had reported that the hydropho bic interaction of sacubitrilat with phe544 was towards the shal low s1 pocket of nep protein the charge positive interac tion with arg717 and polar interaction with asn542 were found to be common in sacubitrilat and selected eight drugs even in this study all the eight drugs showed hydrophobic interactions with phe544 sacubitrilat also showed interactions with asn542 arg717 arg110 and arg102 our eight selected drugs showed in teractions with atleast two of the aforementioned residues insilico docking studies also showed that all the eight drugs showed in teraction with his711 which then formed a hydrogen bond with zinc causing the stabilization of zinc transition state this in teraction with zinc and its stabilization might result in decreased catalytic activity of nep as it is a zinc dependent endopeptidase nep degrades various peptide substrates at the amino sides of hydrophobic amino acids according to the reports the pro tein structure of nep consists of a large hydrophobic pocket con taining the side chains ala543 ile558 phe563 met579 val580 0c r sankhe e rathi and s manandhar of molecular structure his583 val692 and trp693 the cocrystalized ligand sacu bitrilat showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 the eight se lected drugs also showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 but the hydrophobic interaction with ile558 met579 and trp693 were missing in interactions of zinc0 zinc0 and zinc0 respectively sacubitrilat and the selected eight drugs showed polar pipi stacking and cation interaction with his583 the interactions with side chains of ala543 ile558 phe563 met579 val580 his583 val692 and trp693 may con tribute to inhibition of the peptidase activity of nep according to previous reports amino acid residue glu584 is important for peptidase activity and residues such as ala543 and asn542 are important for nep inhibition in the current study all eight selected drugs possess interaction with glu584 asn542 and ala543 the 2d interaction diagrams with a summary of all non bonding interactions are given in table free ligand binding energy calculation the primemmgbsa was employed to calculate the binding en ergy of the top eight drugs with selected docked poses all the 01g bind eight drugs showed stability in the docked pose with 01g bind ing energy kcalmol described in table the ing energy of cocrystallized drug sacubitrilat was found to be 9651kcalmol the cocrystalized ligand and the eight drugs were found to be stable with docked pose this finding indicates that the selected drugs may act as nep inhibitors induced fit docking ifdsp after the virtual docking studies based on the ligand interac tion and binding energy of the eight drugs four ligands showing good values were taken forward for induced fit docking ifd in virtual docking protocol the interactions occur between the bind ing site of the rigid protein and the flexible ligand but this is not the case with the actual ligandprotein interactions in the body where the target protein undergoes backbone or sidechain move ments after binding with ligands this induces alteration in binding sites of the protein also in the body the ligand binding site on the proteins conforms to the ligand shape and binding mode ifd was conducted to resolve the shortcomings of rigid docking pro tocols ifd has two main applications first it generates the most accurate active complex structure of ligand which is not possi ble in virtual molecular docking with rigid protein structure sec ond ifd avoids falsenegative results of virtual docking in virtual docking screening of the ligands was done with the single confor mation of ligands however in ifd confirmers were generated for each ligand hence ifdsp was carried for zinc0 zinc0 zinc0 and zinc0 and a maximum of conformers were generated for each ligand based on molecular docking and binding energy further the ifd score and ligand interaction were analyzed for selected drugs the ifd score and 3d ligand interactions are given in fig zinc0 showed similar nonbonding interactions as predicted in xp docking the zinc0 exhibits a new hbond interaction with his711 with similar nonbonding interactions as observed in xp docking in ligand interactions of zinc0 the new hbond interaction was observed with his711 and lost with glu584 the hydrophobic interaction with ala543 val580 met579 phe689 val692 trp693 phe563 and phe106 was also lost similarly new hydrophobic interaction was observed with ile718 and lost with ile558 and phe544 the new pipi stacking interactions were observed with trp693 and phe106 and missing with amino acid residue his583 the pipi cation interaction with arg717 was retained and lost with arg110 as predicted in xp docking zinc0 retained hbond interaction with his711 and glu584 showed new hbond inter action with trp693 and lost hbond interaction with arg717 the new pipi stacking interaction was observed with phe106 zinc0 also showed new hydrophobic interaction with phe689 and met579 and hydrophobic interaction missing with tyr545 it also showed similar hydrophobic interaction patterns with other amino acid residues as predicted in xp docking adme analysis adme properties of the four drugs were analyzed using the quikprop module the adme profile was assessed using vari ous descriptor calculations such as molecular weight qplogherg qplogpow qppcaco human oral absorption psa and lipinski rule of five given in table all the selected drugs obey the lip inski rule of five molecular dynamics md simulation molecular dynamics is used to simulate ligandprotein com plexes in presence of systems with biological relevance it includes the explicit solvent representation with the entire protein the main advantage of md stimulation is that it represents the actual conditions of the biological system it provides a highly dynamic protein structure and the ligandprotein complex is solvated with water as happens in the biological system ifd however pro vides limited flexibility which is insufficient to mimic the actual conditions of a biological system hence md simulation studies were carried out to get insights into the top four drugs in terms of binding stability and nonbonding interactions with crucial amino acid within the drugbinding pocket of nep protein in a dynamic state in md simulation the frame was captured for 20ps which results in the generation of frames for 20ns stimulation time and saved in a trajectory further rmsd root mean square devi ation for nep protein and ˜lig fit prot™ for the ligands were com puted to estimate the stability of ligandprotein complex based on molecular docking score binding energy and ifd score the md simulation was carried out for four ligand protein complexes viz zinc0 01427nep docked complex complex zinc0 01533877nep docked complex complex zinc0 0601283nep docked complex complex and zinc0 03831594nep docked complex complex for com plex rmsd values for protein and ligand were found to be ˚a and ˚a respectively the rmsd values were found to be in the acceptable range ˚a but the drift in the ligandprotein complex was observed for a period of 05ns20ns in case of complex the ligandprotein stabilization was observed from 022ns and 59ns respectively and drift was observed for 720ns in complex the rmsd values are ˚a and ˚a for protein and ligand respectively for complex the rmsd values were found to be ˚a for both the complex was initially stable but there was drift for 313ns and eventually stabilization was observed for 1320ns according to the results obtained from md simulation complex is possibly more stable than complex and similarly complex showed rmsd value of ˚a for both the protein and the ligand the com plex showed initial drift from to 13ns but eventually stabi lized from 1320ns overall better stability in protein and ligand was observed in complex and compared to complexes and the rmsd plot of selected ligandprotein complexes are given in fig further the binding pattern and nonbonding interactions were analyzed for all four complexes the binding pattern was found to be different for all four complexes in complex the signifi 0cr sankhe e rathi and s manandhar of molecular structure table 2d interaction diagrams of top eight drugs with a summary of all nonbonding interactions sr no drug 2d ligand interaction diagrams nonbonding interaction sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 hbond glu584 his711 arg717 arg102 asn542 hydrophobic met579 val580 ile558 phe689 val692 trp693 phe563 phe106 ile718 ala543 phe544 polar his583 his587 asn542 salt bridge zn806 arg102 pipi stacking trp693 his583 charged positive arg102 his711 arg717 arg110 charged negative asp650 glu646 glu584 hbond arg717 glu584 ala543 asn542 hydrophobic ile718 phe689 val692 trp693 ala543 phe544 met579 val580 phe106 ile558 phe563 polar thr721 his587 his583 asn542 salt bridge zn806 his711 arg110 pipi cation his583 charged positive his711 arg717 arg110 charged negative glu646 asp650 glu584 hbond ala543 his711 glu584 hydrophobic ile558 phe544 ala543 val580 met579 ile718 phe689 val680 trp693 phe563 phe106 polar asn542 his583 his587 salt bridge zn806 pipi stacking his583 trp693 charged positive arg717 his711 charged negative asp650 glu646 hbond glu584 his711 ala543 trp693 hydrophobic ile718 ile558 ala543 phe544 phe689 ala690 val692 trp693 met579 val580 phe563 phe106 polar thr721 his587 his583 asn542 salt bridge zn806 pipi stacking trp693 charged positive arg717 his711 arg110 charged negative asp650 glu646 glu584 zinc000000402909 hbond his711 glu584 hydrophobic ile718 ala543 phe544 phe689 val692 trp693 met579 val580 phe106 phe563 polar his587 his583 asn542 pipi stacking phe106 his583 salt bridge zn806 charged positive arg717 his711 charged negative asp650 glu646 glu584 continued on next page 0c r sankhe e rathi and s manandhar of molecular structure table continued sr no drug 2d ligand interaction diagrams nonbonding interaction zinc000000601283 zinc000000000797 hbond his711 glu584 hydrophobic phe544 ala543 trp693 val692 phe689 val580 met579 phe106 ile558 phe563 polar his587 his583 asn542 salt bridge zn806 pipi stacking his583 pipi cation arg717 arg110 charged positive arg102 arg110 his711 arg717 charged negative asp709 glu646 glu584 asp650 hbond asn542 hydrophobic ile718 val580 met579 phe689 val692 trp693 ile558 ala543 phe544 phe563 phe106 polar his587 his583 asn542 salt bridge zn806 pipi stacking his711 phe544 his583 pipi cation his711 charged positive arg717 his711 charged negative glu646 glu584 asp650 zinc000003831594 hbond glu584 his711 arg717 hydrophobic val580 ala543 phe544 tyr545 phe106 phe563 ile558 trp693 val692 polar his587 his583 asn542 salt bridge zn806 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 zinc000028973441 hbond glu584 his711 hydrophobic met579 val580 phe544 ala543 phe106 trp693 val692 phe563 ile558 polar his587 his583 asn542 salt bridge zn806 pipi stacking phe106 pipi cation arg110 his711 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 asp650 0cr sankhe e rathi and s manandhar of molecular structure fig 3d ifd ligand interactions and scores of the top four selected drugs ligand interaction of a zinc0 b zinc0 czinc0 0601283d zinc0 with different amino acid residues of nep fig rmsd plot of ligandprotein complexes rmsd plot of a zinc0 b zinc0 c zinc0 d zinc0 with the active site of nep 0c r sankhe e rathi and s manandhar of molecular structure table adme analysis of top four selected drugs using qikprop compound id molecular weight qplogp ow qplogherg qplogs qppcaco oral absorption psa rule of five sacubitrilat zinc000001533877 zinc000000001427 zinc000000601283 zinc000003831594 fig ligandprotein interaction diagram obtained after md stimulation ligand interaction of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep cant hbond interactions were observed with amino acid residues glu584 ala543 and his711 and pipi interaction with his583 and trp693 as predicted in xp docking the hydrophobic interac tions with ala543 trp693 met579 and phe689 were retained in md simulation on the other hand hydrophobic interactions with ile558 phe544 and phe563 were missing in md simulation the hydrophobic interaction with ala543 val580 ile718 val692 and phe106 was weaker affecting the stability of the ligand protein complex similarly the water bridgetype interaction with glu584 was observed in complex strong hbond interaction was shown by asn542 arg717 glu584 and ala543 additional hbond interactions were also observed with his711 and glu646 the hydrophobic interaction with ala543 ile718 phe689 trp693 met579 val580 ile558 phe106and phe563 were weakly con tributing to the stability of ligandprotein complex and the inter action was lost with the amino acid residue phe544 additional water bridge type of interaction was shown by asn542 glu646 and ala543 the pipi cation interactions were retained with his583 as predicted in xp docking in complex hbond interac tion was retained with glu584 and his711 and new hbond inter action was observed with asp709 and arg110 in md simulation complex showed weak hydrophobic interaction with ala543 phe544 val580 trp693 phe563 ile558 and phe106the hy drophobic interaction was lost with amino acid residues met579 phe689 and val692 the new pipi stacking interaction was ob served with his711 however pipi stacking interaction was missing with his583 the new pipi cation interaction was observed with arg717 and pipi cation interaction was missing with arg110 as compared to xp docking the additional water bridge type of inter action was shown by asp709 and glu584 in complex hbond interaction was retained with his711 and arg717 new hbond in teractions were found with trp693 and ala543 whereas hbond interaction was lost with glu584 complex showed strong hy drophobic interaction with trp693 and ala543 whereas weak hy drophobic interaction with val680 phe106 phe563 ile558 and val692 in contrast to xp docking similarly hydrophobic interac tion was missing with amino acid residues phe544 and tyr545 the additional water bridge type of interaction was observed with ala543 among all four complexes complexes and were found to more stable the additional hbond interactions in complexes and may contribute to the stability of the ligandprotein com plexes the ligandprotein md interaction diagrams and histograms of selected complexes are given figs and bioisostere replacement the zinc0 indomethacin a nonsteroidal anti ‚ammatory drug and zinc0 tyropanoic acid a ra diocontrast agent were found to be more stable in md simulation for 20ns the zinc0 is anti‚ammatory antipyretic 0cr sankhe e rathi and s manandhar of molecular structure fig histogram of ligandprotein complexes histogram of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep and analgesic in nature it is commonly used in rheuma toid arthritis acute shoulder pains osteoarthritis spondylitis and acute gouty arthritis zinc0 is known as sodium tyropanoate which is employed in xray diagnosis and imaging of gallstones though they exhibit good binding affinity for nep one of the major disadvantages of zinc0 is its rapid elimination from the body [ ] therefore bioisostere re placement of zinc0 and zinc0 was per formed to enhance biological activity and surpass rapid excretion bioisosteres are the molecules which are generated by replace ment an atom or a group of atoms from the parent drug with other functional groups two main advantages associated with bioisostere replacement are first it will result in generation of new bioisostere molecules with similar biological characteristics of the parent drug second bioisosteres can overcome various prob lems associated with the parent drug™s activity pharmacokinetics and toxicity during the bioisosteric replacement and bioisosteric structures of zinc0 and zinc0 respec tively were generated out of these the top two bioisosteres were identified based on the ligand interactions with the crucial amino acid residues of nep docking score the binding energy calculated employing mmgbsa and adme parameters the top two selected bioisosteres of zinc0 and zinc0 are il lustrated by fig the docking scores of the bioisosteres of zinc0 structure structure are and with binding en ergies and kcalmol respectively similarly the dock ing scores of structure and of zinc0 were found to be and with binding energies and Ï Ïkcalmol respectively table further assessment was done based on the ligand interactions with crucial amino acid residues of the protein compared to the parent drugs table structure of zinc0 Answer:

Colon_Cancer

covid19 has had an impact on the provision of colorectal cancer care the aim of the crccovid study is to describe the changes in colorectal cancer services in the uk and usa in response to thepandemic and to understand the longterm impactmethods and analysis this study comprises phases phase is a survey of colorectal units that aims toevaluate adherences and deviations from the best practice guidelines during the covid19 pandemicphase is a monthly prospective data collection of service provision that aims to determine the impactof the service modifications on the longterm cancer specific outcomes compared to the national standards phase aims to predict costs attributable to the modifications of the crc services and additionalresources required to treat patients whose treatment has been affected by the pandemic phase aims tocompare the impact of covid19 on the nhs and usa model of healthcare in terms of service provisionand cost and to propose a standardised model of delivering colorectal cancer services for future outbreaksethics and dissemination this study is a service evaluation and does not require hra approval or ethicalapproval in the uk local service evaluation registration is required for each participating centre in theusa ethical approval was granted by the research and development committee the results of thisstudy will be disseminated to stakeholders submitted for peer review publications conference presentations and circulated via social mediaregistration details nil the authors published by elsevier ltd on behalf of surgical associates ltd this is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 introductionthe covid19 pandemic has had a significant impact on theprovision of healthcare worldwide as of the 29th june covid19 has resulted in confirmed cases and ‡‘ corresponding author at department of surgery and cancer imperial collegelondon chelsea and westminster and the royal marsden campus unitedkingdomemail address ckontovounisiosimperialacuk c kontovounisiosdeaths in the uk at a local level hospitals have been forcedto make a number of workforce modifications and changes toserviceprovision to combat the crisis and maintain standards ofcare for our patients facetoface consultations have beendissolved or minimized in favour of telephone or virtual clinicsprovision of investigations including ct scans and endoscopieshave been significantly reduced and all benign surgical procedurespostponed furthermore the treatment algorithm for confirmed colorectal cancer cases has proved challenging101016jisjp20200700524683574 the authors published by elsevier ltd on behalf of surgical associates ltdthis is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 0ca courtney international of surgery protocols “in the uk over forty thousand patients are diagnosed with colorectal cancer each year deviation from nice colorectal cancercrc guidelines may lead to significantly poorer outcomes however the current model of cancer services delivery cannot be maintained because of both resource limitation and the potential risksto patients and staff during the pandemic there is a lack of highdependency beds which are being utilized for covid19 patientsthere is the risk of exposing colorectal cancer patients the majority of whom are elderly and have significant comorbidities to thevirus during their treatment within the hospital patients requiringneoadjuvant or adjuvant therapy are at particular risk finallystaff safety must also be considered particularly around aerosolgenerating procedures such as endoscopy and laparoscopic surgery intercollegiate general surgery guidance on covid19 outlinedgeneral principles on the provision of a safe surgical service duringthe pandemic however there has been no specific guidance todate on how to best modify colorectal cancer crc service provision during the pandemic in the absence of a national consensusthe onus is on individual hospital trusts and multidisciplinaryteams to make very challenging decisions about individual patientcare lack of a unified approach may have important consequencesat patient and healthcare institution levelsdelay in cancer diagnosis or treatment due to service modification is likely to create an increased demand in resources once thecrisis has passed predicting the economic impact and planningfor this is essentialhow hospitals approach the new constraints on crc care andallocate resources may vary between the uk and usa it is hopedthat gaining insights from both perspectives will improve the problem solving methods and analysis aims and objectivesthe aim of the crc covid study is to describe the changes incolorectal cancer services in the uk and usa in response to thecovid19 pandemic and to understand the longterm impactour primary and secondary objectives relevant to each phase ofthe study are listed in table study designthis is a multicentre service evaluation conducted through aresearch collaborative with the support of the crc covid steeringcommittee all colorectal units continuing to provide cancer services in the uk ireland and the usa have been invited to participate all study and recruitment information is available on thetable primary and secondary study objectiveswebsite crccovid this service evaluation has been endorsedby the royal college of surgeons of england rcsthis service evaluation will be carried out in phases fig phase uses a questionnaire to assess the modificationsadopted by each colorectal unit in order to continue provision ofthe colorectal cancer services during the covid19 pandemic ithas been developed using an iterative process after research ofall relevant guidelines to construct the standard against which services would be evaluatedthe following guidelines relevant to the management of colorectal cancer have been used as standards for this serviceevaluation nice guidelines colorectal cancer [ng151] nice guideline suspected cancer recognition and referral[ng12] association of coloproctology of great britain irelandacpgbi guidelines for the management of cancer of thecolon rectum and anus [“] british society of gastroenterologyassociation of coloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillanceguidelines informal consultations with consultants nurse specialists andpatients have been used to develop the tool and then it has beenmodified after clinician review for face validity flow and relevance the final instrument comprises questionsphase investigates the provision of colorectal cancer servicesduring the covid19 pandemic by evaluating the performance ofeach unit against the national bowel cancer audit outcomes all centres participating in phase will be required to registerthis service evaluation as per local protocol prior to commencement of data collection on redcap this will be the responsibilityof the local leadphase of the study will develop a prediction model of the economic burden of the modifications in cancer service delivery thismodel will be designed jointly by two international businessschools based on previous publications and national statisticsphase will evaluate and compare the impact of the covid19pandemic on the nhs and the usa healthcare using data collectedduring phase and the predictive mode utilized in phase specificdifferences in modifications of crc services will be examined recruitmentphase survey has been distributed to all colorectal consultantsin the uk and ireland through personalised emails social mediaand the rcs the recruitment of colorectal cancer units in thephasephase phase phase phase primary objectiveevaluate adherences and deviations from best practiceguidelines on colorectal cancer during covid19pandemicative crc service deliverysecondary objectives 0f describe modifications to screening process for crc 0f describe modifications to preoperative intraoperative and postoper 0f demonstrate global effect of covid19 pandemic on crc service provi 0f outline consensus recommendations for sustainable modifications to 0f predict the impact of modifications on the incidence and prevalence of 0f plan adjustments to crc service provision after the end of pandemicsion irrespective of the type of healthcare systemdetermine the impact of crc service provision followingmodifications on longterm cancer specific outcomescompared to national standards 0f predict the costs attributable to modifications of crc services during covid19 pandemic 0f predict additional resources required to treat patients whose treatment has been affected by covid19 0f compare the impact of covid19 on the nhs and usa model of healthcare in terms of service provision and cost 0f propose a standardised model of delivering colorectal cancer services for future outbreaksdifferent crc stages in monthscrc services 0ca courtney international of surgery protocols “fig phases of crc covidusa will use similar approach all units recruited into phase arerecruited into phase participation in phase is not mandatory inorder to participate in phase data collectionall surveys and data collection follow the gdpr requirementsand comply with caldicott principles individual patient identifiable data is not collected in this study study data is collectedand managed using redcap electronic data capture tool hostedat the kennedy institute of rheumatology at the university ofoxford redcap research electronic data capture is asecure webbased software platform designed to support datacapture for research studies providing an intuitive interfacefor validated data capture audit trails for tracking data manipulation and export procedures automated export procedures forseamless data downloads to common statistical packages and procedures for data integration and interoperability with externalsources after the close of the phase data collection period all data setswill be checked for missing data where possible centres will begiven an opportunity to rectify missing data centres where of data is missing will be excluded from data analysis and localleads will be notified a nominated data validator will need toensure data accuracy prior to submission if during this processmajor discrepancy is identified within the data set the centre™sdata will be excluded completely from the analysisfor details of the data collected in phase and phase pleaserefer to supplement data analysisphase responses to the survey pertaining to deviations fromdiagnostic and treatment protocols during the covid19 pandemicsee supplement will be converted to a numerical scale where will denote no deviation and will denote complete cessation ofservice provision the scores will be summarized using appropriatesummary statistics and analyzed using unsupervised learningkmeans and hierarchical clustering to identify clusters of homogeneous response to the pandemicphase every month participating centres will report theirdiagnostic and treatment activity see supplement to determine the impact of covid19 on colorectal cancer activity we willuse timeseries methods and data on historical activity and patientoutcomes to estimate a baseline of expected monthly activity that would have taken place in the absence of the pandemicthe baseline and a confidence interval will be estimated atthe national regional and individual nhs trust level the baselineestimate will then be compared to the actual activity as reportedby publicly available data and data collected by this studythe difference between expected and actual activity will providean estimate of the reduction in activityto quantify the impact on patient outcomes associated with theestimated reduction in activity and deviation from standardizedcare protocols we will use estimates of disease progression available in peerreviewed literature [“] a similar methodologywill be used to predict the impact of the pandemic on the incidenceand prevalence of different colorectal cancer stages in the following months under different scenarios predictions will be madeat the regional and national level and depending on data granularity at the trust levelphase will estimate the financial costs of modifications to thecrc service provision due to the covid19 pandemic this willallow prediction of the expenditure and the additional resourcesrequired to resume routine services we will base this on the literature regarding the price of treatment at different disease stages and the information about the cost of resource utilizationconsultations diagnostic tests operating theatre time and hospital stay phase will compare the results from phases “ in theuk with those in the us discussioncancer care and maintaining high standards of diagnosis andtreatment has long been a priority of the nhs and internationalhealth care systems the pandemic has shifted this focus awayfrom the cancer services colorectal cancer patients are particularly 0ca courtney international of surgery protocols “vulnerable to the disruption of their care as diagnosis throughendoscopy was stopped due to concerns about virus aerosolysation this study is important because it is the first study to ask howindividual units had to modify their services and adapt to the newconstraintsin addition to describing the changes and understandingwhether different units had different approaches we wish to gofurther by understanding the effects of diagnostic and treatmentdelay by prospective data collection of cancer cases referrals diagnosis staging and treatment and comparing them to nationally collected audit data these data will allow us to model the economic impact of thedelay and what resources are required to restore cancer servicesto precovid19 standardsthe strengths of this study are in the multimodal approach tothe issues international collaboration and support from the royalcollege of surgeons our diverse team of management and business academics colorectal surgeons nurse specialists and patientadvisors enable us to have a range of approaches to collect andanalyse the datathe main limitation to the study is nonresponder or samplingbias as we require voluntary participation from colorectal teamswe will ensure that we adjust statistical analysis for any underrepresentation we expect that even with minimal participationuseful models can be generated to understand future resourcerequirements at an individual hospital level the methodologyemployed by other units will demonstrate the utility of the modelin summary this is a novel and important multiphase studythat is vital to understand how to best care for cancer patientsand ensure that the effects of the pandemic are mitigated ethics and disseminationthis study is a service evaluation and does not require hraapproval or ethical approval in the uk departmental approvalhas been granted by the university each participating centre mustseek local permission from their local audit department prior tocommencement of data collection in the usa ethical approvalwas granted by the research and development committeedata for phase will be submitted for publication as soon as theresults become available interim data analysis will be presented tothe royal college of surgeons covid19 research collaborativedata for other phases will be submitted for publication once thedata collection has been completed which is anticipated to be afterthe routine service provision resumes all data will be presented atnational and international conferences circumstances permitting guarantornone research registration numbernoneethical approvalnoneauthor contributionsac designed the study wrote the initial proposal drafted themanuscript based on the study proposal and is part of the auditadvisory groupamh ns nd ow sm sr gm nt mg td bs jee ad ptadvised on the study design and the protocol and is part of thesteering committeeck is a project piall authors read commented on and approved the study designthe protocol and the final manuscriptfundingthis research received no specific grant from any fundingagency in the public commercial or notforprofit sectorsdeclaration of competing interestthe authors declare that they have no known competing financial interests or personal relationships that could have appearedto influence the work reported in this paperreferences world health anization the united kingdom who coronovirus diseasecovid19 dashboard covid19whointregioneurocountrygbaccessed june covidsurg collaborative global guidance for surgical care during the covid pandemic br j surg a spinelli g pellino covid19 pandemic perspectives on an unfolding crisisbr j surg “ british society of gastroenterology endoscopy activity and covid19 bsgand jag guidance apr wwwbsgukcovid19adviceendoscopyactivityandcovid19bsgandjagguidance accessed may nhs england nhs improvement letter to chief executives of all nhs trustsand foundation trusts ccg accountable officers gp practices and primary carenetworks and providers of community health services mar wwwenglandnhsukcoronaviruswpcontentuploadssites52202003urgentnextstepsonnhsresponsetocovid19lettersimonstevenspdfaccessed may research uk cancerincidencecancerbowelstatisticswwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancerincidenceheadingzeroaccessed may royal college of surgeons of england updated intercollegiate general surgeryguidance on covid19 apr wwwrcsengacukcoronavirusjointguidanceforsurgeonsv2 accessed may national institute for health and care excellence nice colorectal cancernice guideline ng151 wwwniceukguidanceng151accessed accessed march national institute for health and care excellence nice suspected cancerrecognition and referral nice guideline ng12 wwwniceukguidanceng12 accessed accessed march c cunningham k leong s clark a plumb s taylor i geh s karandikar bmoran association of coloproctology of great britain ireland acpgbiguidelines for the management of cancer of the colon rectum and anus diagnosis investigations and screening colorectal dis suppl “ s gollins b moran r adams c cunningham s bach as myint a renehans karandikar v goh d prezzi g langman s ahmedzai i geh association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement ofmultidisciplinary management colorectal dis suppl “rectum and anusthe coloncancer of glangman m loughrey n shepherd p quirke association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement of cancer of the colon rectum and anus pathologystandards and datasets colorectal dis suppl “ k leong j hartley s karandikar association of coloproctology of greatbritain ireland acpgbi guidelines for the management of cancer of thecolon rectum and anus follow uplifestyle and survivorshipcolorectal dis suppl “ b moran c cunningham t singh p sagar j bradbury i geh s karandikarassociation of coloproctology of great britain ireland acpgbi guidelinesfor the management of cancer of the colon rectum and anus surgicalmanagement colorectal dis suppl “ md rutter j east cj rees n cripps j docherty s dolwani pv kaye kjmonahan mr novelli a plumb bp saunders s thomasgibson djmtolan s whyte s bonnington a scope r wong b hibbert j marsh bmoores a cross l sharp british society of gastroenterologyassociation ofcoloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillance guidelines gut “ 0ca courtney international of surgery protocols “ healthcare quality improvement partnership hqip national bowel canceraudit annual report an audit of the care received by people with bowelcancer in england and wales v20 wwwnbocaukcontentuploads202001nboca2019v20pdf accessed june pa harris r taylor r thielke j payne n gonzalez jg conde researchelectronic data capture redcap“a metadatadriven methodology andworkflow process for providing translational research informatics support jbiomed inform “ pa harris r taylor bl minor v elliott m fernandez l o™neal l mcleodg delacqua f delacqua j kirby sn duda re consortium the redcapconsortium building an international community of software platformpartners j biomed inform nhs england and nhs improvement cancer waiting times wwwenglandnhsukstatisticsstatisticalworkareascancerwaitingtimesaccessed june national bowel cancer audit nboca datagov weblink accessed junewwwnbocaukresourcesnbocadatagovweblink cancer research uk incisive health saving lives averting costs an analysis ofthe financial implications of achieving earlier diagnosis of colorectal lung andovarian cancer wwwcancerresearchuksitesdefaultfilessaving_lives_averting_costspdf accessed may s sun f klebaner t tian a new model of time scheme for progression ofcolorectal cancer bmc syst biol suppl s2 j emery p vedsted new nice guidance on diagnosing cancer in generalpractice br j gen pract “ hb keshava je rosen mr deluzio aw kim fc detterbeck dj boffawhat if i do nothing the natural history of operable cancer of the alimentarytract eur j surg oncol “ yh lee pt kung yh wang wy kuo sl kao wc tsai effect of length oftime from diagnosis to treatment on colorectal cancer survival a populationbased study plos one e0210465 d roder cs karapetis i olver d keefe r padbury j moore r joshi dwattchow dl worthley cl miller c holden e buckley k powell dburanyitrevarton k fusco t price time from diagnosis to treatment ofcolorectal cancer in a south australian clinical registry cohort how it variesand relates to survival bmj open e031421 cancer research uk bowel cancersurvivalstatistics wwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancersurvival accessed june kk turaga s girotra are we harming cancer patients by delaying theircancer surgery during the covid19 pandemic ann surg 0c'

cancer7530

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: covid19 has had an impact on the provision of colorectal cancer care the aim of the crccovid study is to describe the changes in colorectal cancer services in the uk and usa in response to thepandemic and to understand the longterm impactmethods and analysis this study comprises phases phase is a survey of colorectal units that aims toevaluate adherences and deviations from the best practice guidelines during the covid19 pandemicphase is a monthly prospective data collection of service provision that aims to determine the impactof the service modifications on the longterm cancer specific outcomes compared to the national standards phase aims to predict costs attributable to the modifications of the crc services and additionalresources required to treat patients whose treatment has been affected by the pandemic phase aims tocompare the impact of covid19 on the nhs and usa model of healthcare in terms of service provisionand cost and to propose a standardised model of delivering colorectal cancer services for future outbreaksethics and dissemination this study is a service evaluation and does not require hra approval or ethicalapproval in the uk local service evaluation registration is required for each participating centre in theusa ethical approval was granted by the research and development committee the results of thisstudy will be disseminated to stakeholders submitted for peer review publications conference presentations and circulated via social mediaregistration details nil the authors published by elsevier ltd on behalf of surgical associates ltd this is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 introductionthe covid19 pandemic has had a significant impact on theprovision of healthcare worldwide as of the 29th june covid19 has resulted in confirmed cases and ‡‘ corresponding author at department of surgery and cancer imperial collegelondon chelsea and westminster and the royal marsden campus unitedkingdomemail address ckontovounisiosimperialacuk c kontovounisiosdeaths in the uk at a local level hospitals have been forcedto make a number of workforce modifications and changes toserviceprovision to combat the crisis and maintain standards ofcare for our patients facetoface consultations have beendissolved or minimized in favour of telephone or virtual clinicsprovision of investigations including ct scans and endoscopieshave been significantly reduced and all benign surgical procedurespostponed furthermore the treatment algorithm for confirmed colorectal cancer cases has proved challenging101016jisjp20200700524683574 the authors published by elsevier ltd on behalf of surgical associates ltdthis is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 0ca courtney international of surgery protocols “in the uk over forty thousand patients are diagnosed with colorectal cancer each year deviation from nice colorectal cancercrc guidelines may lead to significantly poorer outcomes however the current model of cancer services delivery cannot be maintained because of both resource limitation and the potential risksto patients and staff during the pandemic there is a lack of highdependency beds which are being utilized for covid19 patientsthere is the risk of exposing colorectal cancer patients the majority of whom are elderly and have significant comorbidities to thevirus during their treatment within the hospital patients requiringneoadjuvant or adjuvant therapy are at particular risk finallystaff safety must also be considered particularly around aerosolgenerating procedures such as endoscopy and laparoscopic surgery intercollegiate general surgery guidance on covid19 outlinedgeneral principles on the provision of a safe surgical service duringthe pandemic however there has been no specific guidance todate on how to best modify colorectal cancer crc service provision during the pandemic in the absence of a national consensusthe onus is on individual hospital trusts and multidisciplinaryteams to make very challenging decisions about individual patientcare lack of a unified approach may have important consequencesat patient and healthcare institution levelsdelay in cancer diagnosis or treatment due to service modification is likely to create an increased demand in resources once thecrisis has passed predicting the economic impact and planningfor this is essentialhow hospitals approach the new constraints on crc care andallocate resources may vary between the uk and usa it is hopedthat gaining insights from both perspectives will improve the problem solving methods and analysis aims and objectivesthe aim of the crc covid study is to describe the changes incolorectal cancer services in the uk and usa in response to thecovid19 pandemic and to understand the longterm impactour primary and secondary objectives relevant to each phase ofthe study are listed in table study designthis is a multicentre service evaluation conducted through aresearch collaborative with the support of the crc covid steeringcommittee all colorectal units continuing to provide cancer services in the uk ireland and the usa have been invited to participate all study and recruitment information is available on thetable primary and secondary study objectiveswebsite crccovid this service evaluation has been endorsedby the royal college of surgeons of england rcsthis service evaluation will be carried out in phases fig phase uses a questionnaire to assess the modificationsadopted by each colorectal unit in order to continue provision ofthe colorectal cancer services during the covid19 pandemic ithas been developed using an iterative process after research ofall relevant guidelines to construct the standard against which services would be evaluatedthe following guidelines relevant to the management of colorectal cancer have been used as standards for this serviceevaluation nice guidelines colorectal cancer [ng151] nice guideline suspected cancer recognition and referral[ng12] association of coloproctology of great britain irelandacpgbi guidelines for the management of cancer of thecolon rectum and anus [“] british society of gastroenterologyassociation of coloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillanceguidelines informal consultations with consultants nurse specialists andpatients have been used to develop the tool and then it has beenmodified after clinician review for face validity flow and relevance the final instrument comprises questionsphase investigates the provision of colorectal cancer servicesduring the covid19 pandemic by evaluating the performance ofeach unit against the national bowel cancer audit outcomes all centres participating in phase will be required to registerthis service evaluation as per local protocol prior to commencement of data collection on redcap this will be the responsibilityof the local leadphase of the study will develop a prediction model of the economic burden of the modifications in cancer service delivery thismodel will be designed jointly by two international businessschools based on previous publications and national statisticsphase will evaluate and compare the impact of the covid19pandemic on the nhs and the usa healthcare using data collectedduring phase and the predictive mode utilized in phase specificdifferences in modifications of crc services will be examined recruitmentphase survey has been distributed to all colorectal consultantsin the uk and ireland through personalised emails social mediaand the rcs the recruitment of colorectal cancer units in thephasephase phase phase phase primary objectiveevaluate adherences and deviations from best practiceguidelines on colorectal cancer during covid19pandemicative crc service deliverysecondary objectives 0f describe modifications to screening process for crc 0f describe modifications to preoperative intraoperative and postoper 0f demonstrate global effect of covid19 pandemic on crc service provi 0f outline consensus recommendations for sustainable modifications to 0f predict the impact of modifications on the incidence and prevalence of 0f plan adjustments to crc service provision after the end of pandemicsion irrespective of the type of healthcare systemdetermine the impact of crc service provision followingmodifications on longterm cancer specific outcomescompared to national standards 0f predict the costs attributable to modifications of crc services during covid19 pandemic 0f predict additional resources required to treat patients whose treatment has been affected by covid19 0f compare the impact of covid19 on the nhs and usa model of healthcare in terms of service provision and cost 0f propose a standardised model of delivering colorectal cancer services for future outbreaksdifferent crc stages in monthscrc services 0ca courtney international of surgery protocols “fig phases of crc covidusa will use similar approach all units recruited into phase arerecruited into phase participation in phase is not mandatory inorder to participate in phase data collectionall surveys and data collection follow the gdpr requirementsand comply with caldicott principles individual patient identifiable data is not collected in this study study data is collectedand managed using redcap electronic data capture tool hostedat the kennedy institute of rheumatology at the university ofoxford redcap research electronic data capture is asecure webbased software platform designed to support datacapture for research studies providing an intuitive interfacefor validated data capture audit trails for tracking data manipulation and export procedures automated export procedures forseamless data downloads to common statistical packages and procedures for data integration and interoperability with externalsources after the close of the phase data collection period all data setswill be checked for missing data where possible centres will begiven an opportunity to rectify missing data centres where of data is missing will be excluded from data analysis and localleads will be notified a nominated data validator will need toensure data accuracy prior to submission if during this processmajor discrepancy is identified within the data set the centre™sdata will be excluded completely from the analysisfor details of the data collected in phase and phase pleaserefer to supplement data analysisphase responses to the survey pertaining to deviations fromdiagnostic and treatment protocols during the covid19 pandemicsee supplement will be converted to a numerical scale where will denote no deviation and will denote complete cessation ofservice provision the scores will be summarized using appropriatesummary statistics and analyzed using unsupervised learningkmeans and hierarchical clustering to identify clusters of homogeneous response to the pandemicphase every month participating centres will report theirdiagnostic and treatment activity see supplement to determine the impact of covid19 on colorectal cancer activity we willuse timeseries methods and data on historical activity and patientoutcomes to estimate a baseline of expected monthly activity that would have taken place in the absence of the pandemicthe baseline and a confidence interval will be estimated atthe national regional and individual nhs trust level the baselineestimate will then be compared to the actual activity as reportedby publicly available data and data collected by this studythe difference between expected and actual activity will providean estimate of the reduction in activityto quantify the impact on patient outcomes associated with theestimated reduction in activity and deviation from standardizedcare protocols we will use estimates of disease progression available in peerreviewed literature [“] a similar methodologywill be used to predict the impact of the pandemic on the incidenceand prevalence of different colorectal cancer stages in the following months under different scenarios predictions will be madeat the regional and national level and depending on data granularity at the trust levelphase will estimate the financial costs of modifications to thecrc service provision due to the covid19 pandemic this willallow prediction of the expenditure and the additional resourcesrequired to resume routine services we will base this on the literature regarding the price of treatment at different disease stages and the information about the cost of resource utilizationconsultations diagnostic tests operating theatre time and hospital stay phase will compare the results from phases “ in theuk with those in the us discussioncancer care and maintaining high standards of diagnosis andtreatment has long been a priority of the nhs and internationalhealth care systems the pandemic has shifted this focus awayfrom the cancer services colorectal cancer patients are particularly 0ca courtney international of surgery protocols “vulnerable to the disruption of their care as diagnosis throughendoscopy was stopped due to concerns about virus aerosolysation this study is important because it is the first study to ask howindividual units had to modify their services and adapt to the newconstraintsin addition to describing the changes and understandingwhether different units had different approaches we wish to gofurther by understanding the effects of diagnostic and treatmentdelay by prospective data collection of cancer cases referrals diagnosis staging and treatment and comparing them to nationally collected audit data these data will allow us to model the economic impact of thedelay and what resources are required to restore cancer servicesto precovid19 standardsthe strengths of this study are in the multimodal approach tothe issues international collaboration and support from the royalcollege of surgeons our diverse team of management and business academics colorectal surgeons nurse specialists and patientadvisors enable us to have a range of approaches to collect andanalyse the datathe main limitation to the study is nonresponder or samplingbias as we require voluntary participation from colorectal teamswe will ensure that we adjust statistical analysis for any underrepresentation we expect that even with minimal participationuseful models can be generated to understand future resourcerequirements at an individual hospital level the methodologyemployed by other units will demonstrate the utility of the modelin summary this is a novel and important multiphase studythat is vital to understand how to best care for cancer patientsand ensure that the effects of the pandemic are mitigated ethics and disseminationthis study is a service evaluation and does not require hraapproval or ethical approval in the uk departmental approvalhas been granted by the university each participating centre mustseek local permission from their local audit department prior tocommencement of data collection in the usa ethical approvalwas granted by the research and development committeedata for phase will be submitted for publication as soon as theresults become available interim data analysis will be presented tothe royal college of surgeons covid19 research collaborativedata for other phases will be submitted for publication once thedata collection has been completed which is anticipated to be afterthe routine service provision resumes all data will be presented atnational and international conferences circumstances permitting guarantornone research registration numbernoneethical approvalnoneauthor contributionsac designed the study wrote the initial proposal drafted themanuscript based on the study proposal and is part of the auditadvisory groupamh ns nd ow sm sr gm nt mg td bs jee ad ptadvised on the study design and the protocol and is part of thesteering committeeck is a project piall authors read commented on and approved the study designthe protocol and the final manuscriptfundingthis research received no specific grant from any fundingagency in the public commercial or notforprofit sectorsdeclaration of competing interestthe authors declare that they have no known competing financial interests or personal relationships that could have appearedto influence the work reported in this paperreferences world health anization the united kingdom who coronovirus diseasecovid19 dashboard covid19whointregioneurocountrygbaccessed june covidsurg collaborative global guidance for surgical care during the covid pandemic br j surg a spinelli g pellino covid19 pandemic perspectives on an unfolding crisisbr j surg “ british society of gastroenterology endoscopy activity and covid19 bsgand jag guidance apr wwwbsgukcovid19adviceendoscopyactivityandcovid19bsgandjagguidance accessed may nhs england nhs improvement letter to chief executives of all nhs trustsand foundation trusts ccg accountable officers gp practices and primary carenetworks and providers of community health services mar wwwenglandnhsukcoronaviruswpcontentuploadssites52202003urgentnextstepsonnhsresponsetocovid19lettersimonstevenspdfaccessed may research uk cancerincidencecancerbowelstatisticswwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancerincidenceheadingzeroaccessed may royal college of surgeons of england updated intercollegiate general surgeryguidance on covid19 apr wwwrcsengacukcoronavirusjointguidanceforsurgeonsv2 accessed may national institute for health and care excellence nice colorectal cancernice guideline ng151 wwwniceukguidanceng151accessed accessed march national institute for health and care excellence nice suspected cancerrecognition and referral nice guideline ng12 wwwniceukguidanceng12 accessed accessed march c cunningham k leong s clark a plumb s taylor i geh s karandikar bmoran association of coloproctology of great britain ireland acpgbiguidelines for the management of cancer of the colon rectum and anus diagnosis investigations and screening colorectal dis suppl “ s gollins b moran r adams c cunningham s bach as myint a renehans karandikar v goh d prezzi g langman s ahmedzai i geh association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement ofmultidisciplinary management colorectal dis suppl “rectum and anusthe coloncancer of glangman m loughrey n shepherd p quirke association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement of cancer of the colon rectum and anus pathologystandards and datasets colorectal dis suppl “ k leong j hartley s karandikar association of coloproctology of greatbritain ireland acpgbi guidelines for the management of cancer of thecolon rectum and anus follow uplifestyle and survivorshipcolorectal dis suppl “ b moran c cunningham t singh p sagar j bradbury i geh s karandikarassociation of coloproctology of great britain ireland acpgbi guidelinesfor the management of cancer of the colon rectum and anus surgicalmanagement colorectal dis suppl “ md rutter j east cj rees n cripps j docherty s dolwani pv kaye kjmonahan mr novelli a plumb bp saunders s thomasgibson djmtolan s whyte s bonnington a scope r wong b hibbert j marsh bmoores a cross l sharp british society of gastroenterologyassociation ofcoloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillance guidelines gut “ 0ca courtney international of surgery protocols “ healthcare quality improvement partnership hqip national bowel canceraudit annual report an audit of the care received by people with bowelcancer in england and wales v20 wwwnbocaukcontentuploads202001nboca2019v20pdf accessed june pa harris r taylor r thielke j payne n gonzalez jg conde researchelectronic data capture redcap“a metadatadriven methodology andworkflow process for providing translational research informatics support jbiomed inform “ pa harris r taylor bl minor v elliott m fernandez l o™neal l mcleodg delacqua f delacqua j kirby sn duda re consortium the redcapconsortium building an international community of software platformpartners j biomed inform nhs england and nhs improvement cancer waiting times wwwenglandnhsukstatisticsstatisticalworkareascancerwaitingtimesaccessed june national bowel cancer audit nboca datagov weblink accessed junewwwnbocaukresourcesnbocadatagovweblink cancer research uk incisive health saving lives averting costs an analysis ofthe financial implications of achieving earlier diagnosis of colorectal lung andovarian cancer wwwcancerresearchuksitesdefaultfilessaving_lives_averting_costspdf accessed may s sun f klebaner t tian a new model of time scheme for progression ofcolorectal cancer bmc syst biol suppl s2 j emery p vedsted new nice guidance on diagnosing cancer in generalpractice br j gen pract “ hb keshava je rosen mr deluzio aw kim fc detterbeck dj boffawhat if i do nothing the natural history of operable cancer of the alimentarytract eur j surg oncol “ yh lee pt kung yh wang wy kuo sl kao wc tsai effect of length oftime from diagnosis to treatment on colorectal cancer survival a populationbased study plos one e0210465 d roder cs karapetis i olver d keefe r padbury j moore r joshi dwattchow dl worthley cl miller c holden e buckley k powell dburanyitrevarton k fusco t price time from diagnosis to treatment ofcolorectal cancer in a south australian clinical registry cohort how it variesand relates to survival bmj open e031421 cancer research uk bowel cancersurvivalstatistics wwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancersurvival accessed june kk turaga s girotra are we harming cancer patients by delaying theircancer surgery during the covid19 pandemic ann surg 0c' Answer:

Colon_Cancer

since the late a novel coronavirus officially named as severe acute respiratory syndrome coronavirus sarscov2 was identified as the pathogen to cause pneumonia as a member of the betacoronavirus genus sarscov2 has genomic nucleotides identity with human severe acute respiratory syndrome coronavirus sarscov and shares amino acid sequence identity with sarscov the world health anization who named the disease caused by sarscov2 as coronavirus disease covid19 until april the virus has swept through countries more than million cases with covid19 have been confirmed and more than cases died which has been posing significant threats to public health sarscov2 can cause respiratory diseases and may lead to acute respiratory distress syndrome ards multiple an failure and even death in severe cases in addition to typical symptoms such as cough and fever some patients developed the symptoms in multiple systems such as cardiovascular system digestive system and abbreviations ace2 angiotensin converting enzyme aki acute kidney injury ali acute liver injury alp alkaline phosphatase als artificial liver system alt alanine aminotransferase ami acute myocardial infarction ards acute respiratory distress syndrome ast aspartate aminotransferase at2 alveolar cells bun blood urea nitrogen ccle cancer cell line encyclopedia cns central nervous system covid19 coronavirus disease geo gene expression omnibus ggt gammaglutamyltransferase gi gastrointestinal injury gtex genotypetissue expression icu intensive care unit mcs mechanical circulatory support np nucleoprotein pci percutaneous coronary intervention sarscov2 severe acute respiratory syndrome coronavirus scr serum creatinine scrnaseq single cell rna sequencing stemi stelevation myocardial infarction tbil total bilirubin tem transmission electronic microscope tmprss2 transmembrane protease serine vv venousvenous who world health anization corresponding authors at department of infectious disease and institute of hepatology qingdao municipal hospital qingdao university digestive disease key laboratory of qingdao qingdao china email addresses xinyongning9812163com y xin zlk0823163com l zhuang 101016jbiopha2020110678 received june received in revised form august accepted august biomedicinepharmacotherapy1312020110678availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0cexpressed not only in the cells and tissues of lung but also in extrapulmonary ans [“] fig in this section the expression levels of ace2 and tmprss2 in extrapulmonary ans including heart kidney liver digestive tract brain and other ans were reviewed heart m dong nervous system in the early stages of covid19 which brings more challenges to the timely diagnosis of patients angiotensin converting enzyme ace2 as a metalloproteinase is a carboxyterminal dipeptidyl peptidase the primary physiological role of ace2 is involved in the regulation of vasoconstriction and blood pressure [“] transmembrane protease serine type2 tmprss2 belonging to the type ii transmembrane serine protease family could cleave the coronavirus spike s protein [“] it was demonstrated that ace2 and tmprss2 were crucial for the entry of sarscov and sarscov2 into the host cells cell entry of sarscov2 depends on binding of the s protein to the specific cellular receptor and s protein priming by host cell proteases as shown in fig each s protein of sarscov2 consists of two subunits a globular s1 domain at the nterminal region and the membraneproximal s2 domain sarscov2 utilizes receptorbinding domain within the s1 domain to bind to the cellular receptor ace2 which could trigger the effects of tmprss2 on the cleavage of protein s at the s1 and s2 sites and priming cell membrane fusion for viral entry as receptors and mediators of virus entry are important for determining viral host and an the route of sarscov2 infection and the infected an may depend on the expression and distribution of ace2 and tmprss2 studies have shown that ace2 and tmprss2 are expressed not only in lung tissues but also in extrapulmonary ans including heart kidney liver colon esophagus brain gallbladder and testis suggesting that sarscov2 may also affect extrapulmonary ans [“] in this review the distributions of ace2 and tmprss2 in extrapulmonary ans and the characteristics and clinical managements of extrapulmonary an injury caused by sarscov2 were summarized we believe that this will be important in understanding on the infection of extrapulmonary ans in patients with covid19 the mrna expressions of ace2 and tmprss2 in extrapulmonary ans the mrna expressions of ace2 in different human ans were analyzed and the results showed that ace2 was expressed in the heart furthermore chen analyzed the feature of ace2 expressions among cardiac cell types and found that ace2 was specifically expressed in pericyte moreover rna sequencing from patients with failing hearts and normal donors revealed that myocardial ace2 expressions were significantly increased in patients with heart failure which was further validated at the protein level by proteomics profiling from heart failure and normal donors another study also showed that the expression of ace2 in heart tissues of patients with underlying heart disease was higher than that in normal heart tissues these two studies suggested that the expression of ace2 in heart tissue of patients with underlying heart disease was higher than that in normal heart tissue guo et al analyzed the mrna expression of tmprss2 from the genotypetissue expression gtex database and the results showed that tmprss2 is also expressed in the heart by singlecell rna sequencing scrnaseq to profile the gene expression landscapes of cardiac cells from human embryos qi revealed that the cardiomyocytes from the heart contain ace2expressed cells and tmprss2expressed cells and the cardiovascular progenitor cells and cells tmprss2expressed cells respectively these data showed that both ace2 and tmprss2 were expressed in the heart contain ace2expressed kidney studying the viral susceptibility of extrapulmonary ans is important for a deeper understanding for the pathogenesis of sarscov infection studies have shown that ace2 and tmprss2 were expression analysis from the gtex database showed that kidney displayed the fifth high expression of ace2 to investigate the expression of ace2 in kidney lin analyzed the public singlecell transcriptome dataset of normal kidneys from healthy donors the fig entry of sarscov2 into host cells sarscov2 infected the host cells by the spike protein of the virus and the functions of ace2 and tmprss2 in host cells biomedicinepharmacotherapy13120201106782 0cm dong fig tissue distributions of ace2 and tmprss2 in human a“b the schematic diagram of the expressions of ace2 a and tmprss2 b in multiple human tissues the colour strength is corresponding to the gene expression level ace2 and tmprss2 were expressed in the brain and heart ace2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while tmprss2 is expressed in the hepatocytes and cholangiocytes ace2 and tmprss2 were highly expressed in kidney and intestinal epithelial cells both ace2 and tmprss2 were also expressed in the esophagus stomach nose testis pancreas breast prostate and thyroid results showed that the ace2 was distributed across multiple cell types and was mostly enriched in proximal tubule cells fan et al confirmed the specific ace2 expression in tubular cells from the gene expression omnibus geo dataset while it was not observed in immune cells and glomerular parietal epithelial cells rna and protein expression data of ace2 in different human tissues and cancer cell lines were obtained from three online datasets including the cancer cell line encyclopedia ccle gtex database and the human protein atlas dataset and the results indicated that both mrna and protein expression levels of ace2 were relatively high in kidney cells especially in renal tubular cells meanwhile suryawanshi analyzed the data of kidney tissues in scrnaseq datasets and found that either proximal tubular cells or tubular progenitor cells in the kidney coexpressed ace2 and tmprss2 the data of the scrnaseq from geo dataset gse134355 showed that ace2 and tmprss2 expression levels were high in nephron epithelial cells epithelial cells endothelial cells and mesangial cells of the kidney recently pan also found that the tmprss2 gene was coexpressed with ace2 in kidney podocytes these data showed that both ace2 and tmprss2 were highly expressed in tissues and cells of kidney liver chai et al analyzed the scrnaseq data from geo database gse124395 to evaluate ace2 gene expression in liver the results showed that ace2 was highly expressed in cholangiocytes which level was about times higher than that in hepatocytes the gtex database also showed that both ace2 and tmprss2 were expressed in the liver zhou identified that tmprss2 is highly expressed in hepatocytes from human cell atlas database recently wen indicated that ace2 and tmprss2 are specifically coexpression in trop2 liver progenitors of human liver tissue using scrna sequencing these data indicate that ace2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while tmprss2 is expressed in hepatocytes digestive tract a previous study showed that ace2 could be found in the upper esophagus and it could be detected in stratified epithelial cells and absorptive enterocytes of the ileum and colon quantitative mrna expression profiling of ace2 across human tissues by harmer showed that ace2 was expressed at a high level in gastrointestinal tissues zhang et al analyzed datasets with singlecell transcriptomes of esophagus gastric ileum colon and lung and the data showed that ace2 was not only highly expressed in the type ii alveolar cells at2 of lung but also in the stratified epithelial cells ileum absorptive enterocytes cells and colon enterocytes similarly the immunofluorescent staining of esophagus stomach duodenum and rectum showed that ace2 was stained mainly in the cytoplasm of gastrointestinal epithelial cells besides the scrnaseq data showed that ace2 was significantly elevated in the proximal and distal enterocytes guo et al suggested that tmprss2 was highly expressed in almost all ans of the digestive tract including colon stomach small intestine and esophagus using published scrnaseq data and seven inhouse normal colon samples lee reported that the coexpressions of ace2 and tmprss2 transcripts were mainly observed in the small intestine and colon the highest expressions of tmprss2 and ace2 were found in enterocytes among the intestinal cell types these data showed that tmprss2 and aec2 are highly expressed in the digestive tract nervous system analysis using the gtex database showed that both tmprss2 and ace2 are expressed at relatively low levels in the brain cortex chen found that ace2 was relatively highly expressed in some important brain areas such as the substantia nigra and brain ventricles using seven brain transcriptome databases ace2 was expressed at high level in the piriform cortex of human brain and its expression could also be detected in many neurons including both excitatory and inhibitory neurons and some nonneuron cells including astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex qi analyzed the scrnaseq data of substantia nigra biomedicinepharmacotherapy13120201106783 0cclinical classification of acute cardiac injury nonicucases icu cases nonicucases icucases nonsevere cases severecases1965 recoveredcases died cases nonicucases icucases survivor cases nonsurvivor cases chen hong zhou china korea china m dong and cortex of brain from geo database the results showed that both ace2 and tmprss2 were expressed in the oligodendrocyte precursor cells and the astrocytes of the substantia nigra and cortex there are limited reports on the expressions of ace2 and tmprss2 in peripheral nervous system brann analyzed the ace2 and tmprss2 expression in different cell type from human scrnaseq dataset gse139522 and found that neither olfactory sensory neurons nor olfactory bulb neurons expressed these two genes while ace2 and tmprss2 were expressed in the nonneuronal cells including the sustentacular cells and olfactory bulb pericytes these data showed that ace2 and tmprss2 could also be coexpressed in the nervous system other ans or tissues table characteristics of acute cardiac injury after sarscid0 cov2 infection study basic heart disease acute cardiac injury country subject china wang china na huang li china moreover ace2 and tmprss2 were also reported to be coexpressed in some other ans it has been revealed that both ace2 and tmprss2 are expressed in testis by scrna sequencing and expression profile analysis indicating that testicular cells might be the potential targets of sarscov2 another report revealed that multiple kinds of cells in the nose including nasal brushing epithelial cells nasal turbinate epithelial cells and nasal airway epithelial cells contained ace2expressed and tmprss2expressed cell clusters moreover ace2 and tmprss2 were also expressed in pancreas breast prostate and thyroid and these ans might also be the targets of sarscov2 infection of sarscov2 and extrapulmonary an injury of patients with covid19 sarscov2 infection and cardiac injury recently autopsy analysis by fox revealed that the histopathology of the heart was consistent with the typical pattern of viral myocarditis sarscov2 rna was detected in the cardiac tissues of the patients with covid19 these data suggested that sarscov2 may directly infect heart the epidemiology of covid19 reported that cardiac injury was one of the most severe an damages the clinical manifestations of cardiac injury in covid19 patients are complex and could present with heart failure arrhythmias or acute myocardial infarction ami [ ] inciardi reported the first case who had the symptom of heart failure at first and later the patient was positive for sarscov2 using nucleic acid test cardiac injury is a common symptom in patients with covid19 shi reported that patients with covid19 had cardiac injury moreover there were patients with acute cardiac injury in a cohort including covid19 patients and of patients with acute cardiac injury in the intensive care unit icu furthermore a study by wang showed that there were patients with acute cardiac injury and patients presented with arrhythmia of covid19 patients while acute cardiac injury was observed in of patients with civid19 in the icu these cases suggested that sarscov2 may cause serious heart damage which should be widespreadly concerned furthermore acute cardiac injury is more prevalent in severe cases with covid19 [“] table and it has been reported that covid19 patients with cardiac injury had higher mortality than those without cardiac injury in this review we also summarized the possible relationship between basic heart disease and further cardiac injury [“] table in a cohort of covid19 patients from renmin hospital of wuhan university china shi demonstrated that cardiac injury occurred in patients during hospitalization of which had basic heart disease including coronary heart disease and chronic heart failure and only patients with basic heart disease of covid19 patients without cardiac injury similarly liu suggested that patients with basic heart disease in covid19 patients had table comorbidity with cardiac injury in covid19 patients with basic heart disease subjects with covid19 proportion of basic heart disease patients with cardiacinjury study shi liu xu ma guo patients with basic heart disease with cardiac injury without cardiac injury cardiac injury compared with patients with basic cardiovascular diseases of covid19 patients without cardiac injury other studies also indicated that the patients with basic cardiovascular disease are more likely to present heat injury in covid19 patients [ ] in view of the points above covid19 patients with underlying cardiac conditions seem to have higher rates of cardiac injury sarscov2 infection and kidney injury recently autopsy analysis on six covid19 patients showed that varying degrees of acute tubular necrosis were observed in all the renal specimens nucleoprotein np antigens and np positive inclusion body of sarscov2 could be seen in kidney tissues from all the samples moreover viruslike ps were seen in kidney tissues by transmission electronic microscope tem su analyzed kidney abnormalities in autopsies of patients with covid19 and found that diffuse proximal tubular damage with the loss of brush border were biomedicinepharmacotherapy13120201106784 0c sarscov2 infection and liver injury m dong observed further investigation showed that diffuse necrosis can be seen under the light microscope and electron microscopic examination also showed the clusters of coronavirus ps with distinctive spikes in the tubular epithelium and podocytes it was reported that both np antigens and rna of sarscov2 were detected in urine of covid19 patients these data coincide with the finding of the sarscov2 invasion in kidney collectively sarscov2 could directly infect human renal tubules and lead to kidney damage recent studies have shown that the incidence of acute kidney injury aki in covid19 patients ranged from “ and higher frequency of renal function damage with elevated blood urea nitrogen bun or serum creatinine scr was observed in covid19 patients [ ““] table a study of patients with covid19 indicated that levels of bun and scr were increased in and patients with covid19 respectively and routine urine tests were performed on patients among which patients were positive for urinary protein and patients were positive for hematuria another study also showed that about patients with covid19 had abnormal renal function moreover covid19 patients with more severe disease progression have higher rates of aki huang and colleagues reported that of patients with aki in the icu were observed and none of the patients who did not require care in the icu suffered aki xu found that the fatality rate was obviously higher in covid19 patients with aki than those without renal injury furthermore in another study investigating patients with covid19 at hospital admission more severe patients had higher rates of aki and the cox regression analysis also suggested that covid19 patients who developed aki had a significantly higher mortality risk therefore aki is more prevalent in severe cases with covid19 an autopsy report of a 50yearold patient with covid19 showed moderate microvesicular steatosis and mild lobular activity in liver tissues moreover zhao used human liver ductal anoids as a tool to investigate the sarscov2 infection and the tissue damage induced by sarscov2 ex vivo and the results showed that the expression of sarscov2 np was easily detected in the patchy areas of the hepatic duct indicating that liver ductal anoids were susceptible to sarscov2 infection in addition sarscov2 infection could disrupt the barrier and bile acid transporting functions of cholangiocytes which indicated that sarscov2 might directly induce cholangiocyte injury and consequently bile acid accumulation in view of the points above liver damage in the covid19 patients might be directly caused by the viral infection abnormal liver functions were frequently reported in covid19 patients epidemiologic studies showed that almost half of the patients had differing degrees of liver damage [“ “] table chen reported that out of patients had elevated alanine aminotransferase alt patients had elevated aspartate aminotransferase ast and had elevated total bilirubin tbil in wuhan jinyintan hospital wuhan china similarly a nationwide study involving patients with covid19 in china showed that more than of patients had elevated alt and ast and of patients had elevated tbil it was revealed that the levels of direct bilirubin indirect bilirubin alt alkaline phosphatase alp and gammaglutamyltransferase ggt were significantly higher in males than that in females with covid19 and multivariate logistic regression analysis showed that male was an important independent risk factor for predicting acute liver injury ali in covid19 patients these data indicated that male patients with covid19 may be more susceptible to liver injury furthermore table characteristics of acute kidney injury after sarscid0 cov2 infection study chen wang huang guan xu preexisting kidney conditions na na na country china china china china china subject li chen hong cheng xiao richardson wan li qian pei china china korea china china america china china china china na na na na na na scr serum creatinine bun blood urea nitrogen aki acute kidney injury abnormal renal functional indices scr bun na scr scr scr scr bun na scr bun scr na na na scr bun scr na aki na clinical classification of aki na nonicu cases icu cases icu cases nonsevere cases severe cases mild cases severe cases critical ill cases nonsevere cases severe cases recovered cases died cases nonicu cases icu cases na nonsevere cases severe cases cured cases in hospital cases died cases mild cases severe cases nonsevere cases severe cases na moderate cases severe cases critically ill cases biomedicinepharmacotherapy13120201106785 0cm dong table characteristics of liver injury after sarscid0 cov2 infection study country subject china china china china china china china korea america china china chen wang huang guan xu li chen hong richardson et wan li al qian china na patients with preexisting liverconditions na na na patients with abnormal liver functional indices alt ast tbil na ast ast alt tbil alt ast alt ast tbil alt ast alt ast tbil ast alt ast alt ast tbil alt ast abnormal liver functional indices in the nonsevere patients ast alt na na tbil na abnormal liver functional indices in the severe patients alt na tbil na ast na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na nonsevere patients include patients without icu care and recovered patients severe patients include patients with icu care and death alt alanine aminotransferase ast aspartate aminotransferase tbil total bilirubin multiple studies found that ast alt and tbil were significantly higher in patients treated in the icu than that in nonicu patients li suggested that among the patients with abnormal liver function moderate and severe types of patients were more likely to have liver injury and respectively fu analyzed the relationship between ali and mortality risk in covid19 patients and the results showed that ali is more common in the critically ill patients and ali at the early stage increased death risk of covid19 patients together abnormal liver functions might be associated with the severity of patients with covid19 sarscov2 infection and digestive tract injury epithelial cells of the esophagus stomach duodenum and rectum in one covid19 patient tested positive for sarscov2 rna and the staining of viral np was also visualized in the cytoplasm of epithelial cells in stomach duodenum and rectum moreover minimally invasive autopsies were performed on three patients died of covid19 and the results showed that some epithelial cells of the gastrointestinal mucosa were degenerated necrotic and detached these studies strongly supported that sarscov2 may directly infect the epithelial cells of digestive tract table sarscid0 cov2 detection in gastrointestinal specimens study subject xiao zhang tan xing young holshue lescure tang wang xu gastrointestinal samples stool anal swabs rectal swab stool stool stool stool stool stool rectal swabs tested positive in gastrointestinal specimens the positive time in gastrointestinal specimens days na 6cid0 1cid0 5cid0 na 3cid0 positive time for gastrointestinal samples after respiratory samples were negative days na na 8cid0 na na na na na 2cid0 biomedicinepharmacotherapy13120201106786 0cm dong multiple studies have identified that the sarscov2 rna was detected in anal swabs rectal swabs and stool specimens [ ] of covid19 patients it has been demonstrated that sarscov2 rna could be detected in feces from more than half of covid19 patients in another study xing reported that sarscov2 rna was detected in the feces of three pediatric cases with covid19 in qingdao china and the persistence of sarscov2 in the digestive tract lasted for 6cid0 days the possibility of fecaloral transmission of sarscov2 infection needs to be taken into account furthermore as shown in table long duration of sarscov2 detection in digestive tract by rtpcr has been reported and viral rna remained detectable in the digestive tract for 2cid0 days after nucleic acid turned negative in respiratory samples [“] the studies suggested that sarscov2 could be detected from respiratory tract specimens during the early period to digestive tract specimens during the late period and viral nucleic acid tests in both the respiratory and digestive tract are necessary to confirm the complete clearance of virus some covid19 patients presented gastrointestinal symptoms such as diarrhea nausea vomiting and abdominal pain holshue reported the first case of covid19 patient in the usa which had nausea and vomiting before admission multiple studies found that gastrointestinal symptoms including diarrhea “ nausea and vomiting “ and abdominal pain “ were common at presentation in covid19 patients [ ““] table in a cohort of patients with covid19 in wuhan china gastrointestinal symptoms were described in up to moreover sun showed that critically ill patients with covid19 had gastrointestinal injury gi during hospital stay and the survival curves showed that the mortalities of patients with gi was greater than that of patients without gi jin also found that the rate of the severe type was markedly higher in covid19 patients with gi symptoms than that in those without gi symptoms these data suggested that gi is one of the common extrapulmonary an injuries in covid19 patients and may be related to the severity of the disease on the other hand many studies showed that patients with covid19 could present initially with the typical gastrointestinal symptoms and diarrhea may even occur earlier than pyrexia or respiratory symptom in some cases with covid19 [“] luo reported that of covid19 cases presented initially only with gastrointestinal symptoms the covid19 patients initially only with gastrointestinal symptoms are more difficult to diagnose and might be overlooked which could lead to potentially serious consequences together digestive tract symptoms especially diarrhea are the main complications of covd19 patients which should be noticed during the outbreak of covid19 sarscov2 infection and nervous system injury transmission electron microscopy of autopsy sections showed the presence of sarscov2 virallike ps in frontal lobe brain and neural cell bodies moreover researchers confirmed the presence of sarscov2 in cerebrospinal fluid by genome sequencing the pathological mechanism may be the invasion of sarscov2 into the nervons system the virallike ps in brain capillary endothelium was also observed which suggested that hematogenous route might act as the pathway for sarscov2 to the brain in addition study using the mouse model have shown that sarscov can lead to neuroinvasion via disruption of the nasal epithelium and subsequent neuronal dissemination which suggested that coronavirus may use the olfactory nerve to enter the brain the symptoms from nervous system of covid19 patients including headache dizziness anosmia and dysgeusia have been observed in the clinic [““] table disturbance of consciousness and seizures can occur as complications in the cases with severe covid19 it was convincing enough that the neurological deficits of patients with covid19 could be ongoing if it did not get noticed it was indicated that sarscov2 can cause nervous system damage the neurological deficits meningoencephalitis and acute myelitis in covid19 patients have been reported in the usa switzerland and china [“] according to a recent study out of covid19 patients had central nervous system cns symptoms including dizziness headache impaired consciousness ataxia and epilepsy up to of patients with covid19 have headache and clinical manifestations of dizziness were found from “ of the patients with covid19 [ ] olfactory and gustatory disorders are prevalent peripheral nervous system pns symptoms in covid19 patients in patients with mild and moderate covid19 a high proportion of patients presented olfactory and gustatory dysfunctions and olfactory dysfunction appeared prior to the other symptoms in some cases [“ ] these studies showed that the damage of neurological system may also act as a significant feature of covid19 table gastrointestinal symptoms after sarscid0 cov2 infection study chen wang liu xiao huang guan li chen zhou wan li [

cancer7531

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: since the late a novel coronavirus officially named as severe acute respiratory syndrome coronavirus sarscov2 was identified as the pathogen to cause pneumonia as a member of the betacoronavirus genus sarscov2 has genomic nucleotides identity with human severe acute respiratory syndrome coronavirus sarscov and shares amino acid sequence identity with sarscov the world health anization who named the disease caused by sarscov2 as coronavirus disease covid19 until april the virus has swept through countries more than million cases with covid19 have been confirmed and more than cases died which has been posing significant threats to public health sarscov2 can cause respiratory diseases and may lead to acute respiratory distress syndrome ards multiple an failure and even death in severe cases in addition to typical symptoms such as cough and fever some patients developed the symptoms in multiple systems such as cardiovascular system digestive system and abbreviations ace2 angiotensin converting enzyme aki acute kidney injury ali acute liver injury alp alkaline phosphatase als artificial liver system alt alanine aminotransferase ami acute myocardial infarction ards acute respiratory distress syndrome ast aspartate aminotransferase at2 alveolar cells bun blood urea nitrogen ccle cancer cell line encyclopedia cns central nervous system covid19 coronavirus disease geo gene expression omnibus ggt gammaglutamyltransferase gi gastrointestinal injury gtex genotypetissue expression icu intensive care unit mcs mechanical circulatory support np nucleoprotein pci percutaneous coronary intervention sarscov2 severe acute respiratory syndrome coronavirus scr serum creatinine scrnaseq single cell rna sequencing stemi stelevation myocardial infarction tbil total bilirubin tem transmission electronic microscope tmprss2 transmembrane protease serine vv venousvenous who world health anization corresponding authors at department of infectious disease and institute of hepatology qingdao municipal hospital qingdao university digestive disease key laboratory of qingdao qingdao china email addresses xinyongning9812163com y xin zlk0823163com l zhuang 101016jbiopha2020110678 received june received in revised form august accepted august biomedicinepharmacotherapy1312020110678availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0cexpressed not only in the cells and tissues of lung but also in extrapulmonary ans [“] fig in this section the expression levels of ace2 and tmprss2 in extrapulmonary ans including heart kidney liver digestive tract brain and other ans were reviewed heart m dong nervous system in the early stages of covid19 which brings more challenges to the timely diagnosis of patients angiotensin converting enzyme ace2 as a metalloproteinase is a carboxyterminal dipeptidyl peptidase the primary physiological role of ace2 is involved in the regulation of vasoconstriction and blood pressure [“] transmembrane protease serine type2 tmprss2 belonging to the type ii transmembrane serine protease family could cleave the coronavirus spike s protein [“] it was demonstrated that ace2 and tmprss2 were crucial for the entry of sarscov and sarscov2 into the host cells cell entry of sarscov2 depends on binding of the s protein to the specific cellular receptor and s protein priming by host cell proteases as shown in fig each s protein of sarscov2 consists of two subunits a globular s1 domain at the nterminal region and the membraneproximal s2 domain sarscov2 utilizes receptorbinding domain within the s1 domain to bind to the cellular receptor ace2 which could trigger the effects of tmprss2 on the cleavage of protein s at the s1 and s2 sites and priming cell membrane fusion for viral entry as receptors and mediators of virus entry are important for determining viral host and an the route of sarscov2 infection and the infected an may depend on the expression and distribution of ace2 and tmprss2 studies have shown that ace2 and tmprss2 are expressed not only in lung tissues but also in extrapulmonary ans including heart kidney liver colon esophagus brain gallbladder and testis suggesting that sarscov2 may also affect extrapulmonary ans [“] in this review the distributions of ace2 and tmprss2 in extrapulmonary ans and the characteristics and clinical managements of extrapulmonary an injury caused by sarscov2 were summarized we believe that this will be important in understanding on the infection of extrapulmonary ans in patients with covid19 the mrna expressions of ace2 and tmprss2 in extrapulmonary ans the mrna expressions of ace2 in different human ans were analyzed and the results showed that ace2 was expressed in the heart furthermore chen analyzed the feature of ace2 expressions among cardiac cell types and found that ace2 was specifically expressed in pericyte moreover rna sequencing from patients with failing hearts and normal donors revealed that myocardial ace2 expressions were significantly increased in patients with heart failure which was further validated at the protein level by proteomics profiling from heart failure and normal donors another study also showed that the expression of ace2 in heart tissues of patients with underlying heart disease was higher than that in normal heart tissues these two studies suggested that the expression of ace2 in heart tissue of patients with underlying heart disease was higher than that in normal heart tissue guo et al analyzed the mrna expression of tmprss2 from the genotypetissue expression gtex database and the results showed that tmprss2 is also expressed in the heart by singlecell rna sequencing scrnaseq to profile the gene expression landscapes of cardiac cells from human embryos qi revealed that the cardiomyocytes from the heart contain ace2expressed cells and tmprss2expressed cells and the cardiovascular progenitor cells and cells tmprss2expressed cells respectively these data showed that both ace2 and tmprss2 were expressed in the heart contain ace2expressed kidney studying the viral susceptibility of extrapulmonary ans is important for a deeper understanding for the pathogenesis of sarscov infection studies have shown that ace2 and tmprss2 were expression analysis from the gtex database showed that kidney displayed the fifth high expression of ace2 to investigate the expression of ace2 in kidney lin analyzed the public singlecell transcriptome dataset of normal kidneys from healthy donors the fig entry of sarscov2 into host cells sarscov2 infected the host cells by the spike protein of the virus and the functions of ace2 and tmprss2 in host cells biomedicinepharmacotherapy13120201106782 0cm dong fig tissue distributions of ace2 and tmprss2 in human a“b the schematic diagram of the expressions of ace2 a and tmprss2 b in multiple human tissues the colour strength is corresponding to the gene expression level ace2 and tmprss2 were expressed in the brain and heart ace2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while tmprss2 is expressed in the hepatocytes and cholangiocytes ace2 and tmprss2 were highly expressed in kidney and intestinal epithelial cells both ace2 and tmprss2 were also expressed in the esophagus stomach nose testis pancreas breast prostate and thyroid results showed that the ace2 was distributed across multiple cell types and was mostly enriched in proximal tubule cells fan et al confirmed the specific ace2 expression in tubular cells from the gene expression omnibus geo dataset while it was not observed in immune cells and glomerular parietal epithelial cells rna and protein expression data of ace2 in different human tissues and cancer cell lines were obtained from three online datasets including the cancer cell line encyclopedia ccle gtex database and the human protein atlas dataset and the results indicated that both mrna and protein expression levels of ace2 were relatively high in kidney cells especially in renal tubular cells meanwhile suryawanshi analyzed the data of kidney tissues in scrnaseq datasets and found that either proximal tubular cells or tubular progenitor cells in the kidney coexpressed ace2 and tmprss2 the data of the scrnaseq from geo dataset gse134355 showed that ace2 and tmprss2 expression levels were high in nephron epithelial cells epithelial cells endothelial cells and mesangial cells of the kidney recently pan also found that the tmprss2 gene was coexpressed with ace2 in kidney podocytes these data showed that both ace2 and tmprss2 were highly expressed in tissues and cells of kidney liver chai et al analyzed the scrnaseq data from geo database gse124395 to evaluate ace2 gene expression in liver the results showed that ace2 was highly expressed in cholangiocytes which level was about times higher than that in hepatocytes the gtex database also showed that both ace2 and tmprss2 were expressed in the liver zhou identified that tmprss2 is highly expressed in hepatocytes from human cell atlas database recently wen indicated that ace2 and tmprss2 are specifically coexpression in trop2 liver progenitors of human liver tissue using scrna sequencing these data indicate that ace2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while tmprss2 is expressed in hepatocytes digestive tract a previous study showed that ace2 could be found in the upper esophagus and it could be detected in stratified epithelial cells and absorptive enterocytes of the ileum and colon quantitative mrna expression profiling of ace2 across human tissues by harmer showed that ace2 was expressed at a high level in gastrointestinal tissues zhang et al analyzed datasets with singlecell transcriptomes of esophagus gastric ileum colon and lung and the data showed that ace2 was not only highly expressed in the type ii alveolar cells at2 of lung but also in the stratified epithelial cells ileum absorptive enterocytes cells and colon enterocytes similarly the immunofluorescent staining of esophagus stomach duodenum and rectum showed that ace2 was stained mainly in the cytoplasm of gastrointestinal epithelial cells besides the scrnaseq data showed that ace2 was significantly elevated in the proximal and distal enterocytes guo et al suggested that tmprss2 was highly expressed in almost all ans of the digestive tract including colon stomach small intestine and esophagus using published scrnaseq data and seven inhouse normal colon samples lee reported that the coexpressions of ace2 and tmprss2 transcripts were mainly observed in the small intestine and colon the highest expressions of tmprss2 and ace2 were found in enterocytes among the intestinal cell types these data showed that tmprss2 and aec2 are highly expressed in the digestive tract nervous system analysis using the gtex database showed that both tmprss2 and ace2 are expressed at relatively low levels in the brain cortex chen found that ace2 was relatively highly expressed in some important brain areas such as the substantia nigra and brain ventricles using seven brain transcriptome databases ace2 was expressed at high level in the piriform cortex of human brain and its expression could also be detected in many neurons including both excitatory and inhibitory neurons and some nonneuron cells including astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex qi analyzed the scrnaseq data of substantia nigra biomedicinepharmacotherapy13120201106783 0cclinical classification of acute cardiac injury nonicucases icu cases nonicucases icucases nonsevere cases severecases1965 recoveredcases died cases nonicucases icucases survivor cases nonsurvivor cases chen hong zhou china korea china m dong and cortex of brain from geo database the results showed that both ace2 and tmprss2 were expressed in the oligodendrocyte precursor cells and the astrocytes of the substantia nigra and cortex there are limited reports on the expressions of ace2 and tmprss2 in peripheral nervous system brann analyzed the ace2 and tmprss2 expression in different cell type from human scrnaseq dataset gse139522 and found that neither olfactory sensory neurons nor olfactory bulb neurons expressed these two genes while ace2 and tmprss2 were expressed in the nonneuronal cells including the sustentacular cells and olfactory bulb pericytes these data showed that ace2 and tmprss2 could also be coexpressed in the nervous system other ans or tissues table characteristics of acute cardiac injury after sarscid0 cov2 infection study basic heart disease acute cardiac injury country subject china wang china na huang li china moreover ace2 and tmprss2 were also reported to be coexpressed in some other ans it has been revealed that both ace2 and tmprss2 are expressed in testis by scrna sequencing and expression profile analysis indicating that testicular cells might be the potential targets of sarscov2 another report revealed that multiple kinds of cells in the nose including nasal brushing epithelial cells nasal turbinate epithelial cells and nasal airway epithelial cells contained ace2expressed and tmprss2expressed cell clusters moreover ace2 and tmprss2 were also expressed in pancreas breast prostate and thyroid and these ans might also be the targets of sarscov2 infection of sarscov2 and extrapulmonary an injury of patients with covid19 sarscov2 infection and cardiac injury recently autopsy analysis by fox revealed that the histopathology of the heart was consistent with the typical pattern of viral myocarditis sarscov2 rna was detected in the cardiac tissues of the patients with covid19 these data suggested that sarscov2 may directly infect heart the epidemiology of covid19 reported that cardiac injury was one of the most severe an damages the clinical manifestations of cardiac injury in covid19 patients are complex and could present with heart failure arrhythmias or acute myocardial infarction ami [ ] inciardi reported the first case who had the symptom of heart failure at first and later the patient was positive for sarscov2 using nucleic acid test cardiac injury is a common symptom in patients with covid19 shi reported that patients with covid19 had cardiac injury moreover there were patients with acute cardiac injury in a cohort including covid19 patients and of patients with acute cardiac injury in the intensive care unit icu furthermore a study by wang showed that there were patients with acute cardiac injury and patients presented with arrhythmia of covid19 patients while acute cardiac injury was observed in of patients with civid19 in the icu these cases suggested that sarscov2 may cause serious heart damage which should be widespreadly concerned furthermore acute cardiac injury is more prevalent in severe cases with covid19 [“] table and it has been reported that covid19 patients with cardiac injury had higher mortality than those without cardiac injury in this review we also summarized the possible relationship between basic heart disease and further cardiac injury [“] table in a cohort of covid19 patients from renmin hospital of wuhan university china shi demonstrated that cardiac injury occurred in patients during hospitalization of which had basic heart disease including coronary heart disease and chronic heart failure and only patients with basic heart disease of covid19 patients without cardiac injury similarly liu suggested that patients with basic heart disease in covid19 patients had table comorbidity with cardiac injury in covid19 patients with basic heart disease subjects with covid19 proportion of basic heart disease patients with cardiacinjury study shi liu xu ma guo patients with basic heart disease with cardiac injury without cardiac injury cardiac injury compared with patients with basic cardiovascular diseases of covid19 patients without cardiac injury other studies also indicated that the patients with basic cardiovascular disease are more likely to present heat injury in covid19 patients [ ] in view of the points above covid19 patients with underlying cardiac conditions seem to have higher rates of cardiac injury sarscov2 infection and kidney injury recently autopsy analysis on six covid19 patients showed that varying degrees of acute tubular necrosis were observed in all the renal specimens nucleoprotein np antigens and np positive inclusion body of sarscov2 could be seen in kidney tissues from all the samples moreover viruslike ps were seen in kidney tissues by transmission electronic microscope tem su analyzed kidney abnormalities in autopsies of patients with covid19 and found that diffuse proximal tubular damage with the loss of brush border were biomedicinepharmacotherapy13120201106784 0c sarscov2 infection and liver injury m dong observed further investigation showed that diffuse necrosis can be seen under the light microscope and electron microscopic examination also showed the clusters of coronavirus ps with distinctive spikes in the tubular epithelium and podocytes it was reported that both np antigens and rna of sarscov2 were detected in urine of covid19 patients these data coincide with the finding of the sarscov2 invasion in kidney collectively sarscov2 could directly infect human renal tubules and lead to kidney damage recent studies have shown that the incidence of acute kidney injury aki in covid19 patients ranged from “ and higher frequency of renal function damage with elevated blood urea nitrogen bun or serum creatinine scr was observed in covid19 patients [ ““] table a study of patients with covid19 indicated that levels of bun and scr were increased in and patients with covid19 respectively and routine urine tests were performed on patients among which patients were positive for urinary protein and patients were positive for hematuria another study also showed that about patients with covid19 had abnormal renal function moreover covid19 patients with more severe disease progression have higher rates of aki huang and colleagues reported that of patients with aki in the icu were observed and none of the patients who did not require care in the icu suffered aki xu found that the fatality rate was obviously higher in covid19 patients with aki than those without renal injury furthermore in another study investigating patients with covid19 at hospital admission more severe patients had higher rates of aki and the cox regression analysis also suggested that covid19 patients who developed aki had a significantly higher mortality risk therefore aki is more prevalent in severe cases with covid19 an autopsy report of a 50yearold patient with covid19 showed moderate microvesicular steatosis and mild lobular activity in liver tissues moreover zhao used human liver ductal anoids as a tool to investigate the sarscov2 infection and the tissue damage induced by sarscov2 ex vivo and the results showed that the expression of sarscov2 np was easily detected in the patchy areas of the hepatic duct indicating that liver ductal anoids were susceptible to sarscov2 infection in addition sarscov2 infection could disrupt the barrier and bile acid transporting functions of cholangiocytes which indicated that sarscov2 might directly induce cholangiocyte injury and consequently bile acid accumulation in view of the points above liver damage in the covid19 patients might be directly caused by the viral infection abnormal liver functions were frequently reported in covid19 patients epidemiologic studies showed that almost half of the patients had differing degrees of liver damage [“ “] table chen reported that out of patients had elevated alanine aminotransferase alt patients had elevated aspartate aminotransferase ast and had elevated total bilirubin tbil in wuhan jinyintan hospital wuhan china similarly a nationwide study involving patients with covid19 in china showed that more than of patients had elevated alt and ast and of patients had elevated tbil it was revealed that the levels of direct bilirubin indirect bilirubin alt alkaline phosphatase alp and gammaglutamyltransferase ggt were significantly higher in males than that in females with covid19 and multivariate logistic regression analysis showed that male was an important independent risk factor for predicting acute liver injury ali in covid19 patients these data indicated that male patients with covid19 may be more susceptible to liver injury furthermore table characteristics of acute kidney injury after sarscid0 cov2 infection study chen wang huang guan xu preexisting kidney conditions na na na country china china china china china subject li chen hong cheng xiao richardson wan li qian pei china china korea china china america china china china china na na na na na na scr serum creatinine bun blood urea nitrogen aki acute kidney injury abnormal renal functional indices scr bun na scr scr scr scr bun na scr bun scr na na na scr bun scr na aki na clinical classification of aki na nonicu cases icu cases icu cases nonsevere cases severe cases mild cases severe cases critical ill cases nonsevere cases severe cases recovered cases died cases nonicu cases icu cases na nonsevere cases severe cases cured cases in hospital cases died cases mild cases severe cases nonsevere cases severe cases na moderate cases severe cases critically ill cases biomedicinepharmacotherapy13120201106785 0cm dong table characteristics of liver injury after sarscid0 cov2 infection study country subject china china china china china china china korea america china china chen wang huang guan xu li chen hong richardson et wan li al qian china na patients with preexisting liverconditions na na na patients with abnormal liver functional indices alt ast tbil na ast ast alt tbil alt ast alt ast tbil alt ast alt ast tbil ast alt ast alt ast tbil alt ast abnormal liver functional indices in the nonsevere patients ast alt na na tbil na abnormal liver functional indices in the severe patients alt na tbil na ast na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na nonsevere patients include patients without icu care and recovered patients severe patients include patients with icu care and death alt alanine aminotransferase ast aspartate aminotransferase tbil total bilirubin multiple studies found that ast alt and tbil were significantly higher in patients treated in the icu than that in nonicu patients li suggested that among the patients with abnormal liver function moderate and severe types of patients were more likely to have liver injury and respectively fu analyzed the relationship between ali and mortality risk in covid19 patients and the results showed that ali is more common in the critically ill patients and ali at the early stage increased death risk of covid19 patients together abnormal liver functions might be associated with the severity of patients with covid19 sarscov2 infection and digestive tract injury epithelial cells of the esophagus stomach duodenum and rectum in one covid19 patient tested positive for sarscov2 rna and the staining of viral np was also visualized in the cytoplasm of epithelial cells in stomach duodenum and rectum moreover minimally invasive autopsies were performed on three patients died of covid19 and the results showed that some epithelial cells of the gastrointestinal mucosa were degenerated necrotic and detached these studies strongly supported that sarscov2 may directly infect the epithelial cells of digestive tract table sarscid0 cov2 detection in gastrointestinal specimens study subject xiao zhang tan xing young holshue lescure tang wang xu gastrointestinal samples stool anal swabs rectal swab stool stool stool stool stool stool rectal swabs tested positive in gastrointestinal specimens the positive time in gastrointestinal specimens days na 6cid0 1cid0 5cid0 na 3cid0 positive time for gastrointestinal samples after respiratory samples were negative days na na 8cid0 na na na na na 2cid0 biomedicinepharmacotherapy13120201106786 0cm dong multiple studies have identified that the sarscov2 rna was detected in anal swabs rectal swabs and stool specimens [ ] of covid19 patients it has been demonstrated that sarscov2 rna could be detected in feces from more than half of covid19 patients in another study xing reported that sarscov2 rna was detected in the feces of three pediatric cases with covid19 in qingdao china and the persistence of sarscov2 in the digestive tract lasted for 6cid0 days the possibility of fecaloral transmission of sarscov2 infection needs to be taken into account furthermore as shown in table long duration of sarscov2 detection in digestive tract by rtpcr has been reported and viral rna remained detectable in the digestive tract for 2cid0 days after nucleic acid turned negative in respiratory samples [“] the studies suggested that sarscov2 could be detected from respiratory tract specimens during the early period to digestive tract specimens during the late period and viral nucleic acid tests in both the respiratory and digestive tract are necessary to confirm the complete clearance of virus some covid19 patients presented gastrointestinal symptoms such as diarrhea nausea vomiting and abdominal pain holshue reported the first case of covid19 patient in the usa which had nausea and vomiting before admission multiple studies found that gastrointestinal symptoms including diarrhea “ nausea and vomiting “ and abdominal pain “ were common at presentation in covid19 patients [ ““] table in a cohort of patients with covid19 in wuhan china gastrointestinal symptoms were described in up to moreover sun showed that critically ill patients with covid19 had gastrointestinal injury gi during hospital stay and the survival curves showed that the mortalities of patients with gi was greater than that of patients without gi jin also found that the rate of the severe type was markedly higher in covid19 patients with gi symptoms than that in those without gi symptoms these data suggested that gi is one of the common extrapulmonary an injuries in covid19 patients and may be related to the severity of the disease on the other hand many studies showed that patients with covid19 could present initially with the typical gastrointestinal symptoms and diarrhea may even occur earlier than pyrexia or respiratory symptom in some cases with covid19 [“] luo reported that of covid19 cases presented initially only with gastrointestinal symptoms the covid19 patients initially only with gastrointestinal symptoms are more difficult to diagnose and might be overlooked which could lead to potentially serious consequences together digestive tract symptoms especially diarrhea are the main complications of covd19 patients which should be noticed during the outbreak of covid19 sarscov2 infection and nervous system injury transmission electron microscopy of autopsy sections showed the presence of sarscov2 virallike ps in frontal lobe brain and neural cell bodies moreover researchers confirmed the presence of sarscov2 in cerebrospinal fluid by genome sequencing the pathological mechanism may be the invasion of sarscov2 into the nervons system the virallike ps in brain capillary endothelium was also observed which suggested that hematogenous route might act as the pathway for sarscov2 to the brain in addition study using the mouse model have shown that sarscov can lead to neuroinvasion via disruption of the nasal epithelium and subsequent neuronal dissemination which suggested that coronavirus may use the olfactory nerve to enter the brain the symptoms from nervous system of covid19 patients including headache dizziness anosmia and dysgeusia have been observed in the clinic [““] table disturbance of consciousness and seizures can occur as complications in the cases with severe covid19 it was convincing enough that the neurological deficits of patients with covid19 could be ongoing if it did not get noticed it was indicated that sarscov2 can cause nervous system damage the neurological deficits meningoencephalitis and acute myelitis in covid19 patients have been reported in the usa switzerland and china [“] according to a recent study out of covid19 patients had central nervous system cns symptoms including dizziness headache impaired consciousness ataxia and epilepsy up to of patients with covid19 have headache and clinical manifestations of dizziness were found from “ of the patients with covid19 [ ] olfactory and gustatory disorders are prevalent peripheral nervous system pns symptoms in covid19 patients in patients with mild and moderate covid19 a high proportion of patients presented olfactory and gustatory dysfunctions and olfactory dysfunction appeared prior to the other symptoms in some cases [“ ] these studies showed that the damage of neurological system may also act as a significant feature of covid19 table gastrointestinal symptoms after sarscid0 cov2 infection study chen wang liu xiao huang guan li chen zhou wan li [ Answer:

Colon_Cancer

recently the current pandemic of coronavirus disease covid characterized by a pulmonary infection in humans is caused by a novel virus strain from family coronaviridae known as severe acute respiratory syndrome coronavirus sarscov2 the previous outbreak of severe acute respiratory syndrome sars in “ and middle east respiratory syndrome mers in has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans so far six coronaviruses infecting humans have been identified and the novel coronavirus is the seventh one described to date as being responsible for a respiratory infection sarscov and merscov and the new sarscov2 belong to the betacoronavirus family [“] the coronaviruses have the largest genome around k among the rna viruses sarscov2 was closely related from “ identity to two batderived severe acute respiratory syndrome sarslike coronaviruses batslcovzc45 and batslcovzxc21 but it was more distant from sarscov from “ and merscov about furthermore the performed bioinformatic analysis showed that the nucleotide sequence of sarscov2 is similar to those of other betacoronaviruses with nucleotide identities of ‰¥ there are currently no effective licensed therapies for human coronaviruses hcov infections and existing treatment strategies are generally limited to symptomatic treatment and supportive care email addresses kuzunovahqtchaikapharmacom k uzunova efilipovahqtchaikapharmacom e filipova vpavlovahqtchaikapharmacom corresponding author v pavlova tvekovmuplevenabvbg t vekov 101016jbiopha2020110668 received may received in revised form august accepted august biomedicinepharmacotherapy1312020110668availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0csuch as solidarity who recovery k uzunova in the absence of a specific treatment for this novel virus the effort of researchers is focused on understanding and controlling the disease and on preventing and controlling the replication and spread of the virus to devise therapeutic strategies to counteract the sarscov2 infection numerous potential treatment options are being evaluated in ongoing clinical trials many antiviral and immunological treatments being investigated against coronaviruses are summarized by who in landscape analysis of therapeutics as of march the realtime dashboard of completed ongoing and planned clinical trials for covid includes drugs and promising therapies such as remdesevir lopinavirritonavir hydroxychloroquine il6 inhibitors tocilizumab and sarilumab convalescent plasma therapy stemcell transfusion vaccine candidates traditional chinese medicines which are of top interventions of the presented network among them remdesivir an analogue of adenosine seems to have a more promising future due to proven in vitro and in vivo antiviral efficacy till the beginning of june promising therapies involving lopinavirritonavir and chloroquine or hydroxychloroquine were part of treatment guidelines in many countries but currently they are excluded from covid19 treatment protocols because of uncertainty regarding their risks and benefits and it is recommended that they should be used only in the context of clinical trials [“] in spite of its known in vitroin vivo efficacy and safety profiles some trials evaluating these drugs for covid19 infection treatment uk ntc04381936 and discovery inserm ntc04315948 discontinued hydroxychloroquine and lopinavirritonavir arms the interim trial results showed that hydroxychloroquine and lopinavirritonavir produced little or no reduction in the mortality of hospitalized covid19 patients when compared to standard of care nevertheless some countries worldwide continue to recommend chloroquinehydroxychloroquine as a treatment option [“] the existing drugs that target viral proteins associated with enzymatic activities or blocking viral replication machinery or host proteins involved in viral life cycle regulating the function of the immune system or other cellular processes in host cells have great potential and are available on the market our review aims to highlight the potential molecular mechanisms of the therapeutic options available for the cure of other health conditions and their repurposing for the treatment of this novel coronavirus sars cov2 selected treatments of sarscov2 remdesivir gs5734 “ polymerase inhibitor deltacoronavirus genus pdcov which have the most divergent rdrp of known cov as compared to sars and merscov an in silico test of the covid19 rdrp built model suggested the effectiveness of remdesivir as a potent drug sarscov and sarscov2 both belong to the betacoronaviruses of the b lineage and the rdrp amino acid sequences of the two viruses are identical whereas merscov belongs to the betacoronaviruses of the c lineage and is only identical with sarscov2 another in vitro and in vivo proof came from sheahan who examined if gs5734 could inhibit replication of sarscov and mers cov in primary human airway epithelial hae cell cultures they found out a dosedependent reduction in replication with average ic50 values of μm sarscov and μm merscov moreover the compound inhibits a broad range of diverse cov including circulating human zoonotic bat cov and prepandemic zoonotic cov with both prophylactic and therapeutic 1dpi dosing of gs5734 a reduction in replication below a diseasecausing threshold in mouse model of sars cov pathogenesis was demonstrated therapeutic gs5734 substantially reduced the sarscov induced weight loss in infected animals and significantly suppressed virus lung titers p thus demonstrating that therapeutic administration of gs5734 can reduce disease and suppress replication during an ongoing infection furthermore remdesivir has the potential to block sarscov2 infection in vitro at lowmicromolar concentration and in treatment of merscov and sarscov infections in vivo it demonstrated a significant improvement of pulmonary pathology in mice the rnadependent rna polymerase rdrpmediated mechanism of cov inhibition by gs5734 is proven even in the setting of intact exoribonuclease exonmediated proofreading using the model coronavirus murine hepatitis virus mhv it was demonstrated that gs5734 dramatically inhibited viral replication and viral rna synthesis in wildtype wt virus while an nsp14 exon mutant lacking proofreading demonstrated increased susceptibility to gs5734 45fold more active this suggests that gs5734 is recognized at least partially by a functional exon but that the exon activity is not sufficient to prevent potent inhibition of cov replication the results provide strong evidence that rdrp is the target for remdesivir and support the hypothesis that gs5734 directly inhibits viral rna synthesis the mechanism of inhibition of rdrp of merscov by remdesivir was studied by gordon et al they coexpressed the merscov nonstructural proteins nsp5 nsp7 nsp8 and nsp12 rdrp in insect cells as a part of a polyprotein coexpression of the mers nsp5 protease with nsp7 nsp8 and nsp12 in insect cells yielded a stable complex composed of nsp8 and nsp12 the triphosphate form of the inhibitor rdvtp is utilized as a substrate and competes with its natural counterpart atp and they observed that incorporation of the nucleotide analogue was significantly more efficient once added into the growing rna chain the inhibitor does not cause immediate chain termination the presence of the ²hydroxyl group allows the addition of three more nucleotides until rna synthesis is arrested at position i3 therefore the main possible mechanism of action is delayed rna chain termination recently the same authors obtained almost identical results with sarscov merscov and sarscov2 rdrps they provided evidence that all three coronavirus rdrp complexes terminated rna synthesis at position i3 almost all viruses encode polymerases in the central steps of replication and transcription therefore polymerases are becoming the most attractive and suitable targets for antiviral development there are two major types of polymerase inhibitors i nucleoside and nucleotide substrate analogs and ii allosteric inhibitors nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids to date most of the approved antiviral drugs for antihiv therapy utilize this mechanism remdesivir is a nucleotide analogue with a proved mechanism of action as an inhibitor of rnadependent rna remdesivir rdv is an investigational compound with a broad spectrum of antiviral activities against rna viruses including sarscov and merscov gs5734 was originally developed for the treatment of the ebola virus disease gs5734 the single sp isomer of the 2ethylbutyl lalaninate phosphoramidate prodrug effectively bypasses the rate limiting first phosphorylation step of the nuc nucleoside ribose analogue the mechanism of action of nuc requires intracellular anabolism to the active triphosphate metabolite ntp which is expected to interfere with the activity of viral rnadependent rna polymerases rdrp gs5734 selectively inhibits ebola virus replication by targeting its rdrp and inhibiting viral rna synthesis following efficient intracellular conversion to ntp in nonhuman primates this compound shows a broad spectrum of antiviral activities against several rna viruses including respiratory syncytial virus rsv junín virus lassa fever virus and middle east respiratory syndrome virus but was inactive against alphaviruses or retroviruses furthermore remdesivir dosedependently inhibits endemic human cov229e and covoc43 replications which typically cause upper respiratory infection in children but can cause more severe lower respiratory infection in adults with underlying respiratory conditions ie asthma copd and the elderly as well as a member of the biomedicinepharmacotherapy13120201106682 0c lopinavirritonavir “ protease inhibitor the proteases encoded by most viruses play a crucial role in the viral life cycle the protease inhibitors pis bind competitively to the substrate site of the viral protease this enzyme is responsible for the post translational proteolysis of a polyprotein precursor and the release of functional viral proteins allowing them to function correctly and individually in replicationtranscription and maturation inhibition results in the production of immature virus ps coronavirus proteases of which there are two in sarscov a papainlike cysteine proteinase plpro nsp3 and a 3clike proteinase 3clpro or mpro nsp5 and three in several other coronaviruses cleave the orf1 polypeptide as it is translated enabling the formation of the viral replication complex the substratebinding pockets are highly conserved among cov 3clpro suggesting the possibility for a widespectrum inhibitor design targeting this region in the 3clpro of all covs it is postulated that the 3clproinhibiting activity of lopinavirritonavir contributes at least partially to its anticov effects in silico binding studies of the drugs using the identified crystal structure of mpro and employing the hex program to conduct the docking of the ligands to the sarscov main proteinase revealed that lopinavir and ritonavir could basically bind to the active site of sars main proteinase but the efficacy of lopinavirritonavir was predicted to be poor according to the latest report of the structure of 3clpro from sarscov2 pdb code 6lu7 and the available structure of 3clpro from sarscov pdb code 1uk4 the two main proteases differ by only amino acids comparing ligand binding free energies for the main proteases has confirmed that good binders for sarscov are in general and sarscov k uzunova polymerases this mechanism is probably involved in an antiviral activity against sarscov2 biochemical data provided by gordon suggested a unifying mechanism of inhibition of sarscov merscov and sarscov2 fig and future emerging covs may be similarly susceptible to the inhibition by remdesivir comparable replication with also good binders for sarscov2 3clpro protease inhibitors a class of drugs best known for success against hiv block the final step of virion assembly in the treatment of human immunodeficiency virus infection with proven efficacy the combination of lopinavir with ritonavir is widely used as a boosted protease inhibitor in the treatment of hiv infection because of low oral bioavailability of lopinavir and its extensive metabolism by the cyp3a4 isoenzyme lopinavir needs to be coadministered with ritonavir to achieve drug concentrations high enough to inhibit viral replication [ “] so far the reported results from studies in different cell lines animal models and patients for lopinavirritonavir are not so convincing in their inhibition action in human coronaviruses screening the library of fdaapproved drugs for antimerscov activity in cell culture has identified four compounds chloroquine chlorpromazine loperamide and lopinavir which inhibit merscov replication effective concentration ec50 3cid0 μmoll in vitro lopinavir inhibited mers cov efficacy ec50 μm and a maximal protective effect were observed at a dose of μm it was previously shown that lopinavir but not ritonavir inhibit sarscov chymotrypsinlike 3cl protease at the concentration of μm moreover it was suggested that lopinavir blocks a postentry step in the merscov replicative cycle in vitro the detectable antiviral activities of ribavirin rimantadine lopinavir and baicalin were shown by using the frhk4 cell line and in vero e6 cells infected with sarscov2 lopinavir inhibit replication with ec50 at μm during the sars outbreak treatment with lopinavir in combination with ritonavir was explored with some success in nonrandomized clinical trials patients with sarscov treated with lopinavirritonavir showed a progressive decrease of viral load and reduction of the composite adverse outcome at day recently the antiviral activity of remdesivir and ifn was found to be superior to that of lopinavirritonavirifn against merscov in vitro and in vivo the efficacy of lopinavirritonavir with or without ribavirin is evaluated in sarscov2 patients under randomized control trials currently it was demonstrated that this combination has no benefits in adult patients with severe covid19 although protease inhibitors are a common class of medication used in the treatment of hiv1 infection their efficacy in human coronavirus infections is not convincing moreover several antihiv pis are also known to influence other intracellular pathways it was demonstrated that hiv protease inhibitors indinavir saquinavir and lopinavir independently from any viral infection can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis in view of the weak antiviral activity of protease inhibitors further studies should be done to ascertain whether the clinical benefit could be attributed to their antiapoptotic rather than their antiviral activity hence even if the molecular target of lopinavirritonavir is the main protease 3clpro in sarscov2 infected cells fig there are no biochemical and molecular studies confirming the interaction and associating this with clinical efficacy of the protease inhibitor chloroquinehydroxychloroquine chloroquine chq was introduced into clinical practice in as a prophylactic treatment for malaria hydroxychloroquine hcq differs from chloroquine by the presence of a hydroxyl group at the end of the side chain the nethyl substituent is hydroxylated currently chq and its hydroxyl form hcq are used as antiinflammatory agents for the treatment of rheumatoid arthritis lupus erythematosus and amoebic hepatitis in addition chq has been studied as a potent antiviral agent against hiv1aids [“] hcov229e sarscov [ ] influenza a h5n1virus influenza a and b and many other rna and dna viruses many recent reports and published studies suggested that chq and hcq were associated with reduced fig inhibition of viral infection by lopinavirritonavir and remdesivir biomedicinepharmacotherapy13120201106683 0ck uzunova progression of the covid19 and decreased duration of the symptoms [“] there are in fact overall more than trials currently underway around the world on its impact either as a prophylactic or treatment for covid19 it is noteworthy that the usefulness of hydroxychloroquine and chloroquine is intensively investigated chloroquine was found to exert an antiviral effect during pre and postinfection conditions suggesting to have both prophylactic and therapeutic advantages timeofaddition assay demonstrated that chq functioned at both entry and postentry stages of the sarscov2 infection in vero e6 cells however it did not reduce viral replication in sarscov infected mice hydroxychloroquine is significantly more potent than chq in vitro ec50 values and μm respectively and has a lower potential for drugdrug interactions than chloroquine pharmacokinetic models demonstrate that hydroxychloroquine sulfate is significantly superior days in advance to chloroquine phosphate in inhibiting sarscov2 in vitro and was demonstrated to be much less toxic than chq in animals on the other hand data presented by liu demonstrated that the antiviral effect of hcq against sarscov2 infection was comparable with chq in vitro cc50 μm and μm for chq and hcq respectively moreover they suggested that both chq and hcq blocked the transport of sarscov2 from early endosomes ees to endolysosomes els and caused noticeable sizemorphological changes in ees and els they surmised that endosome maturation might be blocked at intermediate stages of endocytosis resulting in failure of further transport of virions to the ultimate releasing site hydroxychloroquine shares the same mechanism of action as chloroquine apart from the probable role of chq and hcq as antiviral agents their mechanisms of action are not fully understood and it was demonstrated that they have multiple effects on mammalian cells ace2 is known to be a cell receptor for sarscov the high similarities of the amino acid sequences and predicted protein structures of the receptorbinding domain of sarscov2 and sarscov suggest that sarscov2 may efficiently use human ace2 as a receptor for cellular entry and employ the cellular serine protease tmprss2 for s protein priming zhou confirmed that sarscov2 used the ace2 receptor to enter cells and did not use other coronavirus receptors such as aminopeptidase n apn and dipeptidyl peptidase dpp4 so the primary mechanism by which cell infection is prevented by these drugs may be at the stage of binding with the surface receptor and endosomemediated viral entry two independent in vitro studies confirmed that chq inhibits the replication of the sarscov chloroquine inhibits the early stage of sarscov replication in vero e6 cells with a effective concentration of ± μml the antiviral activity of chq was indicative at the time point at virus attachment or penetration vincent established that the drug might interfere with terminal glycosylation of the cellular receptor ace2 when chq was added prior to infection the impairment of terminal glycosylation of ace2 may result in reduced binding affinities between ace2 and sarscov spike protein and negatively influence the initiation of sarscov infection when chq or nh4cl were added after infection these agents could rapidly raise the ph and interrupt ongoing fusion events between the virus and endosomes thus inhibiting the infection on the basis of in vitro experiments they suggested that the primary mechanism by which infection was prevented was the poor affinity of sarscov spike protein to underglycosylated ace2 in vitro studies with hiv infected cells also identified that inhibition of glycosylation might be a possible mechanism of action of chq chq inhibits hiv replication at a postintegration stage resulting in the production of immature virions it was demonstrated that the sole mechanism explaining the antihiv activity of chq was a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2g12 epitope of gp120 according to in vitro results the antiretroviral effects of chq are attributable to the inhibition of viral p glycosylation these effects appeared to be specific since the chq concentrations effective in vitro neither affected any other step in hiv1 replication nor were cytotoxic thus there is direct evidence that chq is an inhibitor of glycosylation of gp120 and these alterations may be responsible for the decreased infectivity of hiv grown in the presence of chloroquine when added after the initiation of infection these drugs might affect the endosomemediated fusion and subsequent virus replication sarscov pseudoviruses may enter cells via receptordependent clathrin and caveolaeindependent phsensitive endocytosis likely through a process involving lipid rafts a later study however suggests that the entry of coronaviruses into the host cells occurs through clathrinmediated endocytosis murine hepatitis virus mhv a prototypic member of the cov family requires trafficking to lysosomes for proteolytic cleavage at the fp proximal position of its spike s protein membrane fusion to occur many authors indicated that s protein cleavage is an important step for fusion activity and subsequent internalization of the sarscov virus genome into cells [“] adding chq prior to infection results to inhibition of endosome maturation and strongly decreased mhv infection and fusion which was not observed when the drug was added at hpi indicating that the compound mainly affects mhv entry chloroquine is a weak base that is known to increase the ph of lysosomal and transgolgi network tgn vesicles leading to the dysfunction of enzymes necessary for proteolytic processing and posttranslational modifications of newly synthesized viral proteins chloroquine is able to prevent the processing of prm protein to m protein in flavivirusinfected mammalian and mosquito cells by raising the ph of the postgolgi vesicles in which this cleavage occurs as a result virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prm protein rather than m protein and this reduced the infectivity of the virus the chloroquinemediated rise in endosomal ph modulates iron metabolism in a variety of cell types decreasing in intracellular concentration of iron affects the function of several cellular enzymes involved in pathways leading to the replication of cellular dna and to the expression of different genes [“] autophagy is a lysosomedependent degradative pathway chq and its analogue hcq are known clinically relevant autophagy inhibitors chq is a weak base that inhibits lysosomal acidification which prevents the fusion of autophagosomes with lysosomes and subsequent autophagic degradation inhibition of autophagy with chq stimulates superoxide generation ubiquitinconjugated protein accumulation and apoptosis in a colon cancer xenograft model chq treatment clearly inhibited autophagy in mouse lung and efficiently ameliorated acute lung injury and dramatically improved the survival rate in mice infected with live avian influenza a h5n1 virus h5n1 virusinduced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase akt the tumor suppressor protein tsc2 and the mammalian target of rapamycin and autophagyblocking agents might be useful as prophylactics and therapeutics against infection of humans by the h5n1 virus furthermore prentice suggested that authophagy was induced by the coronavirus mouse hepatitis virus mhv and was required for formation of double membranebound mhv replication complexes which significantly enhanced the efficiency of replication replication of the virus was impaired in atg5 knockout embryonic stem cells the same authors also examined the sarscovs and found out similar colocalization of the key viral replication proteins with endogenous lc3 a protein marker for autophagosome it could be assumed that autophagy inhibitors like chq could inhibit virus replication at present the exact role of autophagy in cov infection remains debatable and there is much evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sarscovs merscovs and possibly sarscov2 the antiinflammatory properties of chqhcq should also be considered several studies have suggested that multiple an failure biomedicinepharmacotherapy13120201106684 0chas not yet been identified in sarscov2 infected patients and probably multiple pathways could be involved fig conclusion the sarscov2 is the cause of the coronavirus disease covid19 that has been declared a global pandemic by the world health anization who in despite some clinical characteristics that differentiate covid19 from sarscov merscov and seasonal influenza the pathogen sarscov2 has the same phylogenetic similarity to sarscov and mers cov most of the encoded proteins exhibited high sequence identity between sarscov2 and the related batderived coronaviruses batslcovzc45 and batslcovzxc21 a notable difference was a longer spike protein encoded by sarscov2 compared with the bat sarslike coronaviruses sarscov and merscov in addition sarscov2 was distinct from sarscov in a phylogeny of the complete rnadependent rna polymerase rdrp gene moreover the receptorbinding domain of sarscov2 which directly engages the ace2 receptor for cell entry was more closely related to those of sarscovs “ amino acid identity since the outbreak researchers have released many agents that could have potential efficacy against covid19 there is currently no clinically proven specific antiviral agent available for sarscov2 infection like sarscov and merscov certain agents like chloroquine hydroxychloroquine lopinavirritonavir and remdesivir are being used in ongoing clinical trials all over the world with hopes to further delineate their role in the treatment and prophylaxis of covid19 furthermore due to their availability and using for decades and proven safety records it is reasonable to suggest that they may be appropriate for treatment of covid19 remdesivir an adenosine analogue with wellstudied mechanism of action in cov infections can target the rnadependent rna polymerase and block viral rna synthesis and has been a promising antiviral drug antiviral studies in cell culture and animal models the available human safety data as well as the clear mechanism of action characterize rdv as a directacting antiviral since some authors found that lopinavir“ritonavir treatment did not significantly accelerate clinical improvement hence antiviral effects as an inhibitor of the sarscov main 3cl protease should be further investigated although chq and hcq are wellknown drugs for the treatment of k uzunova observed in fatal cases are most likely associated with not only the direct viral infection and destruction of susceptible cells eg endothelial cells but also the effects of proinflammatory cytokines chemokines and other mediators released from infected and activated cells such as monocytes and macrophages the clinical worsening of individuals with sars in week is apparently unrelated to uncontrolled sars coronavirus replication but may be related to immunopathological damage another study reveals that the presence of viral elements within endothelial cells and the accumulation of inflammatory cells led to endotheliitis in several ans as a direct consequence of viral involvement and to host inflammatory response moreover chq has immunomodulatory effects suppressing the productionrelease of tumour necrosis factorα and interleukin6 which mediate the inflammatory complications of several viral diseases chloroquinehcq was reported to inhibit the production of soluble mature tnf in macrophage cell line inhibit tnfα receptor in human histocytic u937 cells inhibit tnfα ifnγ and il6 in peripheral blood mononuclear cells pbmc reduce tnfα production and lipopolysaccharide lpsinduced il1 release in human monocytic cells it is suggested that chq exerts antiinflammatory and immunomodulatory effects predominantly by pretranslational and nonlysosomotropic mechanisms chloroquineinduced inhibition of tnf and il6 production is not mediated through a lysosomotropic mechanism and chloroquine probably acts on tnf secretion by disrupting iron homeostasis besides its antiviral activity and due to its suppressive effects on the productionrelease of tnfα and interleukin chqhcq may be effectively used in the treatment of viral infections characterized by symptoms associated with inflammatory processes andor immunehyperactivation antiinflammatory effects of chq remain poorly understood regulation of proinflammatory cytokines chq can also act on the immune system through cell signaling chq inhibits the activation of p38 mapk in hcov229einfected cells and evokes the activation of erk independently of infection these results suggested that chq may inhibit cov replication by suppressing the p38 activation additionally chq strongly inhibited phosphorylation of mitogenactivated protein kinase mapk p38 and to a lesser extent cjun nterminal kinase and extracellular signalregulated kinase ½ chloroquine could also inhibit innate immune responses trough downregulation of tlr9 signaling pathways requiring endocytosis and acidification of endosomes within plasmacytoid dendritic cells pdcs and act as novel antagonists to chemokine receptor cxcr4 that suppress pancreatic cancer cell proliferation on the other hand another hypothesized mechanism of chq is via the inhibition of antigen degradation and improving the crosspresentation efficiency of dcs in vitro in vivo evidence suggested that a short course of treatment with chq followed by a booster dose of a soluble antigen immunization can efficiently enhance human cd8 t cell responses and single vaccination with inactivated influenza virus combined with chloroquine treatment elicits a higher t cell immunity in mice regulation of nlrp3 inflammasome activation may offer a promising therapeutic approach by inhibiting or slowing down the process of acute respiratory distress syndrome hcq is a known nlrp3 inhibitor and its potential clinical effectiveness is certainly based on the downregulation of il1 expression the major proinflammatory cytokine interleukin1beta il1 is elevated in plasma from hospitalized covid19 patients and its associated signaling pathway seems to drive sarscov2 pathogenicity il1 secretion is primarily initiated by inflamm

cancer7532

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: recently the current pandemic of coronavirus disease covid characterized by a pulmonary infection in humans is caused by a novel virus strain from family coronaviridae known as severe acute respiratory syndrome coronavirus sarscov2 the previous outbreak of severe acute respiratory syndrome sars in “ and middle east respiratory syndrome mers in has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans so far six coronaviruses infecting humans have been identified and the novel coronavirus is the seventh one described to date as being responsible for a respiratory infection sarscov and merscov and the new sarscov2 belong to the betacoronavirus family [“] the coronaviruses have the largest genome around k among the rna viruses sarscov2 was closely related from “ identity to two batderived severe acute respiratory syndrome sarslike coronaviruses batslcovzc45 and batslcovzxc21 but it was more distant from sarscov from “ and merscov about furthermore the performed bioinformatic analysis showed that the nucleotide sequence of sarscov2 is similar to those of other betacoronaviruses with nucleotide identities of ‰¥ there are currently no effective licensed therapies for human coronaviruses hcov infections and existing treatment strategies are generally limited to symptomatic treatment and supportive care email addresses kuzunovahqtchaikapharmacom k uzunova efilipovahqtchaikapharmacom e filipova vpavlovahqtchaikapharmacom corresponding author v pavlova tvekovmuplevenabvbg t vekov 101016jbiopha2020110668 received may received in revised form august accepted august biomedicinepharmacotherapy1312020110668availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0csuch as solidarity who recovery k uzunova in the absence of a specific treatment for this novel virus the effort of researchers is focused on understanding and controlling the disease and on preventing and controlling the replication and spread of the virus to devise therapeutic strategies to counteract the sarscov2 infection numerous potential treatment options are being evaluated in ongoing clinical trials many antiviral and immunological treatments being investigated against coronaviruses are summarized by who in landscape analysis of therapeutics as of march the realtime dashboard of completed ongoing and planned clinical trials for covid includes drugs and promising therapies such as remdesevir lopinavirritonavir hydroxychloroquine il6 inhibitors tocilizumab and sarilumab convalescent plasma therapy stemcell transfusion vaccine candidates traditional chinese medicines which are of top interventions of the presented network among them remdesivir an analogue of adenosine seems to have a more promising future due to proven in vitro and in vivo antiviral efficacy till the beginning of june promising therapies involving lopinavirritonavir and chloroquine or hydroxychloroquine were part of treatment guidelines in many countries but currently they are excluded from covid19 treatment protocols because of uncertainty regarding their risks and benefits and it is recommended that they should be used only in the context of clinical trials [“] in spite of its known in vitroin vivo efficacy and safety profiles some trials evaluating these drugs for covid19 infection treatment uk ntc04381936 and discovery inserm ntc04315948 discontinued hydroxychloroquine and lopinavirritonavir arms the interim trial results showed that hydroxychloroquine and lopinavirritonavir produced little or no reduction in the mortality of hospitalized covid19 patients when compared to standard of care nevertheless some countries worldwide continue to recommend chloroquinehydroxychloroquine as a treatment option [“] the existing drugs that target viral proteins associated with enzymatic activities or blocking viral replication machinery or host proteins involved in viral life cycle regulating the function of the immune system or other cellular processes in host cells have great potential and are available on the market our review aims to highlight the potential molecular mechanisms of the therapeutic options available for the cure of other health conditions and their repurposing for the treatment of this novel coronavirus sars cov2 selected treatments of sarscov2 remdesivir gs5734 “ polymerase inhibitor deltacoronavirus genus pdcov which have the most divergent rdrp of known cov as compared to sars and merscov an in silico test of the covid19 rdrp built model suggested the effectiveness of remdesivir as a potent drug sarscov and sarscov2 both belong to the betacoronaviruses of the b lineage and the rdrp amino acid sequences of the two viruses are identical whereas merscov belongs to the betacoronaviruses of the c lineage and is only identical with sarscov2 another in vitro and in vivo proof came from sheahan who examined if gs5734 could inhibit replication of sarscov and mers cov in primary human airway epithelial hae cell cultures they found out a dosedependent reduction in replication with average ic50 values of μm sarscov and μm merscov moreover the compound inhibits a broad range of diverse cov including circulating human zoonotic bat cov and prepandemic zoonotic cov with both prophylactic and therapeutic 1dpi dosing of gs5734 a reduction in replication below a diseasecausing threshold in mouse model of sars cov pathogenesis was demonstrated therapeutic gs5734 substantially reduced the sarscov induced weight loss in infected animals and significantly suppressed virus lung titers p thus demonstrating that therapeutic administration of gs5734 can reduce disease and suppress replication during an ongoing infection furthermore remdesivir has the potential to block sarscov2 infection in vitro at lowmicromolar concentration and in treatment of merscov and sarscov infections in vivo it demonstrated a significant improvement of pulmonary pathology in mice the rnadependent rna polymerase rdrpmediated mechanism of cov inhibition by gs5734 is proven even in the setting of intact exoribonuclease exonmediated proofreading using the model coronavirus murine hepatitis virus mhv it was demonstrated that gs5734 dramatically inhibited viral replication and viral rna synthesis in wildtype wt virus while an nsp14 exon mutant lacking proofreading demonstrated increased susceptibility to gs5734 45fold more active this suggests that gs5734 is recognized at least partially by a functional exon but that the exon activity is not sufficient to prevent potent inhibition of cov replication the results provide strong evidence that rdrp is the target for remdesivir and support the hypothesis that gs5734 directly inhibits viral rna synthesis the mechanism of inhibition of rdrp of merscov by remdesivir was studied by gordon et al they coexpressed the merscov nonstructural proteins nsp5 nsp7 nsp8 and nsp12 rdrp in insect cells as a part of a polyprotein coexpression of the mers nsp5 protease with nsp7 nsp8 and nsp12 in insect cells yielded a stable complex composed of nsp8 and nsp12 the triphosphate form of the inhibitor rdvtp is utilized as a substrate and competes with its natural counterpart atp and they observed that incorporation of the nucleotide analogue was significantly more efficient once added into the growing rna chain the inhibitor does not cause immediate chain termination the presence of the ²hydroxyl group allows the addition of three more nucleotides until rna synthesis is arrested at position i3 therefore the main possible mechanism of action is delayed rna chain termination recently the same authors obtained almost identical results with sarscov merscov and sarscov2 rdrps they provided evidence that all three coronavirus rdrp complexes terminated rna synthesis at position i3 almost all viruses encode polymerases in the central steps of replication and transcription therefore polymerases are becoming the most attractive and suitable targets for antiviral development there are two major types of polymerase inhibitors i nucleoside and nucleotide substrate analogs and ii allosteric inhibitors nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids to date most of the approved antiviral drugs for antihiv therapy utilize this mechanism remdesivir is a nucleotide analogue with a proved mechanism of action as an inhibitor of rnadependent rna remdesivir rdv is an investigational compound with a broad spectrum of antiviral activities against rna viruses including sarscov and merscov gs5734 was originally developed for the treatment of the ebola virus disease gs5734 the single sp isomer of the 2ethylbutyl lalaninate phosphoramidate prodrug effectively bypasses the rate limiting first phosphorylation step of the nuc nucleoside ribose analogue the mechanism of action of nuc requires intracellular anabolism to the active triphosphate metabolite ntp which is expected to interfere with the activity of viral rnadependent rna polymerases rdrp gs5734 selectively inhibits ebola virus replication by targeting its rdrp and inhibiting viral rna synthesis following efficient intracellular conversion to ntp in nonhuman primates this compound shows a broad spectrum of antiviral activities against several rna viruses including respiratory syncytial virus rsv junín virus lassa fever virus and middle east respiratory syndrome virus but was inactive against alphaviruses or retroviruses furthermore remdesivir dosedependently inhibits endemic human cov229e and covoc43 replications which typically cause upper respiratory infection in children but can cause more severe lower respiratory infection in adults with underlying respiratory conditions ie asthma copd and the elderly as well as a member of the biomedicinepharmacotherapy13120201106682 0c lopinavirritonavir “ protease inhibitor the proteases encoded by most viruses play a crucial role in the viral life cycle the protease inhibitors pis bind competitively to the substrate site of the viral protease this enzyme is responsible for the post translational proteolysis of a polyprotein precursor and the release of functional viral proteins allowing them to function correctly and individually in replicationtranscription and maturation inhibition results in the production of immature virus ps coronavirus proteases of which there are two in sarscov a papainlike cysteine proteinase plpro nsp3 and a 3clike proteinase 3clpro or mpro nsp5 and three in several other coronaviruses cleave the orf1 polypeptide as it is translated enabling the formation of the viral replication complex the substratebinding pockets are highly conserved among cov 3clpro suggesting the possibility for a widespectrum inhibitor design targeting this region in the 3clpro of all covs it is postulated that the 3clproinhibiting activity of lopinavirritonavir contributes at least partially to its anticov effects in silico binding studies of the drugs using the identified crystal structure of mpro and employing the hex program to conduct the docking of the ligands to the sarscov main proteinase revealed that lopinavir and ritonavir could basically bind to the active site of sars main proteinase but the efficacy of lopinavirritonavir was predicted to be poor according to the latest report of the structure of 3clpro from sarscov2 pdb code 6lu7 and the available structure of 3clpro from sarscov pdb code 1uk4 the two main proteases differ by only amino acids comparing ligand binding free energies for the main proteases has confirmed that good binders for sarscov are in general and sarscov k uzunova polymerases this mechanism is probably involved in an antiviral activity against sarscov2 biochemical data provided by gordon suggested a unifying mechanism of inhibition of sarscov merscov and sarscov2 fig and future emerging covs may be similarly susceptible to the inhibition by remdesivir comparable replication with also good binders for sarscov2 3clpro protease inhibitors a class of drugs best known for success against hiv block the final step of virion assembly in the treatment of human immunodeficiency virus infection with proven efficacy the combination of lopinavir with ritonavir is widely used as a boosted protease inhibitor in the treatment of hiv infection because of low oral bioavailability of lopinavir and its extensive metabolism by the cyp3a4 isoenzyme lopinavir needs to be coadministered with ritonavir to achieve drug concentrations high enough to inhibit viral replication [ “] so far the reported results from studies in different cell lines animal models and patients for lopinavirritonavir are not so convincing in their inhibition action in human coronaviruses screening the library of fdaapproved drugs for antimerscov activity in cell culture has identified four compounds chloroquine chlorpromazine loperamide and lopinavir which inhibit merscov replication effective concentration ec50 3cid0 μmoll in vitro lopinavir inhibited mers cov efficacy ec50 μm and a maximal protective effect were observed at a dose of μm it was previously shown that lopinavir but not ritonavir inhibit sarscov chymotrypsinlike 3cl protease at the concentration of μm moreover it was suggested that lopinavir blocks a postentry step in the merscov replicative cycle in vitro the detectable antiviral activities of ribavirin rimantadine lopinavir and baicalin were shown by using the frhk4 cell line and in vero e6 cells infected with sarscov2 lopinavir inhibit replication with ec50 at μm during the sars outbreak treatment with lopinavir in combination with ritonavir was explored with some success in nonrandomized clinical trials patients with sarscov treated with lopinavirritonavir showed a progressive decrease of viral load and reduction of the composite adverse outcome at day recently the antiviral activity of remdesivir and ifn was found to be superior to that of lopinavirritonavirifn against merscov in vitro and in vivo the efficacy of lopinavirritonavir with or without ribavirin is evaluated in sarscov2 patients under randomized control trials currently it was demonstrated that this combination has no benefits in adult patients with severe covid19 although protease inhibitors are a common class of medication used in the treatment of hiv1 infection their efficacy in human coronavirus infections is not convincing moreover several antihiv pis are also known to influence other intracellular pathways it was demonstrated that hiv protease inhibitors indinavir saquinavir and lopinavir independently from any viral infection can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis in view of the weak antiviral activity of protease inhibitors further studies should be done to ascertain whether the clinical benefit could be attributed to their antiapoptotic rather than their antiviral activity hence even if the molecular target of lopinavirritonavir is the main protease 3clpro in sarscov2 infected cells fig there are no biochemical and molecular studies confirming the interaction and associating this with clinical efficacy of the protease inhibitor chloroquinehydroxychloroquine chloroquine chq was introduced into clinical practice in as a prophylactic treatment for malaria hydroxychloroquine hcq differs from chloroquine by the presence of a hydroxyl group at the end of the side chain the nethyl substituent is hydroxylated currently chq and its hydroxyl form hcq are used as antiinflammatory agents for the treatment of rheumatoid arthritis lupus erythematosus and amoebic hepatitis in addition chq has been studied as a potent antiviral agent against hiv1aids [“] hcov229e sarscov [ ] influenza a h5n1virus influenza a and b and many other rna and dna viruses many recent reports and published studies suggested that chq and hcq were associated with reduced fig inhibition of viral infection by lopinavirritonavir and remdesivir biomedicinepharmacotherapy13120201106683 0ck uzunova progression of the covid19 and decreased duration of the symptoms [“] there are in fact overall more than trials currently underway around the world on its impact either as a prophylactic or treatment for covid19 it is noteworthy that the usefulness of hydroxychloroquine and chloroquine is intensively investigated chloroquine was found to exert an antiviral effect during pre and postinfection conditions suggesting to have both prophylactic and therapeutic advantages timeofaddition assay demonstrated that chq functioned at both entry and postentry stages of the sarscov2 infection in vero e6 cells however it did not reduce viral replication in sarscov infected mice hydroxychloroquine is significantly more potent than chq in vitro ec50 values and μm respectively and has a lower potential for drugdrug interactions than chloroquine pharmacokinetic models demonstrate that hydroxychloroquine sulfate is significantly superior days in advance to chloroquine phosphate in inhibiting sarscov2 in vitro and was demonstrated to be much less toxic than chq in animals on the other hand data presented by liu demonstrated that the antiviral effect of hcq against sarscov2 infection was comparable with chq in vitro cc50 μm and μm for chq and hcq respectively moreover they suggested that both chq and hcq blocked the transport of sarscov2 from early endosomes ees to endolysosomes els and caused noticeable sizemorphological changes in ees and els they surmised that endosome maturation might be blocked at intermediate stages of endocytosis resulting in failure of further transport of virions to the ultimate releasing site hydroxychloroquine shares the same mechanism of action as chloroquine apart from the probable role of chq and hcq as antiviral agents their mechanisms of action are not fully understood and it was demonstrated that they have multiple effects on mammalian cells ace2 is known to be a cell receptor for sarscov the high similarities of the amino acid sequences and predicted protein structures of the receptorbinding domain of sarscov2 and sarscov suggest that sarscov2 may efficiently use human ace2 as a receptor for cellular entry and employ the cellular serine protease tmprss2 for s protein priming zhou confirmed that sarscov2 used the ace2 receptor to enter cells and did not use other coronavirus receptors such as aminopeptidase n apn and dipeptidyl peptidase dpp4 so the primary mechanism by which cell infection is prevented by these drugs may be at the stage of binding with the surface receptor and endosomemediated viral entry two independent in vitro studies confirmed that chq inhibits the replication of the sarscov chloroquine inhibits the early stage of sarscov replication in vero e6 cells with a effective concentration of ± μml the antiviral activity of chq was indicative at the time point at virus attachment or penetration vincent established that the drug might interfere with terminal glycosylation of the cellular receptor ace2 when chq was added prior to infection the impairment of terminal glycosylation of ace2 may result in reduced binding affinities between ace2 and sarscov spike protein and negatively influence the initiation of sarscov infection when chq or nh4cl were added after infection these agents could rapidly raise the ph and interrupt ongoing fusion events between the virus and endosomes thus inhibiting the infection on the basis of in vitro experiments they suggested that the primary mechanism by which infection was prevented was the poor affinity of sarscov spike protein to underglycosylated ace2 in vitro studies with hiv infected cells also identified that inhibition of glycosylation might be a possible mechanism of action of chq chq inhibits hiv replication at a postintegration stage resulting in the production of immature virions it was demonstrated that the sole mechanism explaining the antihiv activity of chq was a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2g12 epitope of gp120 according to in vitro results the antiretroviral effects of chq are attributable to the inhibition of viral p glycosylation these effects appeared to be specific since the chq concentrations effective in vitro neither affected any other step in hiv1 replication nor were cytotoxic thus there is direct evidence that chq is an inhibitor of glycosylation of gp120 and these alterations may be responsible for the decreased infectivity of hiv grown in the presence of chloroquine when added after the initiation of infection these drugs might affect the endosomemediated fusion and subsequent virus replication sarscov pseudoviruses may enter cells via receptordependent clathrin and caveolaeindependent phsensitive endocytosis likely through a process involving lipid rafts a later study however suggests that the entry of coronaviruses into the host cells occurs through clathrinmediated endocytosis murine hepatitis virus mhv a prototypic member of the cov family requires trafficking to lysosomes for proteolytic cleavage at the fp proximal position of its spike s protein membrane fusion to occur many authors indicated that s protein cleavage is an important step for fusion activity and subsequent internalization of the sarscov virus genome into cells [“] adding chq prior to infection results to inhibition of endosome maturation and strongly decreased mhv infection and fusion which was not observed when the drug was added at hpi indicating that the compound mainly affects mhv entry chloroquine is a weak base that is known to increase the ph of lysosomal and transgolgi network tgn vesicles leading to the dysfunction of enzymes necessary for proteolytic processing and posttranslational modifications of newly synthesized viral proteins chloroquine is able to prevent the processing of prm protein to m protein in flavivirusinfected mammalian and mosquito cells by raising the ph of the postgolgi vesicles in which this cleavage occurs as a result virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prm protein rather than m protein and this reduced the infectivity of the virus the chloroquinemediated rise in endosomal ph modulates iron metabolism in a variety of cell types decreasing in intracellular concentration of iron affects the function of several cellular enzymes involved in pathways leading to the replication of cellular dna and to the expression of different genes [“] autophagy is a lysosomedependent degradative pathway chq and its analogue hcq are known clinically relevant autophagy inhibitors chq is a weak base that inhibits lysosomal acidification which prevents the fusion of autophagosomes with lysosomes and subsequent autophagic degradation inhibition of autophagy with chq stimulates superoxide generation ubiquitinconjugated protein accumulation and apoptosis in a colon cancer xenograft model chq treatment clearly inhibited autophagy in mouse lung and efficiently ameliorated acute lung injury and dramatically improved the survival rate in mice infected with live avian influenza a h5n1 virus h5n1 virusinduced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase akt the tumor suppressor protein tsc2 and the mammalian target of rapamycin and autophagyblocking agents might be useful as prophylactics and therapeutics against infection of humans by the h5n1 virus furthermore prentice suggested that authophagy was induced by the coronavirus mouse hepatitis virus mhv and was required for formation of double membranebound mhv replication complexes which significantly enhanced the efficiency of replication replication of the virus was impaired in atg5 knockout embryonic stem cells the same authors also examined the sarscovs and found out similar colocalization of the key viral replication proteins with endogenous lc3 a protein marker for autophagosome it could be assumed that autophagy inhibitors like chq could inhibit virus replication at present the exact role of autophagy in cov infection remains debatable and there is much evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sarscovs merscovs and possibly sarscov2 the antiinflammatory properties of chqhcq should also be considered several studies have suggested that multiple an failure biomedicinepharmacotherapy13120201106684 0chas not yet been identified in sarscov2 infected patients and probably multiple pathways could be involved fig conclusion the sarscov2 is the cause of the coronavirus disease covid19 that has been declared a global pandemic by the world health anization who in despite some clinical characteristics that differentiate covid19 from sarscov merscov and seasonal influenza the pathogen sarscov2 has the same phylogenetic similarity to sarscov and mers cov most of the encoded proteins exhibited high sequence identity between sarscov2 and the related batderived coronaviruses batslcovzc45 and batslcovzxc21 a notable difference was a longer spike protein encoded by sarscov2 compared with the bat sarslike coronaviruses sarscov and merscov in addition sarscov2 was distinct from sarscov in a phylogeny of the complete rnadependent rna polymerase rdrp gene moreover the receptorbinding domain of sarscov2 which directly engages the ace2 receptor for cell entry was more closely related to those of sarscovs “ amino acid identity since the outbreak researchers have released many agents that could have potential efficacy against covid19 there is currently no clinically proven specific antiviral agent available for sarscov2 infection like sarscov and merscov certain agents like chloroquine hydroxychloroquine lopinavirritonavir and remdesivir are being used in ongoing clinical trials all over the world with hopes to further delineate their role in the treatment and prophylaxis of covid19 furthermore due to their availability and using for decades and proven safety records it is reasonable to suggest that they may be appropriate for treatment of covid19 remdesivir an adenosine analogue with wellstudied mechanism of action in cov infections can target the rnadependent rna polymerase and block viral rna synthesis and has been a promising antiviral drug antiviral studies in cell culture and animal models the available human safety data as well as the clear mechanism of action characterize rdv as a directacting antiviral since some authors found that lopinavir“ritonavir treatment did not significantly accelerate clinical improvement hence antiviral effects as an inhibitor of the sarscov main 3cl protease should be further investigated although chq and hcq are wellknown drugs for the treatment of k uzunova observed in fatal cases are most likely associated with not only the direct viral infection and destruction of susceptible cells eg endothelial cells but also the effects of proinflammatory cytokines chemokines and other mediators released from infected and activated cells such as monocytes and macrophages the clinical worsening of individuals with sars in week is apparently unrelated to uncontrolled sars coronavirus replication but may be related to immunopathological damage another study reveals that the presence of viral elements within endothelial cells and the accumulation of inflammatory cells led to endotheliitis in several ans as a direct consequence of viral involvement and to host inflammatory response moreover chq has immunomodulatory effects suppressing the productionrelease of tumour necrosis factorα and interleukin6 which mediate the inflammatory complications of several viral diseases chloroquinehcq was reported to inhibit the production of soluble mature tnf in macrophage cell line inhibit tnfα receptor in human histocytic u937 cells inhibit tnfα ifnγ and il6 in peripheral blood mononuclear cells pbmc reduce tnfα production and lipopolysaccharide lpsinduced il1 release in human monocytic cells it is suggested that chq exerts antiinflammatory and immunomodulatory effects predominantly by pretranslational and nonlysosomotropic mechanisms chloroquineinduced inhibition of tnf and il6 production is not mediated through a lysosomotropic mechanism and chloroquine probably acts on tnf secretion by disrupting iron homeostasis besides its antiviral activity and due to its suppressive effects on the productionrelease of tnfα and interleukin chqhcq may be effectively used in the treatment of viral infections characterized by symptoms associated with inflammatory processes andor immunehyperactivation antiinflammatory effects of chq remain poorly understood regulation of proinflammatory cytokines chq can also act on the immune system through cell signaling chq inhibits the activation of p38 mapk in hcov229einfected cells and evokes the activation of erk independently of infection these results suggested that chq may inhibit cov replication by suppressing the p38 activation additionally chq strongly inhibited phosphorylation of mitogenactivated protein kinase mapk p38 and to a lesser extent cjun nterminal kinase and extracellular signalregulated kinase ½ chloroquine could also inhibit innate immune responses trough downregulation of tlr9 signaling pathways requiring endocytosis and acidification of endosomes within plasmacytoid dendritic cells pdcs and act as novel antagonists to chemokine receptor cxcr4 that suppress pancreatic cancer cell proliferation on the other hand another hypothesized mechanism of chq is via the inhibition of antigen degradation and improving the crosspresentation efficiency of dcs in vitro in vivo evidence suggested that a short course of treatment with chq followed by a booster dose of a soluble antigen immunization can efficiently enhance human cd8 t cell responses and single vaccination with inactivated influenza virus combined with chloroquine treatment elicits a higher t cell immunity in mice regulation of nlrp3 inflammasome activation may offer a promising therapeutic approach by inhibiting or slowing down the process of acute respiratory distress syndrome hcq is a known nlrp3 inhibitor and its potential clinical effectiveness is certainly based on the downregulation of il1 expression the major proinflammatory cytokine interleukin1beta il1 is elevated in plasma from hospitalized covid19 patients and its associated signaling pathway seems to drive sarscov2 pathogenicity il1 secretion is primarily initiated by inflamm Answer:

Colon_Cancer

authorsann c gregory olivier zablockiahmed a zayed allison howellbenjamin bolduc matthew b sullivancorrespondencesullivan948osueduin briefat least studies to date have looked atthe human gut virome but with limitedconsistency gregory and zablocki curate and aggregate these data toprovide a systematic virome databaseuse it to assess study biases globalecological patterns and show howviromes evolve throughout the humanlifespanhighlightsd assembly of gut metagenomes from studiesexposed viral populationsd interstudy analyses reveal strong study biases at the viralpopulationleveld viral population detection was higher in bulk versus vlpenriched metagenomesd gut viral diversity is agedependent across healthy westernpeoplegregory cell host microbe “november ª elsevier inc101016jchom202008003ll 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcethe gut virome database reveals agedependentpatterns of virome diversity in the human gutann c gregory145 olivier zablocki134 ahmed a zayed13 allison howell1 benjamin bolduc13and matthew b sullivan12361department of microbiology ohio state university columbus oh usa2department of civil environmental and geodetic engineering ohio state university columbus oh usa3center of microbiome science ohio state university columbus oh usa4these authors contributed equally5present address department of microbiology and immunology rega institute for medical research vibku leuven center formicrobiology leuven belgium6lead contactcorrespondence sullivan948osuedu101016jchom202008003summarythe gut microbiome profoundly affects human health and disease and their infecting viruses are likely asimportant but often missed because of reference database limitations here we built a human gut viromedatabase gvd from viral p or microbial metagenomes from individuals representing countries assess its effectiveness and report a metaanalysis that reveals agedependent patternsacross healthy westerners the gvd contains unique viral populations approximately speciesleveltaxa and improves average viral detection rates over viral refseq and imgvr nearly 182fold and 26foldrespectively gvd metaanalyses show highly personalized viromes reveal that interstudy variability fromtechnical artifacts is larger than any ˜˜disease™™ effect at the population level and document how viral diversitychanges from human infancy into senescence together this compact foundational resource these standardization guidelines and these metaanalysis findings provide a systematic toolkit to help maximize ourunderstanding of viral roles in health and diseaseintroductionthe human gut microbiome is now thought to play an integralrole in health and disease clemente gilbert lynch and pedersen schmidt persistent alterations in the structure diversity and functionof gut microbial communities”dysbiosis”are increasinglyrecognized as key contributors in the establishment and maintenance of a growing number of disease states frank human microbiome project consortium qin including obesity turnbaugh andcancer yoshimoto gut dysbiosis can developfrom complex interplays between host cognate microbiotaand external environmentalfactors mirzaei and maurice shreiner within the gut microbial consortium the bacteriome has been the most extensively studied where significant shifts in population dynamics havebeen observed between healthy and diseased individualszhang however emerging views mirzaei andmaurice ogilvie and jones tetz suggest that the gut virome plays an important role in homeostatic regulation and disease progression through multipleinteraction paths with the cooccurring bacteriome and evendirectly with human immune system components keen anddantas the first step in studying viruses in complex communities isbeing able to detect them problematicallyidentifying viralsequences in large mixedcommunity datasets is notoriouslychallenging because viruses lack a universal viral markerrohwer and edwards as opposed to bacterial 16srrna for example human gut microbiome studies have mostcommonly used sequence homology searches with blast orkraken wood and salzberg against ncbi viral referencesequence database refseqwwwncbinlmnihgovgenomeviruses aclame a mobile element genome database [leplae ] or custom hidden markov modelhmm databases eg prokaryotic virus orthologous groups[pvogs] [grazziotin ] although there is now a suiteof virus identification tools available including deepvirfinderren marvel amgarten vibrantkieft and virsorter roux only the latterhas been used in the human gut microbiome literature to dateand all are dependent upon reference genome databases tosome degree further once viruses are detected there is nostandard applied on how viral contigs translate into ˜˜species™™level sequences that are to be used as a ˜˜working™™ virus poolcell host microbe “ november ª elsevier inc 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcefigure overview of studies comprising the gut virome database gvdglobal heatmap of the world showing the number and distribution of studies per country each white box represents a different continent and contains informationabout the number of individuals sampled represented by the filled human pictograms and percentage of the total gvd sequencing effort for vlpenriched red piecharts and bulk metagenomes yellow pie charts of each country studied within that continentsee also table s1for downstream analysis the lack of viral analysis standardscould partly explain the estimated highly variable “mirzaei and maurice rates of virus detection betweenstudies in addition factors such as differences in sample processing shkoporov broad underrepresentationof viral genome space in reference databases wang lack of culturable host gut microbes wang and interindividual variation add further variability shkoporov further although viral reference datasets are beinggenerated at unprecedented rates roux thesenew data are rarely incorporated for crosscomparisons whichwould ‚ate virus novelty in new datasets andor leaves manyvirus sequences undetected in response to these challengesand to enable viromecentric research in health and diseasewe sought to establish a comprehensive easyaccess databasededicated to human gut viruses this effort would enable futuregut microbiome research by augmenting virus detection andhelping establish processing standards for human gut viruseshere we collected and curated human gut metagenomes previously studied for viruses and published as ofoctober to build the human gut virome database gvd evaluated its utility against the best available databases national center for biotechnology information [ncbi] viral refseqand integrated microbial genomevirus [imgvr] [paezespino ] and used it in metaanalyses to assess methodological effects and establish largescale patterns of gut viromediversity during the course of the human lifespan the gvd™s human gut metagenomic datasets derive from studiesand encompass individuals from countries that originated either from viruslike ps vlps or whole microbialcommunities bulk as well as several datasets that includedrna sequencing data derived from vlps all these datasetswere previously studied for viruses but by using highly variable cell host microbe “ november methods for the gvd we in silico reprocessed these data toidentify viral populations and rigorously remove contaminationthis gvd resource is now available on ivirus bolduc 2017a and will be regularly updatedresults and discussionthe gvd contains unique viral populationsdominated by phagesto build the gvd metagenomic samples from individuals were processed from datasets publicly available as ofdecember n see table s1 along with one unpublished dataset where access was granted prior to publicationthese studies represent tbp of sequence data derivedfrom a spectrum of gut virome study areas including thefollowing healthy gut viromes of infants lim reyes and adults ly manrique minot rampelli aswell as individuals experiencing fecal microbiota transplantfmt for autism and clostridium difficile infection broecker chehoud draper kang zuo ‚ammatory bowel disease ibd fernandes norman pe´ rezbrocal zuo hiv infection monaco type i and ii diabetes aiemjoy kramna´ ma zhao malnutrition reyes and chronic fatigue syndrome giloteaux and hypertension han see table s1 these datasets were globally distributed figure however most of the studies originated from the united states of gvd studies and the highest number of sampled individuals and base pairs bps sequenced came from chinese cohorts of individuals and bp sequenced in the gvd all 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003resourcereads from both bulk and vlp metagenomes and ofthe gvd respectively were processed consistently assembledinto contigs and virallike sequences were identified by usingthree independent methods figure s1 see method detailseach lowscoring prediction was validated by crosscomparisons between methods and subsequently evaluated for falsepositives by detecting whether candidate virus sequences contained benchmarking universal singlecopy orthologs buscorelated host singlecopy genes sima˜ o and for thepresence of viral family proteins vpfspaezespino confidence scores associated with each virus detectionmethod and potential false positives are reported in table s2 toavoid duplicate viral genomes andor partial virus genomesacross the datasets contigs were dereplicated by clusteringsequences according to percentage of average nucleotide identity ani and sequence length multiple reports brum 2015a duhaime and sullivan duhaime gregory 2019a roux have revealed that ani was a suitable threshold for defining a set of closelyrelated discrete ˜˜viral populations™™ followon studies suggestthat this cutoff establishes populations that are largely concordant with a biologically relevant viral species definition bobayand ochman gregory 2019a using this clustering strategy we identified highly variable numbers of uniqueviral populations per study range “ viral populationsmean figure s2a the gvd comprises viralcontigs and viral populations r5 kb or r15 kb and circular contigs n50 bp l50 bp and mostlybacteriophages of gvd for context ncbi™s viral refseq v98 released january database holds viruses of eukaryotes bacteria and archaea from all environments combined specifically for bacteriophages the gvdcontains 12fold more than the entire set of cultured phage isolates in viral refseq to date thus the gvd greatly augments therepertoire of known phages in the human gut importantly due toa lack of negative controls across out of the studies in thegvd there is a chance that some of the viral populationsincluded in the gvd might result from contamination thispaucity of negative controls is currently a limitation to gut viromestudiestaxonomically of gvd viral populations are bacterialviruses ie phages are eukaryotic viruses and are archaeal viruses figure 2a the eukaryotic viruseswere taxonomically diverse from families dominated by singlestranded dna ssdna families anelloviridae genomoviridae and circoviridae all of which havebeen previously reported in the datasets underlying the gvdmonaco with the exception of genomoviridaethree singlestranded positivesense rna virus families weredetected table s3 represented by viral populations of the gvd the human picornaviridae was the most represented parechoviruses coxsackievirus cosaviruses enterovirus and hepatovirus along with plant or fungal viruses ofthe alphaflexviridae and virgaviridae and one putative memberof cruliviridae detection of plant viruses has been reportedbefore zhang and is likely the result of transient passage through dietary habits human picornaviruses associatedwith gastrointestinal tract disorders were to be expected andmost derived from a cameroonian patient cohort selected forllgastroenteritis symptoms in which the study design includedrna sequencing yinda the low number of recovered rna viruses of the gvd see tables s2 and s3 inthe gvd might stem in part from having a few studies out of that included viral rna sequencing more importantly thelikely biggest factor contributing to low rna virus detection isthat de novo rna virus identification method development isan ongoing effort shi starr such thatrna virus diversity in gut viromes and generally in viral metagenomes is likely vastly undersampled and that our detection islimited to homology to wellcharacterized pathogens zhang among the phages did not have internationalcommittee on taxonomy of viruses ictv classification and theremaining fraction comprised of doublestranded dna dsdnatailed phage families siphoviridae myoviridae podoviridae andackermannviridae microviridae and inoviridae see table s2twentyfour unknown archaeal viral populations were detectedbut none with close genome andor gene homology to any of theclassified archaeal viruses notably our naive viral taxonomicassignments using ˜˜a majorityrules approach™™ see method details led to taxonomic assignments that recent literature hasshown are erroneous and due to methodological artefactssuch as phycodnaviridae and mimiviridae sutton so we manually removed such taxa thus given that most ofthe viral populations are represented by fragments of their genomes taxonomic assignments using the ˜˜a majorityrulesapproach™™ willimprove and be refined as more completegenome representatives are sequenced and assembled nonetheless the high number of unclassified phages likely resultsfrom the underrepresentation of human gut phages in referencedatabases and further highlights how much viral diversity remains to be characterized in the human gutto fill this phage and archaeal virus taxonomic classificationgap we used an extensively validated adriaenssens bolduc 2017b jang bin genomebased genesharing network strategy that de novo predictsgenuslevel groupings ˜˜viral clusters™™ [vcs] from viral population data a network figure 2b computed from gvdphage genomes only those kb in length of gvd and reference phage genomes from ncbi viral refseqversion revealed vcs of these vcs were exclusively composed of gvd genomes viral genomes or 18 of gvd genomes whereas vcs contained genomesfrom both refseq and the gvd viral genomes or 18 ofgvd genomes and vcs were exclusively composed of refseq taxa thus the gvd augments the current number of ictvrecognized phage genera approximately 35fold although notexplored here given that our goals focused on taxonomic classification the shared protein content within and between vcscalculated in our network analyses could be used to guideqpcr assays for nextgeneration sequencing validationmonaco and kwon andor tracking of viruses at eitherthe viral population or genera level under changing conditionskramna´ next we sought to link the gvd phage and archaeal viralpopulations to their hosts by using in silico strategies seemethod details in total we were able to identify the hostsdown to the microbial taxonomic family genome taxonomyparks of 18database [gtdb] taxonomycell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcefigure the gut virome database gvda pie charts showing the number of bacteriophages eukaryotic viruses and archaeal viruses in the gvd center and their familial taxonomic composition by thebacteriophages left and the eukaryotic viruses rightb genesharing taxonomic network of the gvd including viral refseq viruses v88 refseq viruses are highlighted in red every node represents a virus genomewhereas connecting edges identify significant genesharing between genomes which form the basis for their clustering in genuslevel taxonomyc concentric pie chart showing the number of annotated bacterial host phyla inner and family outer of the gvd viruses host taxonomy follows thegtdb database taxonomic classifications and putative host information per each viral population is listed in table s2 see also figures s1 and s2 and tables s2s3 and s6n of the viral populations see table s2 the mostcommon identifiable hosts figure 2c across gvd viral populations belonged to the bacterial phyla firmicutes gtdb firmicutes firmicutes_a and firmicutes_c combined andbacteroidetes gtdb bacteroidota consistent withour knowledge that firmicutes and bacteroides are the mostprominent bacterial phyla in the human gastrointestinal tracteckburg notably firmicutes typically outnumberbacteroidetes in unhealthy individuals with metabolic anddigestive disorders broecker chehoud ley nicholson norman ott zhao and gvd metagenomes are biased toward unhealthy individuals of themetagenomes comprising of the bps sequenced whichmight account for the increased firmicutes viral populationsin gvdthe gvd significantly improves virus detection overcurrent viral genome databasesto assess the value of the gvd we quantitatively evaluated virusidentification sensitivity between multiple databases bycomparing the number of viral populations identifiable by readrecruitment against gvd ncbi™s viral refseq v96 doe™s imgvr v4 paezespino and the individual virome databases from each study figure see method details to controlfor assembly improvements since the original metagenome andor virome datasets were published for the latter we individuallyassembled the original viromes into viral populations for read cell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003resourcellfigure gvd as a reference database increases viral population detectionboxplots showing median and quartiles of the number of viral populations detected per study using the individual virome viral refseq v96 jgi imgvr v4 vd databases all pairwise comparisons were performed by using mannwhitney u tests nonsignificant p values are denoted as ˜˜ns™™see also figure s3 and table s4recruitment ncbi viral refseq was the most commonly usedviral genome database across the studies surveyed here beingused in of studies where the specific database used wasdocumented information on the genome database used was unavailable for three studies see table s1 and hosted virusgenomes already dereplicated as of v96 november usedhere in comparison the imgvr database was not documented as being used by any of the studies gathered despitethe latest release v4 july used in this study containingnearly two orders of magnitude more virus genomes andgenome fragments virus contigs though not dereplicated for comparison purposes to the gvd see method details we dereplicated the imgvr contigs the same way aswe did the gvd to obtain viralpopulationlevel genomes thisyielded viral populations for the imgvr databasein out of the total studies tested figure the gvdenabled the detection of significantly more viruses than viral refseq v96 mannwhitney u tests p [average] ± [standard deviation]fold increase and individual viromesmannwhitney u tests p 6fold ± 40fold increasenotably the proportion of the metagenome mapping to thegvd was highly variable between studies figure s2b and asexpected a higher proportion of vlpenriched metagenomesmapped to the gvd than did bulk metagenomes figure s2cthere was a single study reyes in which no viruseswere detected see method details in all databases queried inthis analysis in comparison to imgvr we detected more viruses with the gvd in all studies from total of whichwere in a significant manner mannwhitney u tests p 26fold ± 21fold increase figure five of the remaining fourteen studies had too low of a sample size andor number of detected viruses to statistically compare the gvd and imgvradditionally we tested the ability of the gvd to increase thenumber of viral populations detected in a study not included inthe gvd clooney figure s3a we saw similar results and gvd significantly outperformed viral refseq v96 andthe individual virome while having a nonsignificant higher median number of viral populations detected than imgvrwhen we considered the number of reads that recruitedacross the different databases across all studies significantlymore reads mannwhitney u tests p were recruited tothe gvd than to any other database across out of the studies figure s3b after gvd imgvr was the next best performing database for viral detection in the human gut given thatour tests showed an average of 64fold ± 120fold increase overviral refseq mannwhitney u tests p imgvr was expected to surpass viral refseq because it aggregates both cultivated reference virus genomes from refseq prophages and uncultivated virus genomes andorfragments from many environments including multiple humanbody sites paezespino overall the significant increase in virus detection by the gvd over other databases highlights the low representation of gut viruses in refseq and thusdemonstrates the value of the gvd for sequencebased virusidentification in human gut microbiome datasets thus giventhat the gvd significantly improves viral detection over currentviral genome databases we used the gvd as the database forall remaining analyses in this studythe human gut virome is highly person specificin light of the current hypothesis of a ˜˜core™™ gut virome manrique we were first curious whether any gvd viralcell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcefigure individual viromes study databases and crossstudy comparisonsshown at the top left is a hierarchically clustered heatmap showing the number of viral populations shared within and between studies clustered into four groupsi“iv viral population cooccurrence network per individual within each study per group shown on the bottom right is a hierarchically clustered heatmapshowing the number of viral genera shared within and between studies clustered into three groups viral genus cluster cooccurrence networks per metagenomewithin each study per group colored dots and pictograms next to study names in heatmaps represent metagenome type and a common disease studies acrossall studies in gvd respectivelysee also figure s4population was found across a high percentage or all metagenomes in the gvd on average ± average ± sd range“ viral populations were detected per metagenome butnot a single viral population was found across all metagenomesin fact the most ubiquitous viral population in the gvd wasfound in only of the metagenomes viral populationsoccurred in more than of the metagenomes and most or of the viral populations were only sporadicallydetected at all of the metagenomes figures s4a ands4b table s4 further we specifically looked at the prevalenceof crassphages a wellrecognized multigenera family ofphages known to be widespread in gut viromes guerin figure s4b in total we identified crassphage populations see method details of which had genomes kb cell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003resourcellfigure vlpenriched vlp and bulk metagenomes comparisons for studying viruses in the human guta“c boxplots showing median and quartiles of the number of assembled contigs per base pair sequenced per study a of vlp and bulk metagenomes b ofvlp metagenomes with and without mda and c of the different vlpenrichment methodologies across the studies outlier dots were removed from plot c tobetter show the range of values the n value above each box plot represents the number of studies using each vlpenrichment methodd scatter plot with a linear regression line showing the number of assembled viral contigs per bp sequenced per study with vlp and bulk metagenome studiesidentified by different colors in the inset is a venn diagram showing the number of gvd viral populations that originated from vlp or bulk or both types ofmetagenomese boxplots showing median and quartiles of the number of viral populations detected per bp sequenced per individual of vlp and bulk metagenomesf boxplots showing median and quartiles of the number of assembled contigs per bp sequenced top left and the median contig length top right for vlp andbulk metagenomes processed for the same samples in the shkoporov bottom connected dot plot showing the number of viral populations detectedper bp sequencedby using vlp and bulk metagenomes for each individual in the shkoporov study all pairwise comparisons were performed by usingmannwhitney u tests nonsignificant p values are denoted as ˜˜ns™™see also figure s4that clustered into vcs ie genuslevel grouping bygenomebased genesharing networks jang bin although togetherthese crassphage populations areubiquitous across the gvd samples there was not one crassphage viral population found universally and the most widespread crassphage population occurred in only of samplesthese findings support the mounting evidence for highly personal gut viromes as recently highlighted in twins morenogallego and in ten healthy adults during a yearlongmonitoring period shkoporov although the latterstudy pointed to the potential of a core virome at higher taxonomic levels we failed to recover any universally shared viralvcs approximately genus level taxonomy figures s4c ands4d given that the most ubiquitous vc was only present in of the metagenomes that same study suggesting thepotential of a highertaxon core only looked at ten healthywestern adults and the metagenomes in the gvd representeda diversity of people from different geographical regions andages thus further studies are necessary to resolve whether acore virome does exists at higher taxonomic levelsvaried processing methodology prevents populationlevel bdiversity interstudy comparisonsnext given a systematically processed gvd and its demonstrably improved virus detection capability we sought to determine whether global clustering patterns would emerge via a gvdbdiversity betweensample changes in population composition metaanalysis to this end we performed population cooccurrence analyses at two levels of resolution per study andacross metagenomes within studies and then evaluated whatlevel of metadata best captured the resultant variation methodology disease state etc to assess populationoverlap between studies we counted the number of gvd viral populationsthat recruited reads within and between different gvd studiescell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llie the same viral population was detected in metagenomein both studies compared we expected studies exploringsimilar disease states would share the greatest number of viralpopulations however hierarchical clustering of the studies onthe basis of the number of shared viral populations revealedthat this was rare and mostly not the case and studies exploringthe viromes of diseased individuals eg ibd clostridium difficile infection and diabetes did not cluster together figure top left”heatmap instead we saw that the different studies hierarchically clustered into four groups i“iv and that this clustering was weakly driven around metagenome type many bulkmetagenomes clustered together into group iiibecause the gvd studies did cluster into four distinct groupsfigure top left”heatmap we next tested whether any metadata best captured the resultant variation across the metagenomes within each study within each group using an inversecovariance analysis ie combined partial correlations acrossshared viral population between metagenomes to subclusterthe different metagenomes across the studies in each groupwe found that the metagenomes within a study subclusteredtogether irrespective of geographical origin health status andor diet figure top left”networks notably the group iii metagenomes derived mostly from bulk metagenomes were moreclosely subclustered but they still subclustered strongly bystudy this perstudy subclustering implies that even withinthese grouped studies metagenomes from different studiesare not comparable because the interstudy variation is drivenby methodological impacts these results reveal that althoughmethodology does not affect the number of viral contigs recovered it does affect the recovered types of viruses see upcomingfindings comparing methodological effects interestingly whenwe looked at genuslevelie vclevel cooccurrence wesaw that there are still strong groupings a“c at the study levelbut within each group metagenomes across these studiesshare many vcs figure bottom right thus bdiversitymetaanalysis across all studies exploring the effect of ˜˜disease™™across gvd studies is not possible at the population level butwithin similarly processed studies it might be possible at thegenus levelto enrich or not to enrich viruses recovered from bulkversus viruspenriched metagenomesfrom a pragmatic point of view we next wondered whethergvd datasets could inform experimental design specifically cell host microbe “ november resourcefigure more gut viruses are temperatephages than in the soil and oceanspie charts showing the percentages of temperatephages found in the human gut gvd datasetsoils isogenie dataset and oceans globaloceans viromes datasetto study viruses is sequencing effort better put into metagenomes of bulk or purified vlps the gvd™s gut metagenomes are roughly evenly divided acrossthese two metagenome types with bulkand vlp metagenomes contributing tbp 18 of gvd and tbp 18 of gvd of data respectively although most samplesonly have one or the other data one study samples shkoporov provided both bulk and vlp metagenomesfor sampleswe first assessed whether there was a difference in de novoviral recovery between vlp and bulk metagenomes figure 5awe measured viral recovery by using the number of viral contigs kb or kb and circular in length ie not dereplicatedviral populations assembled per bp sequenced per study giventhat the viral contigs assembled from samples within the samestudy are often pooled these analyses revealed no significantdifference mannwhitney u test p in the number of viralcontigs assembled per bp sequenced between vlp and bulkmetagenomes which contrasts viralrecovery results frompermafrost soils where vlp metagenomes outperform bulkmetagenomes by 2fold trubl however viral recovery from the gvd™s vlp metagenomes was heterogeneous sowe evaluated how vlp methodology affected viral recoveryfirst although multiple displacement amplification mdaisknown to provide nonquantitative metagenomic datasets withboth systematic and stochastic biases solonenko yilmaz we found no significant difference mannwhitney u test p in viral recovery between nonmdaand mdatreated metagenomes figure 5b nonetheless itwas notable that mdatreated vlp studies were significantly enriched in eukaryotic ssdna viruses mannwhitney u tests p a known bias of mda figure s5a second we tested theeffect of vlp enrichment strategies which ranged fromremoving human and bacterial cells to enrich for vlps centrifugation filtration cscl gradients and nucleases to concentrating the vlps centricon concentration and peg precipitationagain we found no significant difference in the number of viralcontigs recovered kruskalwallis test p across

cancer7533

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: authorsann c gregory olivier zablockiahmed a zayed allison howellbenjamin bolduc matthew b sullivancorrespondencesullivan948osueduin briefat least studies to date have looked atthe human gut virome but with limitedconsistency gregory and zablocki curate and aggregate these data toprovide a systematic virome databaseuse it to assess study biases globalecological patterns and show howviromes evolve throughout the humanlifespanhighlightsd assembly of gut metagenomes from studiesexposed viral populationsd interstudy analyses reveal strong study biases at the viralpopulationleveld viral population detection was higher in bulk versus vlpenriched metagenomesd gut viral diversity is agedependent across healthy westernpeoplegregory cell host microbe “november ª elsevier inc101016jchom202008003ll 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcethe gut virome database reveals agedependentpatterns of virome diversity in the human gutann c gregory145 olivier zablocki134 ahmed a zayed13 allison howell1 benjamin bolduc13and matthew b sullivan12361department of microbiology ohio state university columbus oh usa2department of civil environmental and geodetic engineering ohio state university columbus oh usa3center of microbiome science ohio state university columbus oh usa4these authors contributed equally5present address department of microbiology and immunology rega institute for medical research vibku leuven center formicrobiology leuven belgium6lead contactcorrespondence sullivan948osuedu101016jchom202008003summarythe gut microbiome profoundly affects human health and disease and their infecting viruses are likely asimportant but often missed because of reference database limitations here we built a human gut viromedatabase gvd from viral p or microbial metagenomes from individuals representing countries assess its effectiveness and report a metaanalysis that reveals agedependent patternsacross healthy westerners the gvd contains unique viral populations approximately speciesleveltaxa and improves average viral detection rates over viral refseq and imgvr nearly 182fold and 26foldrespectively gvd metaanalyses show highly personalized viromes reveal that interstudy variability fromtechnical artifacts is larger than any ˜˜disease™™ effect at the population level and document how viral diversitychanges from human infancy into senescence together this compact foundational resource these standardization guidelines and these metaanalysis findings provide a systematic toolkit to help maximize ourunderstanding of viral roles in health and diseaseintroductionthe human gut microbiome is now thought to play an integralrole in health and disease clemente gilbert lynch and pedersen schmidt persistent alterations in the structure diversity and functionof gut microbial communities”dysbiosis”are increasinglyrecognized as key contributors in the establishment and maintenance of a growing number of disease states frank human microbiome project consortium qin including obesity turnbaugh andcancer yoshimoto gut dysbiosis can developfrom complex interplays between host cognate microbiotaand external environmentalfactors mirzaei and maurice shreiner within the gut microbial consortium the bacteriome has been the most extensively studied where significant shifts in population dynamics havebeen observed between healthy and diseased individualszhang however emerging views mirzaei andmaurice ogilvie and jones tetz suggest that the gut virome plays an important role in homeostatic regulation and disease progression through multipleinteraction paths with the cooccurring bacteriome and evendirectly with human immune system components keen anddantas the first step in studying viruses in complex communities isbeing able to detect them problematicallyidentifying viralsequences in large mixedcommunity datasets is notoriouslychallenging because viruses lack a universal viral markerrohwer and edwards as opposed to bacterial 16srrna for example human gut microbiome studies have mostcommonly used sequence homology searches with blast orkraken wood and salzberg against ncbi viral referencesequence database refseqwwwncbinlmnihgovgenomeviruses aclame a mobile element genome database [leplae ] or custom hidden markov modelhmm databases eg prokaryotic virus orthologous groups[pvogs] [grazziotin ] although there is now a suiteof virus identification tools available including deepvirfinderren marvel amgarten vibrantkieft and virsorter roux only the latterhas been used in the human gut microbiome literature to dateand all are dependent upon reference genome databases tosome degree further once viruses are detected there is nostandard applied on how viral contigs translate into ˜˜species™™level sequences that are to be used as a ˜˜working™™ virus poolcell host microbe “ november ª elsevier inc 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcefigure overview of studies comprising the gut virome database gvdglobal heatmap of the world showing the number and distribution of studies per country each white box represents a different continent and contains informationabout the number of individuals sampled represented by the filled human pictograms and percentage of the total gvd sequencing effort for vlpenriched red piecharts and bulk metagenomes yellow pie charts of each country studied within that continentsee also table s1for downstream analysis the lack of viral analysis standardscould partly explain the estimated highly variable “mirzaei and maurice rates of virus detection betweenstudies in addition factors such as differences in sample processing shkoporov broad underrepresentationof viral genome space in reference databases wang lack of culturable host gut microbes wang and interindividual variation add further variability shkoporov further although viral reference datasets are beinggenerated at unprecedented rates roux thesenew data are rarely incorporated for crosscomparisons whichwould ‚ate virus novelty in new datasets andor leaves manyvirus sequences undetected in response to these challengesand to enable viromecentric research in health and diseasewe sought to establish a comprehensive easyaccess databasededicated to human gut viruses this effort would enable futuregut microbiome research by augmenting virus detection andhelping establish processing standards for human gut viruseshere we collected and curated human gut metagenomes previously studied for viruses and published as ofoctober to build the human gut virome database gvd evaluated its utility against the best available databases national center for biotechnology information [ncbi] viral refseqand integrated microbial genomevirus [imgvr] [paezespino ] and used it in metaanalyses to assess methodological effects and establish largescale patterns of gut viromediversity during the course of the human lifespan the gvd™s human gut metagenomic datasets derive from studiesand encompass individuals from countries that originated either from viruslike ps vlps or whole microbialcommunities bulk as well as several datasets that includedrna sequencing data derived from vlps all these datasetswere previously studied for viruses but by using highly variable cell host microbe “ november methods for the gvd we in silico reprocessed these data toidentify viral populations and rigorously remove contaminationthis gvd resource is now available on ivirus bolduc 2017a and will be regularly updatedresults and discussionthe gvd contains unique viral populationsdominated by phagesto build the gvd metagenomic samples from individuals were processed from datasets publicly available as ofdecember n see table s1 along with one unpublished dataset where access was granted prior to publicationthese studies represent tbp of sequence data derivedfrom a spectrum of gut virome study areas including thefollowing healthy gut viromes of infants lim reyes and adults ly manrique minot rampelli aswell as individuals experiencing fecal microbiota transplantfmt for autism and clostridium difficile infection broecker chehoud draper kang zuo ‚ammatory bowel disease ibd fernandes norman pe´ rezbrocal zuo hiv infection monaco type i and ii diabetes aiemjoy kramna´ ma zhao malnutrition reyes and chronic fatigue syndrome giloteaux and hypertension han see table s1 these datasets were globally distributed figure however most of the studies originated from the united states of gvd studies and the highest number of sampled individuals and base pairs bps sequenced came from chinese cohorts of individuals and bp sequenced in the gvd all 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003resourcereads from both bulk and vlp metagenomes and ofthe gvd respectively were processed consistently assembledinto contigs and virallike sequences were identified by usingthree independent methods figure s1 see method detailseach lowscoring prediction was validated by crosscomparisons between methods and subsequently evaluated for falsepositives by detecting whether candidate virus sequences contained benchmarking universal singlecopy orthologs buscorelated host singlecopy genes sima˜ o and for thepresence of viral family proteins vpfspaezespino confidence scores associated with each virus detectionmethod and potential false positives are reported in table s2 toavoid duplicate viral genomes andor partial virus genomesacross the datasets contigs were dereplicated by clusteringsequences according to percentage of average nucleotide identity ani and sequence length multiple reports brum 2015a duhaime and sullivan duhaime gregory 2019a roux have revealed that ani was a suitable threshold for defining a set of closelyrelated discrete ˜˜viral populations™™ followon studies suggestthat this cutoff establishes populations that are largely concordant with a biologically relevant viral species definition bobayand ochman gregory 2019a using this clustering strategy we identified highly variable numbers of uniqueviral populations per study range “ viral populationsmean figure s2a the gvd comprises viralcontigs and viral populations r5 kb or r15 kb and circular contigs n50 bp l50 bp and mostlybacteriophages of gvd for context ncbi™s viral refseq v98 released january database holds viruses of eukaryotes bacteria and archaea from all environments combined specifically for bacteriophages the gvdcontains 12fold more than the entire set of cultured phage isolates in viral refseq to date thus the gvd greatly augments therepertoire of known phages in the human gut importantly due toa lack of negative controls across out of the studies in thegvd there is a chance that some of the viral populationsincluded in the gvd might result from contamination thispaucity of negative controls is currently a limitation to gut viromestudiestaxonomically of gvd viral populations are bacterialviruses ie phages are eukaryotic viruses and are archaeal viruses figure 2a the eukaryotic viruseswere taxonomically diverse from families dominated by singlestranded dna ssdna families anelloviridae genomoviridae and circoviridae all of which havebeen previously reported in the datasets underlying the gvdmonaco with the exception of genomoviridaethree singlestranded positivesense rna virus families weredetected table s3 represented by viral populations of the gvd the human picornaviridae was the most represented parechoviruses coxsackievirus cosaviruses enterovirus and hepatovirus along with plant or fungal viruses ofthe alphaflexviridae and virgaviridae and one putative memberof cruliviridae detection of plant viruses has been reportedbefore zhang and is likely the result of transient passage through dietary habits human picornaviruses associatedwith gastrointestinal tract disorders were to be expected andmost derived from a cameroonian patient cohort selected forllgastroenteritis symptoms in which the study design includedrna sequencing yinda the low number of recovered rna viruses of the gvd see tables s2 and s3 inthe gvd might stem in part from having a few studies out of that included viral rna sequencing more importantly thelikely biggest factor contributing to low rna virus detection isthat de novo rna virus identification method development isan ongoing effort shi starr such thatrna virus diversity in gut viromes and generally in viral metagenomes is likely vastly undersampled and that our detection islimited to homology to wellcharacterized pathogens zhang among the phages did not have internationalcommittee on taxonomy of viruses ictv classification and theremaining fraction comprised of doublestranded dna dsdnatailed phage families siphoviridae myoviridae podoviridae andackermannviridae microviridae and inoviridae see table s2twentyfour unknown archaeal viral populations were detectedbut none with close genome andor gene homology to any of theclassified archaeal viruses notably our naive viral taxonomicassignments using ˜˜a majorityrules approach™™ see method details led to taxonomic assignments that recent literature hasshown are erroneous and due to methodological artefactssuch as phycodnaviridae and mimiviridae sutton so we manually removed such taxa thus given that most ofthe viral populations are represented by fragments of their genomes taxonomic assignments using the ˜˜a majorityrulesapproach™™ willimprove and be refined as more completegenome representatives are sequenced and assembled nonetheless the high number of unclassified phages likely resultsfrom the underrepresentation of human gut phages in referencedatabases and further highlights how much viral diversity remains to be characterized in the human gutto fill this phage and archaeal virus taxonomic classificationgap we used an extensively validated adriaenssens bolduc 2017b jang bin genomebased genesharing network strategy that de novo predictsgenuslevel groupings ˜˜viral clusters™™ [vcs] from viral population data a network figure 2b computed from gvdphage genomes only those kb in length of gvd and reference phage genomes from ncbi viral refseqversion revealed vcs of these vcs were exclusively composed of gvd genomes viral genomes or 18 of gvd genomes whereas vcs contained genomesfrom both refseq and the gvd viral genomes or 18 ofgvd genomes and vcs were exclusively composed of refseq taxa thus the gvd augments the current number of ictvrecognized phage genera approximately 35fold although notexplored here given that our goals focused on taxonomic classification the shared protein content within and between vcscalculated in our network analyses could be used to guideqpcr assays for nextgeneration sequencing validationmonaco and kwon andor tracking of viruses at eitherthe viral population or genera level under changing conditionskramna´ next we sought to link the gvd phage and archaeal viralpopulations to their hosts by using in silico strategies seemethod details in total we were able to identify the hostsdown to the microbial taxonomic family genome taxonomyparks of 18database [gtdb] taxonomycell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcefigure the gut virome database gvda pie charts showing the number of bacteriophages eukaryotic viruses and archaeal viruses in the gvd center and their familial taxonomic composition by thebacteriophages left and the eukaryotic viruses rightb genesharing taxonomic network of the gvd including viral refseq viruses v88 refseq viruses are highlighted in red every node represents a virus genomewhereas connecting edges identify significant genesharing between genomes which form the basis for their clustering in genuslevel taxonomyc concentric pie chart showing the number of annotated bacterial host phyla inner and family outer of the gvd viruses host taxonomy follows thegtdb database taxonomic classifications and putative host information per each viral population is listed in table s2 see also figures s1 and s2 and tables s2s3 and s6n of the viral populations see table s2 the mostcommon identifiable hosts figure 2c across gvd viral populations belonged to the bacterial phyla firmicutes gtdb firmicutes firmicutes_a and firmicutes_c combined andbacteroidetes gtdb bacteroidota consistent withour knowledge that firmicutes and bacteroides are the mostprominent bacterial phyla in the human gastrointestinal tracteckburg notably firmicutes typically outnumberbacteroidetes in unhealthy individuals with metabolic anddigestive disorders broecker chehoud ley nicholson norman ott zhao and gvd metagenomes are biased toward unhealthy individuals of themetagenomes comprising of the bps sequenced whichmight account for the increased firmicutes viral populationsin gvdthe gvd significantly improves virus detection overcurrent viral genome databasesto assess the value of the gvd we quantitatively evaluated virusidentification sensitivity between multiple databases bycomparing the number of viral populations identifiable by readrecruitment against gvd ncbi™s viral refseq v96 doe™s imgvr v4 paezespino and the individual virome databases from each study figure see method details to controlfor assembly improvements since the original metagenome andor virome datasets were published for the latter we individuallyassembled the original viromes into viral populations for read cell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003resourcellfigure gvd as a reference database increases viral population detectionboxplots showing median and quartiles of the number of viral populations detected per study using the individual virome viral refseq v96 jgi imgvr v4 vd databases all pairwise comparisons were performed by using mannwhitney u tests nonsignificant p values are denoted as ˜˜ns™™see also figure s3 and table s4recruitment ncbi viral refseq was the most commonly usedviral genome database across the studies surveyed here beingused in of studies where the specific database used wasdocumented information on the genome database used was unavailable for three studies see table s1 and hosted virusgenomes already dereplicated as of v96 november usedhere in comparison the imgvr database was not documented as being used by any of the studies gathered despitethe latest release v4 july used in this study containingnearly two orders of magnitude more virus genomes andgenome fragments virus contigs though not dereplicated for comparison purposes to the gvd see method details we dereplicated the imgvr contigs the same way aswe did the gvd to obtain viralpopulationlevel genomes thisyielded viral populations for the imgvr databasein out of the total studies tested figure the gvdenabled the detection of significantly more viruses than viral refseq v96 mannwhitney u tests p [average] ± [standard deviation]fold increase and individual viromesmannwhitney u tests p 6fold ± 40fold increasenotably the proportion of the metagenome mapping to thegvd was highly variable between studies figure s2b and asexpected a higher proportion of vlpenriched metagenomesmapped to the gvd than did bulk metagenomes figure s2cthere was a single study reyes in which no viruseswere detected see method details in all databases queried inthis analysis in comparison to imgvr we detected more viruses with the gvd in all studies from total of whichwere in a significant manner mannwhitney u tests p 26fold ± 21fold increase figure five of the remaining fourteen studies had too low of a sample size andor number of detected viruses to statistically compare the gvd and imgvradditionally we tested the ability of the gvd to increase thenumber of viral populations detected in a study not included inthe gvd clooney figure s3a we saw similar results and gvd significantly outperformed viral refseq v96 andthe individual virome while having a nonsignificant higher median number of viral populations detected than imgvrwhen we considered the number of reads that recruitedacross the different databases across all studies significantlymore reads mannwhitney u tests p were recruited tothe gvd than to any other database across out of the studies figure s3b after gvd imgvr was the next best performing database for viral detection in the human gut given thatour tests showed an average of 64fold ± 120fold increase overviral refseq mannwhitney u tests p imgvr was expected to surpass viral refseq because it aggregates both cultivated reference virus genomes from refseq prophages and uncultivated virus genomes andorfragments from many environments including multiple humanbody sites paezespino overall the significant increase in virus detection by the gvd over other databases highlights the low representation of gut viruses in refseq and thusdemonstrates the value of the gvd for sequencebased virusidentification in human gut microbiome datasets thus giventhat the gvd significantly improves viral detection over currentviral genome databases we used the gvd as the database forall remaining analyses in this studythe human gut virome is highly person specificin light of the current hypothesis of a ˜˜core™™ gut virome manrique we were first curious whether any gvd viralcell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llresourcefigure individual viromes study databases and crossstudy comparisonsshown at the top left is a hierarchically clustered heatmap showing the number of viral populations shared within and between studies clustered into four groupsi“iv viral population cooccurrence network per individual within each study per group shown on the bottom right is a hierarchically clustered heatmapshowing the number of viral genera shared within and between studies clustered into three groups viral genus cluster cooccurrence networks per metagenomewithin each study per group colored dots and pictograms next to study names in heatmaps represent metagenome type and a common disease studies acrossall studies in gvd respectivelysee also figure s4population was found across a high percentage or all metagenomes in the gvd on average ± average ± sd range“ viral populations were detected per metagenome butnot a single viral population was found across all metagenomesin fact the most ubiquitous viral population in the gvd wasfound in only of the metagenomes viral populationsoccurred in more than of the metagenomes and most or of the viral populations were only sporadicallydetected at all of the metagenomes figures s4a ands4b table s4 further we specifically looked at the prevalenceof crassphages a wellrecognized multigenera family ofphages known to be widespread in gut viromes guerin figure s4b in total we identified crassphage populations see method details of which had genomes kb cell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003resourcellfigure vlpenriched vlp and bulk metagenomes comparisons for studying viruses in the human guta“c boxplots showing median and quartiles of the number of assembled contigs per base pair sequenced per study a of vlp and bulk metagenomes b ofvlp metagenomes with and without mda and c of the different vlpenrichment methodologies across the studies outlier dots were removed from plot c tobetter show the range of values the n value above each box plot represents the number of studies using each vlpenrichment methodd scatter plot with a linear regression line showing the number of assembled viral contigs per bp sequenced per study with vlp and bulk metagenome studiesidentified by different colors in the inset is a venn diagram showing the number of gvd viral populations that originated from vlp or bulk or both types ofmetagenomese boxplots showing median and quartiles of the number of viral populations detected per bp sequenced per individual of vlp and bulk metagenomesf boxplots showing median and quartiles of the number of assembled contigs per bp sequenced top left and the median contig length top right for vlp andbulk metagenomes processed for the same samples in the shkoporov bottom connected dot plot showing the number of viral populations detectedper bp sequencedby using vlp and bulk metagenomes for each individual in the shkoporov study all pairwise comparisons were performed by usingmannwhitney u tests nonsignificant p values are denoted as ˜˜ns™™see also figure s4that clustered into vcs ie genuslevel grouping bygenomebased genesharing networks jang bin although togetherthese crassphage populations areubiquitous across the gvd samples there was not one crassphage viral population found universally and the most widespread crassphage population occurred in only of samplesthese findings support the mounting evidence for highly personal gut viromes as recently highlighted in twins morenogallego and in ten healthy adults during a yearlongmonitoring period shkoporov although the latterstudy pointed to the potential of a core virome at higher taxonomic levels we failed to recover any universally shared viralvcs approximately genus level taxonomy figures s4c ands4d given that the most ubiquitous vc was only present in of the metagenomes that same study suggesting thepotential of a highertaxon core only looked at ten healthywestern adults and the metagenomes in the gvd representeda diversity of people from different geographical regions andages thus further studies are necessary to resolve whether acore virome does exists at higher taxonomic levelsvaried processing methodology prevents populationlevel bdiversity interstudy comparisonsnext given a systematically processed gvd and its demonstrably improved virus detection capability we sought to determine whether global clustering patterns would emerge via a gvdbdiversity betweensample changes in population composition metaanalysis to this end we performed population cooccurrence analyses at two levels of resolution per study andacross metagenomes within studies and then evaluated whatlevel of metadata best captured the resultant variation methodology disease state etc to assess populationoverlap between studies we counted the number of gvd viral populationsthat recruited reads within and between different gvd studiescell host microbe “ november 0cplease cite this in press as gregory the gut virome database reveals agedependent patterns of virome diversity in the human gutcell host microbe 101016jchom202008003llie the same viral population was detected in metagenomein both studies compared we expected studies exploringsimilar disease states would share the greatest number of viralpopulations however hierarchical clustering of the studies onthe basis of the number of shared viral populations revealedthat this was rare and mostly not the case and studies exploringthe viromes of diseased individuals eg ibd clostridium difficile infection and diabetes did not cluster together figure top left”heatmap instead we saw that the different studies hierarchically clustered into four groups i“iv and that this clustering was weakly driven around metagenome type many bulkmetagenomes clustered together into group iiibecause the gvd studies did cluster into four distinct groupsfigure top left”heatmap we next tested whether any metadata best captured the resultant variation across the metagenomes within each study within each group using an inversecovariance analysis ie combined partial correlations acrossshared viral population between metagenomes to subclusterthe different metagenomes across the studies in each groupwe found that the metagenomes within a study subclusteredtogether irrespective of geographical origin health status andor diet figure top left”networks notably the group iii metagenomes derived mostly from bulk metagenomes were moreclosely subclustered but they still subclustered strongly bystudy this perstudy subclustering implies that even withinthese grouped studies metagenomes from different studiesare not comparable because the interstudy variation is drivenby methodological impacts these results reveal that althoughmethodology does not affect the number of viral contigs recovered it does affect the recovered types of viruses see upcomingfindings comparing methodological effects interestingly whenwe looked at genuslevelie vclevel cooccurrence wesaw that there are still strong groupings a“c at the study levelbut within each group metagenomes across these studiesshare many vcs figure bottom right thus bdiversitymetaanalysis across all studies exploring the effect of ˜˜disease™™across gvd studies is not possible at the population level butwithin similarly processed studies it might be possible at thegenus levelto enrich or not to enrich viruses recovered from bulkversus viruspenriched metagenomesfrom a pragmatic point of view we next wondered whethergvd datasets could inform experimental design specifically cell host microbe “ november resourcefigure more gut viruses are temperatephages than in the soil and oceanspie charts showing the percentages of temperatephages found in the human gut gvd datasetsoils isogenie dataset and oceans globaloceans viromes datasetto study viruses is sequencing effort better put into metagenomes of bulk or purified vlps the gvd™s gut metagenomes are roughly evenly divided acrossthese two metagenome types with bulkand vlp metagenomes contributing tbp 18 of gvd and tbp 18 of gvd of data respectively although most samplesonly have one or the other data one study samples shkoporov provided both bulk and vlp metagenomesfor sampleswe first assessed whether there was a difference in de novoviral recovery between vlp and bulk metagenomes figure 5awe measured viral recovery by using the number of viral contigs kb or kb and circular in length ie not dereplicatedviral populations assembled per bp sequenced per study giventhat the viral contigs assembled from samples within the samestudy are often pooled these analyses revealed no significantdifference mannwhitney u test p in the number of viralcontigs assembled per bp sequenced between vlp and bulkmetagenomes which contrasts viralrecovery results frompermafrost soils where vlp metagenomes outperform bulkmetagenomes by 2fold trubl however viral recovery from the gvd™s vlp metagenomes was heterogeneous sowe evaluated how vlp methodology affected viral recoveryfirst although multiple displacement amplification mdaisknown to provide nonquantitative metagenomic datasets withboth systematic and stochastic biases solonenko yilmaz we found no significant difference mannwhitney u test p in viral recovery between nonmdaand mdatreated metagenomes figure 5b nonetheless itwas notable that mdatreated vlp studies were significantly enriched in eukaryotic ssdna viruses mannwhitney u tests p a known bias of mda figure s5a second we tested theeffect of vlp enrichment strategies which ranged fromremoving human and bacterial cells to enrich for vlps centrifugation filtration cscl gradients and nucleases to concentrating the vlps centricon concentration and peg precipitationagain we found no significant difference in the number of viralcontigs recovered kruskalwallis test p across Answer:

Colon_Cancer

] we have filtered only research s published in english language and selected the following keywords air pollution and covid19 or sarscov2 particulate matter or pm and covid19 or sarscov2 nitrogen dioxide or no2 and covid19 or sarscov2 we choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported covid19 cases andor deaths and air pollution data related to pm25 pm10 and no2 thus excluding any letter opinion commentary review or nonrelevant s we obtained a total of eligible published research s in their final version and paper in its preprint version for some of them we chose to include only principal findings that clearly fit the aim this review particulate matter and covid19 atmospheric particulate matter pm is originated by a wide range of anthropogenic and natural sources kim it consists of a heterogeneous mixture of solid and liquid ps suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals who it has been associated with increased respiratory morbidity and mortality liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis li rhee in vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections becker and soukup recently the research group of setti gave first preliminary evidence that sarscov2 rna can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of pm it could represent a potential early indicator of covid19 although it does not give information regarding covid19 progression or severity several observations report a significant association between ambient concentrations of pm25 adhikari and yin bashir fattorini and regoli frontera jiang li vasquezapestegui wu yao zhu zoran 2020a and pm10 bashir coccia 2020b fattorini and regoli jiang li yao zhu zoran 2020a with covid19 pandemic across the most affected countries china italy and usa see table first evidences on the temporal association between air pollution and covid19 were reported in china where the outbreak was first identified zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in china the authors included over of dailyconfirmed new cases in the whole of china between january 23rd and february 29th they applied a generalized additive model gam to examine the effects of meteorological factors and air pollution on covid19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders they observed that the effect of pm25 on daily confirmed cases was greater than pm10 in particular they found that a 10μgm3 increase lag0“ in pm25 and pm10 was associated with a ci to and ci to increase in the daily counts of covid19 confirmed cases respectively jiang focused their attention on three most affected cities of china wuhan xiaogan and huanggang collecting data of daily cases and ambient air pollutant from jan 25th to feb 29th the authors by applying a multivariate poisson regression revealed a significant temporal association between pm25 increased and covid19 incidence in all the considered cities especially in huanggang wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ conversely an increase in pm10 concentrations was associated with a decrease of covid19 incidence these results were partially confirmed by findings of li who conducted a simple linear regression to compare covid19 incidence with pm concentrations in wuhan and xiaogan from jan 26th to feb 29th in they found that an increase in pm25 was correlated with an increase of covid19 incidence in both cities wuhan r2 p xiaogan r2 p while for pm10 only in xiaogan r2 p the spatial distribution of particulate matter and case fatality rate cfr of covid19 was studied by yao in cities of china including wuhan collecting data up to march 22nd first they found a significantly positive global spatial autocorrelation of covid19 cfr global moran™s index i p highlighting a high cfr clustering located in hubei province with a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product gdp per capita hospital beds per capita local indicators of spatial association lisa map values city size and population or proportion of people older than years it was found that for every μgm3 increase in pm25 and pm10 the cfr increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of pm25 and pm10 in “ respectively some studies describe the association between air pollution and covid19 across italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other european countries the 28th of july italy recorded more than total confirmed cases and deaths who most of which were distributed in the regions of northern italy especially the lombardy it is recognized as one the most air polluted areas of europe eea where the frequent pm10 annual exceedances of the who threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year baccini bontempi 2020bfocused the attention on two of the most affected regions of northern italy lombardy and piedmont the authors based on pm10 daily exceedances and covid19 confirmed cases on march 12th thus before the italian sanitary crisis observed that pm10 concentration was exceeded only few times among the lombard cities that at the beginning of the epidemic were most affected on the contrary among some piedmont cities suffering of severe pm10 pollution events covid19 incidence was lower based on their results the authors concluded that covid19 diffusion by airborne pm10 is hard to demonstrate nevertheless several research revealed how pm in particular pm25 could had a role in accelerate and vast diffusion of covid19 in northern italy for example coccia 2020b by analyzed data on italian province capitals and data of infected individuals up to april 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for pm10 in previous years and covid19 diffusion in particular cities with more than days of pm10 exceedances showed a very high average number of infected individual about infected individuals on 7th april whereas cities having less than days of pm10 exceedances showed a lower average number of infected about infected individuals frontera gave also evidences on the role of pm25 as a contributing factor of covid19 outbreak in northern italy where environmentalresearch19120201101293 0cc copat table summary table reporting reviewed results on the association between covid19 casesdeaths and air pollution pm25 pm10 and no2 references zhu data analysis generalized additive model gam aim temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 spatial association between fatality rate and air pollution pm25 and pm10 spatial association between deaths counts and air pollution no2 temporal association between total cases daily confirmed cases and total deaths and air pollution pm25 and pm10 temporal association between total cases daily confirmed cases and total deaths and air pollution no2 spatial description of pm10 exceedances versus covid19 cases multivariate poisson regression simple linear regression multiple linear regression descriptive analysis percentage of deaths in three no2 μmol m2concentration range “ “ “ pearson coefficient correlation pearson coefficient correlation descriptive analysis number of days of pm10 exceeding μgm3 and covid19 incidence area of study cities of china period from jan 23rd to feb 29th jiang li yao ogen zoran 2020a zoran 2020b bontempi 2020b from jan 25th to feb 29th from jan 26th to feb 29th in data up to march 22nd data up to the end of feb from jan 1st to apr 30th from jan 1st to apr 30th from feb 10th to march 12th wuhan xiaogan and huanggang china wuhan and xiaogan cities of china administrative regions in italy spain france and germany milan italy milan italy provinces of lombardy italy provinces of piedmont italy coccia 2020b data up to april 7th italian provinces fattorini and regoli data up to april 27th italian provinces pm25 a 10μgm3 pm25 increase lag0“ was associated with a increase of daily confirmed new cases pm10 a 10μgm3 pm10 increase lag0“ was associated with a increase of daily confirmed new cases wuhan rr ci1032“ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p χ2 p a μgm3 increase in pm25 was associated with a “ increase in fatality rate χ2 p a μgm3 increase in pm10 was associated with a “ increase in fatality rate no2 a 10μgm3 no2 increase lag0“ was associated with a increase in daily confirmed new cases wuhan rr ci “ xiaogan rr ci “ huanggang no association found wuhan r2 p xiaogan r2 p of fatality cases are associated with no2 μmolm2 r cid0 r r cid0 r cid0 r r cid0 r cid0 r cid0 r cid0 lombardy pm10 exceeding between and covid19 incidence between and piedmont pm10 exceeding between and covid19 incidence between and covid19 in north italy has a high association with air pollution of cities measured with days exceeding the limits set for pm10 r2 p r2 p continued on next page hierarchical multiple regression model pearson regression coefficient analysis r2 p spatial association between confirmed cases and air pollution pm10 spatial association between total confirmed cases and air pollution pm25 pm10 and no2 environmentalresearch19120201101294 0cc copat table continued references frontera frontera wu adhikari and yin bashir bashir vasquezapestegui vasquezapestegui vasquezapestegui period data up to 31st march data up to 31st march data up to april 04th from march 1st to apr 20th from march 4th to april 24th from march 4th to april 24th data up to june 12th data up to june 12th data up to june 12th area of study italian regions italian regions counties in the usa queens county new york usa california california districts of lima perù districts of lima perù districts of lima perù aim spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 prediction of risk of covid19 deaths in the long term average exposure to fine particulate matter pm25 temporal association between daily confirmed cases and total deaths and air pollution pm25 association between confirmed cases and air pollution pm25 pm10 and no2 association between deaths and air pollution pm25 pm10 and no2 spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 spatial association between case fatality rate and air pollution pm25 data analysis pearson regression coefficient analysis pm25 r2 p pm10 pearson regression coefficient analysis r2 p longterm exposure increase of μgm3 in pm25 is associated with a increase in the covid19 death rate estimate on cases values cid0 ci “ estimate on deaths value cid0 ci “ kendall r cid0 spearman r cid0 zeroinflated negative binomia models negative binomial regression model spearman and kendall correlation tests spearman and kendall correlation tests no2 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 multivariate regression model crude coefficient p multivariate regression model crude coefficient p multivariate regression model crude coefficient cid0 p mortality was found significantly higher than less polluted italian regions by collecting data up to march 31st for all italian regions and performing a pearson correlation analysis they found a strong positive association both with the total number of confirmed cases r and deaths r other than with hospitalized cases r the italian situation was further highlighted by the study of fattorini and regoli in italian provinces they explored the spatial association between air pollution and covid19 cases with data up to april 27th by applying the pearson regression coefficient analysis they revealed a positive association both with pm25 and pm10 r2 p and r2 p respectively a focus on the most affected city of italy milan was conducted by zoran 2020a this city is located in the po valley basin known hotspot for atmospheric pollution at the continental scale eea the authors performed a temporal association between covid19 total cases daily new positive cases and total deaths and particulate matter from jan 1st and apr 30th by applying a person correlation in accordance with other studied they found a positive association between daily confirmed cases and pm25 r and pm10 r although they did not consider any delay time from infection to covid19 onset nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships to date the usa have more than million confirmed cases and thousand deaths who here ambient concentrations of pm and o3 were found responsible to cause between and premature deaths fann the association between air pollutants and covid19 cases and deaths was studied by bashir in the state of california from march 4th to april 24th corresponding to the beginning of the covid19 outbreak in usa based on their significant correlation found the authors state that a limited human exposure to these pollutants will contribute to defeating covid19 this conclusion seems unclear because they found a negative correlation with pm25 and pm10 environmentalresearch19120201101295 0cc copat by applying both the kendall rank correlation and spearman™s one and it is not clear if they normalized covid19 cases by population size and if they performed a day by day association or a spatial association across the country a focus on the queen county new york usa was provided by adhikari and yin they retrieved data of pm daily concentrations from two ground monitoring stations and collected data of confirmed covid19 cases and numbers of related deaths from usafacts in the period from march to april the authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of pm25 on disease outcomes over the past days they found a significant negative association among pm25 and new daily confirmed covid19 cases cid0 ci “ and deaths cid0 ci “ low pm concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as italy where pm25 monthly concentrations reached values higher than μgm3 fattorini and regoli frontera or in china where pm25 monthly concentrations reached values higher than μgm3 zhu jiang as said by the authors other gaseous pollutants such as no2 and so2 could have influenced transmission and pathogenesis of covid19 in the united states wu investigated whether longterm average exposure to fine particulate matter pm25 increases the risk of covid19 deaths by considering approximately counties in the united states of the population with an exposure prediction model the authors calculated the county level longterm exposure to pm25 averaged for to and collected covid19 deaths counts up to april 04th they conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors they found that a small longterm exposure increase of only μgm3 in pm25 is associated with a increase in the covid19 death rate confidence interval ci vasquezapestegui recently reported first evidences on the spatial relationship between particulate matter and covid19 outbreak from latin america the authors described the situation occurred in districts of lima located in the second most affected country of latin america peru in particular by applying a multivariate regression model they evaluated the association between the population exposure to pm25 concentrations in the previous years “ and cases deaths and casefatality rates of covid19 with data up to june 12th a significant association has been found both with cases and deaths crude coefficient with p and with p respectively but not with case fatality rate all these studies highlight the role of pm in triggers of the covid19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems nitrogen dioxide no2 and covid19 induced lung damage hence viral infection becomes more common after exposure to no2 zhu furthermore no2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children to increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation bahrami asl kowalska increase of chronic obstructive pulmonary disease copd ghanbari ghozikali pfeffer and increase of pulmonary heart disease related mortality chen a recent study explored the possible role of no2 in interference in angiotensin converting enzyme ace2 the expression of ace2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of covid19 alifano first observations report an association between ambient concentrations of no2 and covid19 pandemic across europe china and usa bashir fattorini and regoli jiang li et al ogen zhu et al zoran et al 2020b conversely to the other papers findings of zoran 2020b and bashir provides different findings reporting no association or a negative one between no2 and daily deaths counts in china zhu by applying the same method explained for pm observed that a 10μgm3 increase lag0“ in no2 is associated with a ci “ increase in the daily counts of covid19 confirmed cases in cities of china these findings are confirmed by jiang and li et a who applied the same method described for pm jiang revealed a significant positive association between no2 and covid19 both in wuhan and xiaogan wuhan rr ci1053“ xiaogan rr ci “ but did not found any significant association in huanggang li found a significant linear correlation both in wuhan r2 p and xiaogan r2 p ogen presented evidences on the relationship between exposure to no2 including the months of january and february shortly before the covid19 spread in europe and novel coronavirus fatality in the most affected european countries concluding that longterm exposure to no2 may be a potential contributor to mortality caused by sarscov2 he collected data concerning the number of fatality cases from administrative regions in italy spain france and germany and correlated mortality with tropospheric no2 concentrations measured by the sentinel5 precursor spaceborne satellite the major tropospheric no2 hotspot identified was located in the northern italy in all european regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards results showed that out of the fatality cases by march were in five regions located in north italy and central spain furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum no2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 the methodology used by ogen cannot support a longterm exposure investigation surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust nevertheless the study provide new insights for future investigation the italian situation was further studied by fattorini and regoli who collected data of covid19 incidence up to april 27th from italian provinces they revealed a strong spatial correlation with no2 mean levels concentrations “ pearson coefficient r2 p confirming the northern italy being a hotspot of no2 in addition to urbanized cities of central and southern italy such as rome and naples a focus on the temporal association between ground levels of no2 and covd19 cases total cases daily new positive cases and total deaths was performed by zoran 2020b for the city of milan italy in the period pre and postlockdown measures the authors nitrogen dioxide is a nastysmelling gas formed by reaction in the atmosphere of nitrogen oxides nox with other chemicals nox is naturally produced in atmosphere by lightning kang et al volcanoes oceans and biological decay thurston the major outdoor anthropogenic sources of nox are primarily emissions from transportation and fuel combustion in particular in urban areas they comes from vehicle exhaust gases and domestic heating grange maawa the nitrogen dioxide has mainly effect on the respiratory system because an increase of the outdoor concentration of no2 may significantly increase the risk of respiratory tract infection this phenomenon is particularly evident in children as they are more susceptible to no2 environmentalresearch19120201101296 0cacknowledgments c copat found no2 negative correlated with all the considered epidemiological data but the methodology used has some limitations as the delay time from infection to the covid19 onset or covid19 death was not considered as well the significant reduction of air pollution due to lockdown measures since midmarch in usa the association was also studied by bashir for the state of california as discussed above for pm the authors found a negative correlation also between no2 levels and covid19 cases and mortality nevertheless they stated that this pollutant contributes to the spread of the disease based on these scientific evidences in addition to confirming that exposure to no2 is harmful to human health and increases the risk of incurring respiratory diseases it can be stated that exposure to no2 may be one of the most important trigger for the spread and fatality caused by the covid19 disease declare references adhikari a yin j shortterm effects of ambient ozone pm25 and the authors declare no conflict of interest we have no funding to bontempi e 2020b first data analysis about possible covid19 virus airborne alifano m alifano p fez p iannelli a reninangiotensin system at the meteorological factors on covid19 confirmed cases and deaths in queens new york int j environ res publ health httpsdoi103390 ijerph17114047 heart of covid19 pandemic biochimie httpsdoi101016j biochi202004008 baccini m biggeri a grillo p consonni d bertazzi pa health impact assessment of fine p pollution at the regional level am j epidemiol “ httpsdoi101093ajekwr256 bahrami asl f leili m vaziri y salahshour arian s cristaldi a oliveri conti g ferrante m health impacts quantification of ambient air pollutants using airq model approach in hamadan iran environ res “ httpsdoi 101016jenvres201710050 bashir mf ma bj bilal komal b bashir ma farooq th iqbal n bashir m correlation between environmental pollution indicators and covid19 pandemic a brief study in californian context environ res https doi101016jenvres2020109652 becker s soukup jm exposure to urban air particulates alters the macrophage mediated inflammatory response to respiratory viral infection j toxicol environ health “ httpsdoi101080009841099157539 bontempi e 2020a commercial exchanges instead of air pollution as possible origin of covid19 initial diffusion phase in italy more efforts are necessary to address interdisciplinary research environ res httpsdoi101016j envres2020109775 diffusion due to air particulate matter pm the case of lombardy italy environ res httpsdoi101016jenvres2020109639 bontempi e vergalli s squazzoni f understanding covid19 diffusion requires an interdisciplinary multidimensional approach environ res httpsdoi101016jenvres2020109814 bremner sa anderson hr atkinson rw mcmichael aj strachan dp bland j m bower js shortterm associations between outdoor air pollution and mortality in london occup environ med “ httpsdoi 101136oem564237 cai qc lu j xu qf guo q xu dz sun qw yang h zhao gm jiang qw influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome publ health “ https doi101016jpuhe200609023 carugno m dentali f mathieu g fontanella a mariani j bordini l milani g p consonni d bonzini m bollati v pesatori ac pm10 exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in lombardy italy environ res “ https doi101016jenvres201806016 chen h chen y lian z wen l sun b wang p li x liu q yu x lu y qi y zhao s zhang l yi x liu f pan g 2020a correlation between the migration scale index and the number of new confirmed coronavirus disease cases in china epidemiol infect e99 httpsdoi101017 s0950268820001119 chen j zeng j shi c liu r lu r mao s zhang l associations between shortterm exposure to gaseous pollutants and pulmonary heart diseaserelated mortality among elderly people in chengdu china environ health httpsdoi 101186s1294001905008 chen s prettner k kuhn m geldsetzer p wang c b¨arnighausen t bloom de 2020b covid19 and climate global evidence from countries medrxiv prepr serv health sci httpsdoi1011012020060420121863 coccia m 2020a how high wind speed can reduce negative effects of confirmed cases and total deaths of covid19 infection in society ssrn scholarly paper no id social science research network rochester ny httpsdoi 102139ssrn3603380 coccia m 2020b factors determining the diffusion of covid19 and suggested strategy to prevent future accelerated viral infectivity similar to covid sci total environ httpsdoi101016jscitotenv2020138474 balakrishnan k brunekreef b dandona l dandona r feigin v freedman g hubbell b jobling a kan h knibbs l liu y martin r morawska l pope ca shin h straif k shaddick g thomas m van dingenen r van donkelaar a vos t murray cjl forouzanfar mh estimates and year trends of the global burden of disease attributable to ambient air pollution an analysis of data from the global burden of diseases study lancet lond engl httpsdoi101016s0140673617305056 “ conticini e frediani b caro d can atmospheric pollution be considered a co factor in extremely high level of sarscov2 lethality in northern italy environ pollut barking essex httpsdoi101016jenvpol2020114465 croft dp zhang w lin s thurston sw hopke pk van wijngaarden e squizzato s masiol m utell mj rich dq associations between source cohen aj brauer m burnett r anderson hr frostad j estep k conclusion the scientific evidences collected in the literature highlight the important contribution of chronic exposure to air pollution on the covid19 spread and lethality although the potential effect of airborne virus exposure it has not been still demonstrated in particular it seems that pm25 and no2 are more closely correlated to covid19 than pm10 the lower correlation of pm10 with covid19 incidence and mortality can be due to the impossibility of particulate matter greater than μm to reach type ii alveolar cells where is located the cell entry receptor ace2 for sarscov2 nevertheless differences between countries such as the implementation of different lockdown restrictions stage of infection topographic sociodemographic and socioeconomic characteristics level of air pollution and meteorological factors may have contributed to obtain some contrasting finding although most of the revised studies support the relationship between air pollution and covid19 the manifold limitations of this review are the small number of papers collected and the great diversity of methodologies used sometimes lacking in some parts which makes the results difficult to compare the authors who first investigated this association although with great effort and rapidity of analysis dictated by a global emergency sometimes do not include all confounding factors whenever possible such as control policy urbanization rate availability of medical resources population size weather lifestyles sociodemographic and socioeconomic variables in addition to date incidence data are underestimated in all countries and to a lesser extent mortality data for this reason the cases included in the considered studies cannot be considered conclusive more studies are needed to better clarify the role of air pollution during the covid19 pandemic particularly studies that consider the multiplepollutants to strengthen scientific evidences and support firm conclusions useful to implement pandemic application plans to adequately prevent new health emergencies for a long time we have known that reducing outdoor and indoor air pollution in cities or countries can have a significant effect on health almost immediately and the benefits can far outweigh the costs surely the health emergency that the world is experiencing right now highlights how environmental research is a fundamental reference point to improve the knowledge concerning diseases of infectious origin and how all the intellectual and economic resources are to be spent to accelerate actions aimed to implement environmental policies act to reduce air pollution and develop new urban planning interventions influences or multidisciplinary studies declaration of competing interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper environmentalresearch19120201101297 0cc cop

cancer7534

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: ] we have filtered only research s published in english language and selected the following keywords air pollution and covid19 or sarscov2 particulate matter or pm and covid19 or sarscov2 nitrogen dioxide or no2 and covid19 or sarscov2 we choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported covid19 cases andor deaths and air pollution data related to pm25 pm10 and no2 thus excluding any letter opinion commentary review or nonrelevant s we obtained a total of eligible published research s in their final version and paper in its preprint version for some of them we chose to include only principal findings that clearly fit the aim this review particulate matter and covid19 atmospheric particulate matter pm is originated by a wide range of anthropogenic and natural sources kim it consists of a heterogeneous mixture of solid and liquid ps suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals who it has been associated with increased respiratory morbidity and mortality liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis li rhee in vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections becker and soukup recently the research group of setti gave first preliminary evidence that sarscov2 rna can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of pm it could represent a potential early indicator of covid19 although it does not give information regarding covid19 progression or severity several observations report a significant association between ambient concentrations of pm25 adhikari and yin bashir fattorini and regoli frontera jiang li vasquezapestegui wu yao zhu zoran 2020a and pm10 bashir coccia 2020b fattorini and regoli jiang li yao zhu zoran 2020a with covid19 pandemic across the most affected countries china italy and usa see table first evidences on the temporal association between air pollution and covid19 were reported in china where the outbreak was first identified zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in china the authors included over of dailyconfirmed new cases in the whole of china between january 23rd and february 29th they applied a generalized additive model gam to examine the effects of meteorological factors and air pollution on covid19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders they observed that the effect of pm25 on daily confirmed cases was greater than pm10 in particular they found that a 10μgm3 increase lag0“ in pm25 and pm10 was associated with a ci to and ci to increase in the daily counts of covid19 confirmed cases respectively jiang focused their attention on three most affected cities of china wuhan xiaogan and huanggang collecting data of daily cases and ambient air pollutant from jan 25th to feb 29th the authors by applying a multivariate poisson regression revealed a significant temporal association between pm25 increased and covid19 incidence in all the considered cities especially in huanggang wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ conversely an increase in pm10 concentrations was associated with a decrease of covid19 incidence these results were partially confirmed by findings of li who conducted a simple linear regression to compare covid19 incidence with pm concentrations in wuhan and xiaogan from jan 26th to feb 29th in they found that an increase in pm25 was correlated with an increase of covid19 incidence in both cities wuhan r2 p xiaogan r2 p while for pm10 only in xiaogan r2 p the spatial distribution of particulate matter and case fatality rate cfr of covid19 was studied by yao in cities of china including wuhan collecting data up to march 22nd first they found a significantly positive global spatial autocorrelation of covid19 cfr global moran™s index i p highlighting a high cfr clustering located in hubei province with a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product gdp per capita hospital beds per capita local indicators of spatial association lisa map values city size and population or proportion of people older than years it was found that for every μgm3 increase in pm25 and pm10 the cfr increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of pm25 and pm10 in “ respectively some studies describe the association between air pollution and covid19 across italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other european countries the 28th of july italy recorded more than total confirmed cases and deaths who most of which were distributed in the regions of northern italy especially the lombardy it is recognized as one the most air polluted areas of europe eea where the frequent pm10 annual exceedances of the who threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year baccini bontempi 2020bfocused the attention on two of the most affected regions of northern italy lombardy and piedmont the authors based on pm10 daily exceedances and covid19 confirmed cases on march 12th thus before the italian sanitary crisis observed that pm10 concentration was exceeded only few times among the lombard cities that at the beginning of the epidemic were most affected on the contrary among some piedmont cities suffering of severe pm10 pollution events covid19 incidence was lower based on their results the authors concluded that covid19 diffusion by airborne pm10 is hard to demonstrate nevertheless several research revealed how pm in particular pm25 could had a role in accelerate and vast diffusion of covid19 in northern italy for example coccia 2020b by analyzed data on italian province capitals and data of infected individuals up to april 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for pm10 in previous years and covid19 diffusion in particular cities with more than days of pm10 exceedances showed a very high average number of infected individual about infected individuals on 7th april whereas cities having less than days of pm10 exceedances showed a lower average number of infected about infected individuals frontera gave also evidences on the role of pm25 as a contributing factor of covid19 outbreak in northern italy where environmentalresearch19120201101293 0cc copat table summary table reporting reviewed results on the association between covid19 casesdeaths and air pollution pm25 pm10 and no2 references zhu data analysis generalized additive model gam aim temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 spatial association between fatality rate and air pollution pm25 and pm10 spatial association between deaths counts and air pollution no2 temporal association between total cases daily confirmed cases and total deaths and air pollution pm25 and pm10 temporal association between total cases daily confirmed cases and total deaths and air pollution no2 spatial description of pm10 exceedances versus covid19 cases multivariate poisson regression simple linear regression multiple linear regression descriptive analysis percentage of deaths in three no2 μmol m2concentration range “ “ “ pearson coefficient correlation pearson coefficient correlation descriptive analysis number of days of pm10 exceeding μgm3 and covid19 incidence area of study cities of china period from jan 23rd to feb 29th jiang li yao ogen zoran 2020a zoran 2020b bontempi 2020b from jan 25th to feb 29th from jan 26th to feb 29th in data up to march 22nd data up to the end of feb from jan 1st to apr 30th from jan 1st to apr 30th from feb 10th to march 12th wuhan xiaogan and huanggang china wuhan and xiaogan cities of china administrative regions in italy spain france and germany milan italy milan italy provinces of lombardy italy provinces of piedmont italy coccia 2020b data up to april 7th italian provinces fattorini and regoli data up to april 27th italian provinces pm25 a 10μgm3 pm25 increase lag0“ was associated with a increase of daily confirmed new cases pm10 a 10μgm3 pm10 increase lag0“ was associated with a increase of daily confirmed new cases wuhan rr ci1032“ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p χ2 p a μgm3 increase in pm25 was associated with a “ increase in fatality rate χ2 p a μgm3 increase in pm10 was associated with a “ increase in fatality rate no2 a 10μgm3 no2 increase lag0“ was associated with a increase in daily confirmed new cases wuhan rr ci “ xiaogan rr ci “ huanggang no association found wuhan r2 p xiaogan r2 p of fatality cases are associated with no2 μmolm2 r cid0 r r cid0 r cid0 r r cid0 r cid0 r cid0 r cid0 lombardy pm10 exceeding between and covid19 incidence between and piedmont pm10 exceeding between and covid19 incidence between and covid19 in north italy has a high association with air pollution of cities measured with days exceeding the limits set for pm10 r2 p r2 p continued on next page hierarchical multiple regression model pearson regression coefficient analysis r2 p spatial association between confirmed cases and air pollution pm10 spatial association between total confirmed cases and air pollution pm25 pm10 and no2 environmentalresearch19120201101294 0cc copat table continued references frontera frontera wu adhikari and yin bashir bashir vasquezapestegui vasquezapestegui vasquezapestegui period data up to 31st march data up to 31st march data up to april 04th from march 1st to apr 20th from march 4th to april 24th from march 4th to april 24th data up to june 12th data up to june 12th data up to june 12th area of study italian regions italian regions counties in the usa queens county new york usa california california districts of lima perù districts of lima perù districts of lima perù aim spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 prediction of risk of covid19 deaths in the long term average exposure to fine particulate matter pm25 temporal association between daily confirmed cases and total deaths and air pollution pm25 association between confirmed cases and air pollution pm25 pm10 and no2 association between deaths and air pollution pm25 pm10 and no2 spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 spatial association between case fatality rate and air pollution pm25 data analysis pearson regression coefficient analysis pm25 r2 p pm10 pearson regression coefficient analysis r2 p longterm exposure increase of μgm3 in pm25 is associated with a increase in the covid19 death rate estimate on cases values cid0 ci “ estimate on deaths value cid0 ci “ kendall r cid0 spearman r cid0 zeroinflated negative binomia models negative binomial regression model spearman and kendall correlation tests spearman and kendall correlation tests no2 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 multivariate regression model crude coefficient p multivariate regression model crude coefficient p multivariate regression model crude coefficient cid0 p mortality was found significantly higher than less polluted italian regions by collecting data up to march 31st for all italian regions and performing a pearson correlation analysis they found a strong positive association both with the total number of confirmed cases r and deaths r other than with hospitalized cases r the italian situation was further highlighted by the study of fattorini and regoli in italian provinces they explored the spatial association between air pollution and covid19 cases with data up to april 27th by applying the pearson regression coefficient analysis they revealed a positive association both with pm25 and pm10 r2 p and r2 p respectively a focus on the most affected city of italy milan was conducted by zoran 2020a this city is located in the po valley basin known hotspot for atmospheric pollution at the continental scale eea the authors performed a temporal association between covid19 total cases daily new positive cases and total deaths and particulate matter from jan 1st and apr 30th by applying a person correlation in accordance with other studied they found a positive association between daily confirmed cases and pm25 r and pm10 r although they did not consider any delay time from infection to covid19 onset nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships to date the usa have more than million confirmed cases and thousand deaths who here ambient concentrations of pm and o3 were found responsible to cause between and premature deaths fann the association between air pollutants and covid19 cases and deaths was studied by bashir in the state of california from march 4th to april 24th corresponding to the beginning of the covid19 outbreak in usa based on their significant correlation found the authors state that a limited human exposure to these pollutants will contribute to defeating covid19 this conclusion seems unclear because they found a negative correlation with pm25 and pm10 environmentalresearch19120201101295 0cc copat by applying both the kendall rank correlation and spearman™s one and it is not clear if they normalized covid19 cases by population size and if they performed a day by day association or a spatial association across the country a focus on the queen county new york usa was provided by adhikari and yin they retrieved data of pm daily concentrations from two ground monitoring stations and collected data of confirmed covid19 cases and numbers of related deaths from usafacts in the period from march to april the authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of pm25 on disease outcomes over the past days they found a significant negative association among pm25 and new daily confirmed covid19 cases cid0 ci “ and deaths cid0 ci “ low pm concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as italy where pm25 monthly concentrations reached values higher than μgm3 fattorini and regoli frontera or in china where pm25 monthly concentrations reached values higher than μgm3 zhu jiang as said by the authors other gaseous pollutants such as no2 and so2 could have influenced transmission and pathogenesis of covid19 in the united states wu investigated whether longterm average exposure to fine particulate matter pm25 increases the risk of covid19 deaths by considering approximately counties in the united states of the population with an exposure prediction model the authors calculated the county level longterm exposure to pm25 averaged for to and collected covid19 deaths counts up to april 04th they conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors they found that a small longterm exposure increase of only μgm3 in pm25 is associated with a increase in the covid19 death rate confidence interval ci vasquezapestegui recently reported first evidences on the spatial relationship between particulate matter and covid19 outbreak from latin america the authors described the situation occurred in districts of lima located in the second most affected country of latin america peru in particular by applying a multivariate regression model they evaluated the association between the population exposure to pm25 concentrations in the previous years “ and cases deaths and casefatality rates of covid19 with data up to june 12th a significant association has been found both with cases and deaths crude coefficient with p and with p respectively but not with case fatality rate all these studies highlight the role of pm in triggers of the covid19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems nitrogen dioxide no2 and covid19 induced lung damage hence viral infection becomes more common after exposure to no2 zhu furthermore no2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children to increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation bahrami asl kowalska increase of chronic obstructive pulmonary disease copd ghanbari ghozikali pfeffer and increase of pulmonary heart disease related mortality chen a recent study explored the possible role of no2 in interference in angiotensin converting enzyme ace2 the expression of ace2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of covid19 alifano first observations report an association between ambient concentrations of no2 and covid19 pandemic across europe china and usa bashir fattorini and regoli jiang li et al ogen zhu et al zoran et al 2020b conversely to the other papers findings of zoran 2020b and bashir provides different findings reporting no association or a negative one between no2 and daily deaths counts in china zhu by applying the same method explained for pm observed that a 10μgm3 increase lag0“ in no2 is associated with a ci “ increase in the daily counts of covid19 confirmed cases in cities of china these findings are confirmed by jiang and li et a who applied the same method described for pm jiang revealed a significant positive association between no2 and covid19 both in wuhan and xiaogan wuhan rr ci1053“ xiaogan rr ci “ but did not found any significant association in huanggang li found a significant linear correlation both in wuhan r2 p and xiaogan r2 p ogen presented evidences on the relationship between exposure to no2 including the months of january and february shortly before the covid19 spread in europe and novel coronavirus fatality in the most affected european countries concluding that longterm exposure to no2 may be a potential contributor to mortality caused by sarscov2 he collected data concerning the number of fatality cases from administrative regions in italy spain france and germany and correlated mortality with tropospheric no2 concentrations measured by the sentinel5 precursor spaceborne satellite the major tropospheric no2 hotspot identified was located in the northern italy in all european regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards results showed that out of the fatality cases by march were in five regions located in north italy and central spain furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum no2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 the methodology used by ogen cannot support a longterm exposure investigation surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust nevertheless the study provide new insights for future investigation the italian situation was further studied by fattorini and regoli who collected data of covid19 incidence up to april 27th from italian provinces they revealed a strong spatial correlation with no2 mean levels concentrations “ pearson coefficient r2 p confirming the northern italy being a hotspot of no2 in addition to urbanized cities of central and southern italy such as rome and naples a focus on the temporal association between ground levels of no2 and covd19 cases total cases daily new positive cases and total deaths was performed by zoran 2020b for the city of milan italy in the period pre and postlockdown measures the authors nitrogen dioxide is a nastysmelling gas formed by reaction in the atmosphere of nitrogen oxides nox with other chemicals nox is naturally produced in atmosphere by lightning kang et al volcanoes oceans and biological decay thurston the major outdoor anthropogenic sources of nox are primarily emissions from transportation and fuel combustion in particular in urban areas they comes from vehicle exhaust gases and domestic heating grange maawa the nitrogen dioxide has mainly effect on the respiratory system because an increase of the outdoor concentration of no2 may significantly increase the risk of respiratory tract infection this phenomenon is particularly evident in children as they are more susceptible to no2 environmentalresearch19120201101296 0cacknowledgments c copat found no2 negative correlated with all the considered epidemiological data but the methodology used has some limitations as the delay time from infection to the covid19 onset or covid19 death was not considered as well the significant reduction of air pollution due to lockdown measures since midmarch in usa the association was also studied by bashir for the state of california as discussed above for pm the authors found a negative correlation also between no2 levels and covid19 cases and mortality nevertheless they stated that this pollutant contributes to the spread of the disease based on these scientific evidences in addition to confirming that exposure to no2 is harmful to human health and increases the risk of incurring respiratory diseases it can be stated that exposure to no2 may be one of the most important trigger for the spread and fatality caused by the covid19 disease declare references adhikari a yin j shortterm effects of ambient ozone pm25 and the authors declare no conflict of interest we have no funding to bontempi e 2020b first data analysis about possible covid19 virus airborne alifano m alifano p fez p iannelli a reninangiotensin system at the meteorological factors on covid19 confirmed cases and deaths in queens new york int j environ res publ health httpsdoi103390 ijerph17114047 heart of covid19 pandemic biochimie httpsdoi101016j biochi202004008 baccini m biggeri a grillo p consonni d bertazzi pa health impact assessment of fine p pollution at the regional level am j epidemiol “ httpsdoi101093ajekwr256 bahrami asl f leili m vaziri y salahshour arian s cristaldi a oliveri conti g ferrante m health impacts quantification of ambient air pollutants using airq model approach in hamadan iran environ res “ httpsdoi 101016jenvres201710050 bashir mf ma bj bilal komal b bashir ma farooq th iqbal n bashir m correlation between environmental pollution indicators and covid19 pandemic a brief study in californian context environ res https doi101016jenvres2020109652 becker s soukup jm exposure to urban air particulates alters the macrophage mediated inflammatory response to respiratory viral infection j toxicol environ health “ httpsdoi101080009841099157539 bontempi e 2020a commercial exchanges instead of air pollution as possible origin of covid19 initial diffusion phase in italy more efforts are necessary to address interdisciplinary research environ res httpsdoi101016j envres2020109775 diffusion due to air particulate matter pm the case of lombardy italy environ res httpsdoi101016jenvres2020109639 bontempi e vergalli s squazzoni f understanding covid19 diffusion requires an interdisciplinary multidimensional approach environ res httpsdoi101016jenvres2020109814 bremner sa anderson hr atkinson rw mcmichael aj strachan dp bland j m bower js shortterm associations between outdoor air pollution and mortality in london occup environ med “ httpsdoi 101136oem564237 cai qc lu j xu qf guo q xu dz sun qw yang h zhao gm jiang qw influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome publ health “ https doi101016jpuhe200609023 carugno m dentali f mathieu g fontanella a mariani j bordini l milani g p consonni d bonzini m bollati v pesatori ac pm10 exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in lombardy italy environ res “ https doi101016jenvres201806016 chen h chen y lian z wen l sun b wang p li x liu q yu x lu y qi y zhao s zhang l yi x liu f pan g 2020a correlation between the migration scale index and the number of new confirmed coronavirus disease cases in china epidemiol infect e99 httpsdoi101017 s0950268820001119 chen j zeng j shi c liu r lu r mao s zhang l associations between shortterm exposure to gaseous pollutants and pulmonary heart diseaserelated mortality among elderly people in chengdu china environ health httpsdoi 101186s1294001905008 chen s prettner k kuhn m geldsetzer p wang c b¨arnighausen t bloom de 2020b covid19 and climate global evidence from countries medrxiv prepr serv health sci httpsdoi1011012020060420121863 coccia m 2020a how high wind speed can reduce negative effects of confirmed cases and total deaths of covid19 infection in society ssrn scholarly paper no id social science research network rochester ny httpsdoi 102139ssrn3603380 coccia m 2020b factors determining the diffusion of covid19 and suggested strategy to prevent future accelerated viral infectivity similar to covid sci total environ httpsdoi101016jscitotenv2020138474 balakrishnan k brunekreef b dandona l dandona r feigin v freedman g hubbell b jobling a kan h knibbs l liu y martin r morawska l pope ca shin h straif k shaddick g thomas m van dingenen r van donkelaar a vos t murray cjl forouzanfar mh estimates and year trends of the global burden of disease attributable to ambient air pollution an analysis of data from the global burden of diseases study lancet lond engl httpsdoi101016s0140673617305056 “ conticini e frediani b caro d can atmospheric pollution be considered a co factor in extremely high level of sarscov2 lethality in northern italy environ pollut barking essex httpsdoi101016jenvpol2020114465 croft dp zhang w lin s thurston sw hopke pk van wijngaarden e squizzato s masiol m utell mj rich dq associations between source cohen aj brauer m burnett r anderson hr frostad j estep k conclusion the scientific evidences collected in the literature highlight the important contribution of chronic exposure to air pollution on the covid19 spread and lethality although the potential effect of airborne virus exposure it has not been still demonstrated in particular it seems that pm25 and no2 are more closely correlated to covid19 than pm10 the lower correlation of pm10 with covid19 incidence and mortality can be due to the impossibility of particulate matter greater than μm to reach type ii alveolar cells where is located the cell entry receptor ace2 for sarscov2 nevertheless differences between countries such as the implementation of different lockdown restrictions stage of infection topographic sociodemographic and socioeconomic characteristics level of air pollution and meteorological factors may have contributed to obtain some contrasting finding although most of the revised studies support the relationship between air pollution and covid19 the manifold limitations of this review are the small number of papers collected and the great diversity of methodologies used sometimes lacking in some parts which makes the results difficult to compare the authors who first investigated this association although with great effort and rapidity of analysis dictated by a global emergency sometimes do not include all confounding factors whenever possible such as control policy urbanization rate availability of medical resources population size weather lifestyles sociodemographic and socioeconomic variables in addition to date incidence data are underestimated in all countries and to a lesser extent mortality data for this reason the cases included in the considered studies cannot be considered conclusive more studies are needed to better clarify the role of air pollution during the covid19 pandemic particularly studies that consider the multiplepollutants to strengthen scientific evidences and support firm conclusions useful to implement pandemic application plans to adequately prevent new health emergencies for a long time we have known that reducing outdoor and indoor air pollution in cities or countries can have a significant effect on health almost immediately and the benefits can far outweigh the costs surely the health emergency that the world is experiencing right now highlights how environmental research is a fundamental reference point to improve the knowledge concerning diseases of infectious origin and how all the intellectual and economic resources are to be spent to accelerate actions aimed to implement environmental policies act to reduce air pollution and develop new urban planning interventions influences or multidisciplinary studies declaration of competing interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper environmentalresearch19120201101297 0cc cop Answer:

Colon_Cancer

the ongoing pandemic coronavirus disease outbreak covid2019 stemming from the beta coronavirus class sarscov2 causing severe acute respiratory syndrome sars has created a global health emergency in countries around the globe huang nalla sarscov2 a novel coronavirus strain belonging to the sarbecovirus subgenus genus betacoronavirus family coronaviridae had first appeared in late in wuhan china infecting a large number of hosts million people with a mortality rate of ‰¥ neogi carter such putative etiopathogenic agents associated with the zoonotic viral transmission pathways are responsible for respiratory viral pneumonia and gastrointestinal infections leading to infected people or patients having multiple an failure in a b s t r a c t severe acute respiratory syndrome coronavirus sarscov2 a neoteric virus belonging to the beta coronavirus class has created a global health concern responsible for an outbreak of severe acute respiratory illness the covid19 pandemic infected hosts exhibit diverse clinical features ranging from asymptomatic to severe symptoms in their genital ans respiratory digestive and circulatory systems considering the high transmissibility r0 ‰ compared to middle east respiratory syndrome coronavirus merscov and sarscov the quest for the clinical development of suitable antiviral nanotherapeutics ntps is incessant we are presenting a systematic review of the literature published between and to validate the hypothesis that the pharmacokinetics collateral acutechronic side effects of nano drugs and spike proteins arrangement of coronaviruses can revolutionize the therapeutic approach to cure covid19 our aim is also to critically assess the slow release kinetics and specific target site chemical synthesis influenced competence of ntps and nanotoxicity based antiviral actions which are commonly exploited in the synthesis of modulated nanomedicines the pathogenesis of novel virulent pathogens at the cellular and molecular levels are also considered which is of utmost importance to characterize the emerging nanodrug agents as diagnostics or therapeutics or viral entry inhibitors such types of approaches trigger the scientists and policymakers in the development of a conceptual framework of nanobiotechnology by linking nanoscience and virology to present a smart molecular diagnosis treatment for pandemic viral infections comorbidities till date no therapeutic drug has been discovered for the treatment of sarscov2 infection though the importance of monoclonal antibodies protease and helicase inhibitors and interferons ifnsα treatments have been highlighted in a handful of literature farzin torchilin palmieri and papi development of drugresistant strains host cell toxicitytarget specific actions costs associated with the serological diagnosis and therapeutics limit the widespread application of synthetic drugs nucleoside analogs palmieri and papi torchilin harnessing the potential of nanobiotechnology in the biomedical science development of engineered nanostructured materialsnanomedicines may lead to better drug delivery advanced therapeutics and medical diagnostics at nanoscale in recent years with the advancement of clinical practices the use of metal gold silver zinc and copper corresponding author discipline of earth sciences room no 336a block indian institute of technology gandhinagar gujarat india email addresses manishenvgmailcom manishkumariitgnacin m kumar 101016jenvres2020110119 received may received in revised form july accepted august environmentalresearch1912020110119availableonline23august2020001393512020elsevierincallrightsreserved 0cs mukherjee nanops in magnetic immunoassay viral diagnostics and microfluidic technology has driven the researchers to explore the potential of nanotherapeutics makvandi farzin letko and ahlawat and narayan have investigated the multistrain inhibition of sars coronavirus sarscov herpes simplex virus hsv1 and human immunodeficiency virus hiv of tcells by sulfonated nanops binding they have reported that small p size tunable surface charges biomimetic properties faster encapsulation have made the engineered nanops smart and stable colloidal carriers for the delivery of genes and drugs the mode of action of functionalized nanops can be explained by their covalent linkages with biological substrates such as peptides proteins antibodies and nucleic acids some researchers have reported virusnanops electrostatic nonspecific interactions elsewhere ie influenza a h5n1 strain inhibition alizadeh and khodavandi rothan and byrareddy nanoparticulate drug carriers through the cellular and mechanistic establishment crossed the membranes and with the help of capping agents such as sulfate polysaccharidespolymers undergo multivalent bond interactions with virus glycoproteins ie hemagglutinin ha bachmaier balakrishna such surface proteins often act as a prime inducer of neutralizing antibodies as observed by boulware and chaturvedi and shrivastava where antigenic determinants enable nucleocapsid to take entry into the host cells as reported for in vitro sarscov inhibition of virus replication and the fusion between the viral and host cell endosomal membrane are often facilitated by some emerging nanobased technologies ie silver nanorod array surface enhanced raman spectroscopy sers substrate interferometric biosensor immunoassay chauhan chhikara choudhary the development of such biomolecular detectors enable the molecular binding of virus ps to target specific antigen phase antibodies coated waveguide which help in the fast and reliable detection of viral infections kirchdoerfer and ward cojocaru das considering the high transmissibility r0 ‰ and low to moderate pathogenicity of sarscov2 compared to middle east respiratory syndrome coronavirus merscov and sarscov the quest for clinical development of suitable antiviral nanotherapeutics is need of table a summary of biocompatible nanomaterials and antiviral nanopharmaceuticals commonly used for biomedical drug delivery action as virucidal agents souce of data weiss udugama letko jamshidi gao dong nanocarriers aunps immobilized aunps tio2 nps modified tio2nps agnps engineered agnps sinps mesoporous si fullerenes modified fullerenes fenps engineered fenps acidbasic functionalized nanotube metal functionalized nanotube carbon dots graphene oxide go polystyrene nps virucidal action immunizationviral detection viral detection inactivation of virus by photolysis virus inhibitioninactivation viral entry inhibition viral replication deformation virus detection viral entry inhibition virus destabilization inhibition of virus entry host pathogen interaction viral detectionremoval virus inactivation immunization viral inhibition viral entry hindrance mucosal vaccine development target specific cell bhk21 helacdltr vero cells c636 balbc mice na mdck vero cells human rhabdomyosarcoma vero cells hek293t na supt1 na na ncih292 grass carp pk15 marc145 vero cells na virus types hin1 h3n2 hiv1 h3n2 dengue virus h5n1 h3n2 h5n1 h1n1 h7n3 feline calicivirus influenza papilloma virus hsv1 bacteriophage λ hiv1 wild and resistant type zika virus bacteriophage ms2 h5n2 h3n2 reovirus rsv pseudorabies virus porcine epidemic diarrhea virus hiv1 mode of antiviral activity binding with peroxidasemimic enzymes and viral gp120 antibody mediated inhibition viral capsid protein interaction viral surface protein interaction inhibit cd4based binding viral envelop rupture cell mediated immunenucleic acid inhibition hinder viral attachment viral capsidenvelop attachment and interaction impairing viral polyproteinhinder gag processing viral envelopprotein binding phosphatidylserine inhibit viral tropism photoactivated mediated viral inhibition destabilization vp7dna mediated inhibition type i interferon production inhibited negative single layered sharp edged p interaction with virus viral gp120 antigen binding and mannoselectin specific inhibition action immune system inhibition cd8 t cells inhibition antibodydependent cellmediated phagocytosiscytotoxicity viral transcriptase inhibition gene silencing action viral cell entry inhibition peroxidase inhibition viral entry inhibition coagulation results from virus surface protein interaction magnb mediated enzymeatic signaling inhibition cell cycle inhibition at g2m phase glycosaminoglycan binding affinity and in vitro replication inhibition chitosan coated nps peptide coated nps protein coated nps amide coated nps nanoliposomes nanomicelle aunpscarbon nanotubes polymeric micelle aunpsagnps aufenpscarbon nanotubes nanolipid carriers dendrimer na balbc strain balbc mice neuro 2a cell lines na apc49 huh75 na male wistar rats cell mdck na vk2e6e7 vero cells helfs rabies virus influenza a virus influenza a virus hiv na hepatitis c virus h3n2 hiv h1n1 h3n2 norovirus h1n1 papilloma virus hsv12 immunization nanoparticulate vaccine influenza vaccine antiviral therapy drug delivery immunomodulator antiviral activity and bioavailable vaccines viral detection by colorimetric assay oral bioavailable drugs viral inhibition and drug delivery action viral dna detection nontoxic viral inhibition mrna vaccine na not applicable aunpsgold nanops agnpssilver nanops fenpsiron nanops sinpssilica nanops tio2 titanium nanops gographene oxides environmentalresearch19120201101192 0ccome under anic nanocarrier systems based on the their function as therapeutic agents encapsulated chemical attachment adsorbed or dissolved grein neogi gacem fig demonstrates the comparative size range of nanops whereas for hybrid nanobased systems the molecular composition and target specific action of individual engineered nanoparticulate systems play a vital role in drug delivery actions table the mode of action of such nanobased therapeutic agents can be categorised based on the permeability of vasculature passive targeting for malignancy or attachment of bioactive ligands to the selective nanotherapeutics kirchdoerfer and ward gao gadade and pekamwar role of metallic nanops as in vivo and in vitro drug discharging agents s mukherjee the hour rosenberg nalla dung though it is noteworthy to mention that r0 is not data specific to the covid19 disease rather it depends on the place and the behavior of the population it is associated with social isolation hygienic habits among others and the variation must occur in different ways viceconte and petrosillo li the drug development for antiviral treatment of sarscov2 depends on several factors such as pharmacokinetics drug properties collateral acutechronic side effects and spike proteins s arrangement of coronaviruses virus properties these can act as therapeutic targets to prevent the fusion of the virus to host cell via binding with host cell receptors trimeric scaffolds such as nsp10 a viral transcription factor used along with secondary antigens in vaccines for lung and lower respiratory tract infections these are essential to nucleate and stabilize pseudosubdomains of protein and peptide antigens which are also responsible for cell infection and polycistronic expression that may help in the nanobased therapeutic vaccine development dong grein etman peginterferon α2a2b pegasys ifnα2b show excellent drug delivery actions against hepatitis virus c whereas cationiccrosslinked nanops biodegradable polymers such as mpolyethylene glycol mpeg€“pla clpm are found suitable for hepatitis b and c virus infections synthetic and natural polymeric nanops pnps are widely used since the last decade for the effective control of pathogenic viruses based on their individual biochemical properties immuno biocompatibility biodistribution factors table low to zero toxicity profile high stability against proteinase degradation improved safety efficacy profiles and good internalization properties make such nanozymes effective against chronic infectious diseases and also help in exerting their cytoprotective actions against cytopathic effect polymeric nanops in hybridized forms have found their way in biomedical sectors as therapeutics and diagnostics of human adenovirus influenza virus and hiv therefore the widespread application of hybridized nanoformulation systems have been documented by kerry and kim where functionalized dnazyme along with cellular peptides encoding viral envelopes help in the knockdown of hsv2 hvc and influenza a viruses for the first time the purpose of the present systematic review lays out the framework for a developing a critical understanding of selfassembling metal nanops targeting a variety of fusion proteins for vaccine development b the spatial geometry three fold symmetry axis and radial distributions that drive the rapid antigen processing and render virucidal activity c building up a deep insight for biomarkers research in both prophylactic and therapeutic approaches d the critical assessment of nanops as therapeutics pathways biodegradation this review also provides a guide map for the regulation of metabolic enzymes on selected nanopharmaceuticals through which the multigenerational effects can be evaluated this publication is designed to provide vital information on biocompatible nanocarriers active vs passive targeted drug delivery action of nanomedicines and critically analyze the possible hybrid nanobased therapy for sarscov2 inhibition our current review also highlights the stateofart molecular fingerprinting techniques of virus identification through advanced biosensing methods which critically explore integral surveillance and monitoring of novel viral genotypes mode of action and biomedical applications of biocompatible nanocarriers the presence of multiple surface binding sites in vivo clinical interaction with the targeted sites composition based multiple interactions shape luminescence and large surfacevolume ratio of inanic nanops render their multifarious biomedical applications ignatov dong the virucidal activity of silver nanops agnps allows its wider application in the annihilation or amelioration of several viral infections such as poliovirus type1 coxsackievirus b3 influenza a virus etc the mode of action of such nanops can be described either inhibition of cd4dependent cellular bindingpathogenesis or by covalent linking with sulfhydryl group virion surface hill neogi elsheekh the viral internalization can also be prevented by merging nanops with viral genomecore protein that hinders viral replicationattachment release of viral core into the cytoplasm and governing conformational changes in viruscoreceptor association letko hu this type of viral replication inhibitions are quite effective in curing of dsrna viruses infectious hsv2 and bursal disease viruses iravani jamshidi on the other hand gold nanops aunps may also exert their antiviral efficacy through hindering of gp120 fusion to cd4 that are surrounded by capping agents encapsulated aunps table hybridized and charged aunps render virucidal mechanism after associating with nanobased formulation and mimicking peroxidase enzymatic reaction where inhibited viral entry is facilitated by blocking of transcription within the host cell isida kumar 2020a 2020b 2020c kang on the other hand stabilization of magnetic nanops is often carried out by biocompatible polymers which helps in the translation of magneto responsive nanoparticulate systems in clinical diagnostics bio imaging bioresonanceimaging and cell separation the use of superparamagnetic iron nanops γfe2o3fe2o3fe3o4 is not directly involved in the therapeutics but their indirect application may inhibit viral multiplication even at postentry cellular level as seen for zika virus h5n2 and hcv zhou 2020ab dong photothermal nanotherapy high refractive index photocatalytic activities and high solubility properties of titanium nanops tinps enable them to find their wide antiviral application for bacteriophages and h3n2 viruses grein kar the clinical application of functionalized silica nanops sinps drives the diverse antiviral therapeutics or diagnostics application of nanocarriers in inhibition of hiv hepatitis b virus and other recombinant viruses kim nucleic acid hybridization and fluorescent based virus viral protein detection methods are getting popular based on the antiviral efficacy of sinps though their meso to nanoporous biocompatible surfaces allow hostpathogen interaction the emerging demand for novel antiviral nanotherapeutics triggers the researchers to ponder about immunomodulation and immunization of host cells these are essential to regulate premature drug release or inhibition of viral entry through in vivo biodistribution kalantarzadeh kerry the different forms of nanomaterials that are mostly used as antiviral agents depend largely on the pathway of drug delivery system which provide versatile forms of nanobased carriers starting from complex anic hybrid nanosystems to simple inanic metallic composites nanocapsules nanocages and nanospheres are categorised under inanic nanop based systems though nanocapsules can sometimes through and associated drug metabolism environmentalresearch19120201101193 0cs mukherjee fig schematic of the size range of nanops commonly applied in clinical practice as drug delivery agents for gene and drug delivery system quantum dots qd a type of nanosized crystals have excellent nanobased sensing which allow them to be used as antiviral therapeutics or in vitro diagnosis of virulent pathogens where strong chemical interactionsbonding render their biochemical conjugation with therapeutic molecules kostarelos kumar et al 2020a 2020b 2020c and zhou 2020ab have shown that different metallic composition pb cu ga zn hg based qd showed target specific actions against hiv1 human tlymphotropic virus1 after their binding with nh2 receptorbiotin acceptor some researchers have shown that nanoformulations based qds crossed the bbb in vitro model along with target dna and saquinavir antiviral agent which have been widely utilized as highly active antiretroviral therapy kumar 2020a 2020b 2020c lembo evaluation of anic and hybrid nanops as therapeutics and in drug delivery action anic nanopsnanocarriers onp play an important role in the drug delivery action if the therapeutic compounds are of large sized molecules €“ nm encapsulation of such nanoagents through environmentalresearch19120201101194 0cs mukherjee specific designstructure render offtarget toxicity which is required for target specific action lopez gacem slow release kinetics and specific target site chemical synthesis have direct influence on onps therapeutic competence kumar 2020a 2020b 2020c li polymeric nanops pnps have the clinical potential to carry the target drugs to its core or can coordinate with target molecules on its planar surface mainardes and diedrich nasrollahzadeh biocorona formation ease of biodegradation strong mechanicalthermal properties of carbonbased nanocarriers nanorods nanodots etc render their antiviral activities against respiratory syncytial virus rsv hiv1 ebola virus etc table in spite of having broad spectrum activities against antiviral infections the ratelimiting phase of these carbon materials limits their further biomedical applications nikaeen and nguyen have suggested that drug conjugated nanops having excellent antiviral activities can help in the development of an influenza vaccine with matrix protein m2e and hemagglutinin haamine functionalized gelatin surface coating though more research is required to validate the immunity and protection of cellular genes for such impeccable protein vaccines against infectious virulent pathogens like sarscov2 with the advancement of medical sciences the emergence of multifunctional nanobased lipid nanocarriers opens a new door in the field of novel antiviral nanotherapeutics table read and risitano have demonstrated that podophyllotoxin stearic acidglycol loaded lipid nanocarriers are useful in maintaining in vitro slow drug release nontoxic viral inhibition and hemocompatibility of vk2e6e7 hela cells such solidlipid nanops and other nanolipid carriers can fig a and b schematic of the internalization of nanodrugs through the plasma membrane and targeted drug release a and transcytosis of nanodrugs through cell barriers b nanoparticulate drug carriers through the cellular and mechanistic establishment crossed the membranes bloodbrain barrier and bloodtestis barrier and with the help of capping agents such as sulfate polysaccharidespolymers undergo multivalent bond interactions with virus glycoproteins ie hemagglutinin ha environmentalresearch19120201101195 0cs mukherjee become the exceptional antiviral drug discharging agents for infectious viral pathogens ie hpv hiv and hepatitis c virus nú˜nezdelgado prather hyperbranched monodispersed easily biodegradable anic dendrimers have gained importance for more than a decade in the field of nanomedicine because they act as targeted carriers for biological systems the antiviral activity of dendrimers is still under scientific investigations which may be facilitated through conjugated drug delivery mechanisms patil palestino the efficacy of such nanoparticulated dendrimer system can be observed for the inhibition of hsv1influenza virus where antibody mediated responsecd8 t cell activation and regulation of gene expressions are achieved through small rnas inactivation shang sportelli niosomes the nonionic surfactant based liposome alike anic nanovesicle are getting popular in the advanced biomedical sciences considering their nonimmunogenicity and stable optical properties which make them a suitable carrier of both hydrolipophilic drug molecules excellent bioavailability and controlled release of specific drugs at the targeted sites make such nanocarriers ideal antiviral agents hsv virus where nanoniosome was loaded with suitable antiviral drug acyclovir s´anchezl´opez et al improved drug delivery mechanism and suitable drug release kinetics may allow such nanocarriers to be used in infectious virus diseases nanomicelles fig supramolecular globular micelles exhibit colloidal stability and super encapsulation potential which help such polymeric micelles to show antiviral activity in vitro as observed from curcumin loaded bioavailable nanoformulations hepatitis c virus sivasankarapillai some researchers used graphene oxides conjugated adnps against infectious sars and bursal viruses where the drug resistance event after such antiviral efficacy was mediated by selenium nanops senps andor amantadine am arrangement te velthuis kumar 2020a 2020b 2020c diagnosis or detection of such virulent pathogens have been documented by viceconte and petrosillo and vazquezmunoz and lopez through thiolstabilized gold cluster or enteroviruses labeling with cysteine molecule a brief overview about antiviral nanomedicines rsv virus tremiliosi udugama the size and zeta potential of silver nanops can exert inhibition effects on different human parainfluenza three virus strains or on their replication event the nanocolloidal system of vivagel® is immensely used for the control of zika virus infection tkm130803 is widely used in the treatment of ebola virus utilizing the concept of rnaibased therapy for the lipidbased nanosystem it is well doccumented that for human norovirus treatment the employment of goldcopper sulfide coreshell capsid protein binding results into excellent virucidal activity ziaie on the other hand nanotrap ps are quite often used in the inhibition of infection of target cells by capturing viral rnaviral proteins ie influenza virus treatment zhou 2020ab zhang intrinsic in vivo instability poor immunogenicity and toxicity multiple therapeutic and prophylactic approaches can be overcome by nanovaccinology where cellular and humoral immune response drive the faster uptake of mucosagut associated lymphoid tissue slow controlled release of antigens is facilitated by surface modifications of nanovaccines with antibodiescarbohydrates which results in the target specific immune response by different immune cells additionally their small size and prolonged shelf life help in the faster recognition of the hostreceptor immune system ie hepatitis a virus hav and influenza virus where epaxalexapal is used with immuno targeting agents yu yang and wang nonresponsive immune systems high dose administration coldchain transport of parenteral vaccines limit their widespread application in drug therapy particularly for mucosally administered vaccines the chitosannanop embedded system might be useful for therapeutic proteins or antigens having negative charges which makes its wider application in vaccination against hbv virus through gene delivery systems table the utilization of mouse model employing humoral and mucosal immune responses helped in the liposomebased vaccine development in case of hepaxen used for hepatitis a c and e which further utilized the recombinant surface antigen as a prophylactic vaccine waris wang 2020a 2020b the usages of inflexal v and influvac as standard virosomal vaccine against influenza virus are getting quite popular considering its active biocompatible and immunogenicity wu weiss these types of licensed subunit nanovaccines are found quite useful for older infants and middleaged group people in terms of nanosafety issue as they mimic natural infections as seen in table a cysteineguanine rich oligonucleotide combination with extracellular m2egold conjugates renders molecular protection for pr8h1n1 influenza which was further activated by thiolgold interactions zhang zhou 2020ab from the discussion provided here it is clear that most of the research has been done for influenza virus vaccination but some scanty literature also report several nanomedicines including nanovaccines are under clinical trial or at least in the stage of commercialization for the cure of infectious viral diseases table in general the use of drugs for antiviral therapy is usually employed to target different life cycles of virulent pathogens ie hiv ebola virus or hsv1 valdiglesias and laffon alnrsv01 is a commonly used lipid nanop drug for lower tract respiratory disease which targets the nucleocapsid €œn€ gene of table commercial nanomedicines or under clinical trial for the antiviral therapytreatment souce of data neogi letko dong kalantarzadeh kang nanomedicines influvac plus tkmhbv cervisil doravirine dermavir inflexal v epaxal pegasys geovax novavax fluquit curevac peglntron vivagel a pepreclinical evaluation uceunder clinical evaluation hav hepatitis a virus hiv human immunodeficiency virus hbv hepatitis b virus hpv human papillomavirus hcv hepatitis c virus hsv herpes simplex virus mode of action presence of neuraminidase and hemagglutinin rnai therapeutics gene silencing reverse trancriptase inhibitor nonnucleoside dna immunogen with hiv specific t cell precursor antigens specific on speherical carriers surface natural process mimics peroxidases pegylation control stability of protein ankara€”virus alike drug therapy clinical stage antiviral nanobiotechnology gene silencing mrna technique pegylation control stability of protein dendrimer with sulphonic acid group interaction disease indication influenza hbv hpv hiv hiv influenza hav hbv hcv sarscov2 sarscov2 influenza sarscov2 hcv hsv hiv biomedical application virosome vaccine solidlipid nanop sirna therapeutic nanoparticulate formulation therapeutic vaccine liposome vaccine liposome vaccine pegylated interferon antiviral therapy nanoparticulate therapeutics sirna therapeutic infections virus vaccine pegylated interferon dendrimer yearstage of development uce preclinical evaluation ucea uce pe pe pea pe uce environmentalresearch19120201101196 0cs mukherjee vaccine research for rotanoroebola virus hpv rsv and others dung das metabolic pathway of nanotherapeutics and their limitations in clinical practice pulmonary and hepatotoxicity studies are required to build a safety profile of such nanodelivery therapeutic or diagnostic agents nanobased approach for sarscov2 infection inhibition nanops uptake cellular process in nanotherapeutics are governed by their physicochemical properties along with cellular membrane characteristics which may have direct influence on the rate of administered drug dosages and structure of engineered nanops it is hypothesized that nanops with optimum diameter of ‰ nm and high surface charge density are quite effective in crossing the cellular membranes for hivderived tat cell penetrating peptides weiss waris immunoliposomes and other carbon based nanotubesnanocarriers play an important role in the activation of the complement pathway of host immune systems to deregulate in vitro utilization of nps neogi kumar 2020a 2020b 2020c antibodies that are specifically targeted at polyethylene glycol pegmacrogol polymers and peglike nanostructures can show independent therapeutic efficacy based on their individual immunotoxicology and risk assessment strategies letko read experimental findings wang 2020a 2020b hill with nanobased therapeutic agents reveal the urgent requirement of more rigorous scientific investigations to prove their clinical efficiency in reversing the drug resistance event ie h1n1 virus through seam biodegradation process of nanotherapeutics has gained special attention considering uniform biodistribution kinetics and sustained drug release which are essential for improved drug design process distribution metabolism absorption excretion are important pharmacokinetic features which rate of biochemical features are directly governed by hydrophobichydrophilic profile and tacticity of the nano based formulations at in vitro level patil exocytosis process plays a very important role in the clearance of the foreign nanops out of the cell depending on administered nanocarriers it was hypothesized that ps with nm diameter can

cancer7535

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: the ongoing pandemic coronavirus disease outbreak covid2019 stemming from the beta coronavirus class sarscov2 causing severe acute respiratory syndrome sars has created a global health emergency in countries around the globe huang nalla sarscov2 a novel coronavirus strain belonging to the sarbecovirus subgenus genus betacoronavirus family coronaviridae had first appeared in late in wuhan china infecting a large number of hosts million people with a mortality rate of ‰¥ neogi carter such putative etiopathogenic agents associated with the zoonotic viral transmission pathways are responsible for respiratory viral pneumonia and gastrointestinal infections leading to infected people or patients having multiple an failure in a b s t r a c t severe acute respiratory syndrome coronavirus sarscov2 a neoteric virus belonging to the beta coronavirus class has created a global health concern responsible for an outbreak of severe acute respiratory illness the covid19 pandemic infected hosts exhibit diverse clinical features ranging from asymptomatic to severe symptoms in their genital ans respiratory digestive and circulatory systems considering the high transmissibility r0 ‰ compared to middle east respiratory syndrome coronavirus merscov and sarscov the quest for the clinical development of suitable antiviral nanotherapeutics ntps is incessant we are presenting a systematic review of the literature published between and to validate the hypothesis that the pharmacokinetics collateral acutechronic side effects of nano drugs and spike proteins arrangement of coronaviruses can revolutionize the therapeutic approach to cure covid19 our aim is also to critically assess the slow release kinetics and specific target site chemical synthesis influenced competence of ntps and nanotoxicity based antiviral actions which are commonly exploited in the synthesis of modulated nanomedicines the pathogenesis of novel virulent pathogens at the cellular and molecular levels are also considered which is of utmost importance to characterize the emerging nanodrug agents as diagnostics or therapeutics or viral entry inhibitors such types of approaches trigger the scientists and policymakers in the development of a conceptual framework of nanobiotechnology by linking nanoscience and virology to present a smart molecular diagnosis treatment for pandemic viral infections comorbidities till date no therapeutic drug has been discovered for the treatment of sarscov2 infection though the importance of monoclonal antibodies protease and helicase inhibitors and interferons ifnsα treatments have been highlighted in a handful of literature farzin torchilin palmieri and papi development of drugresistant strains host cell toxicitytarget specific actions costs associated with the serological diagnosis and therapeutics limit the widespread application of synthetic drugs nucleoside analogs palmieri and papi torchilin harnessing the potential of nanobiotechnology in the biomedical science development of engineered nanostructured materialsnanomedicines may lead to better drug delivery advanced therapeutics and medical diagnostics at nanoscale in recent years with the advancement of clinical practices the use of metal gold silver zinc and copper corresponding author discipline of earth sciences room no 336a block indian institute of technology gandhinagar gujarat india email addresses manishenvgmailcom manishkumariitgnacin m kumar 101016jenvres2020110119 received may received in revised form july accepted august environmentalresearch1912020110119availableonline23august2020001393512020elsevierincallrightsreserved 0cs mukherjee nanops in magnetic immunoassay viral diagnostics and microfluidic technology has driven the researchers to explore the potential of nanotherapeutics makvandi farzin letko and ahlawat and narayan have investigated the multistrain inhibition of sars coronavirus sarscov herpes simplex virus hsv1 and human immunodeficiency virus hiv of tcells by sulfonated nanops binding they have reported that small p size tunable surface charges biomimetic properties faster encapsulation have made the engineered nanops smart and stable colloidal carriers for the delivery of genes and drugs the mode of action of functionalized nanops can be explained by their covalent linkages with biological substrates such as peptides proteins antibodies and nucleic acids some researchers have reported virusnanops electrostatic nonspecific interactions elsewhere ie influenza a h5n1 strain inhibition alizadeh and khodavandi rothan and byrareddy nanoparticulate drug carriers through the cellular and mechanistic establishment crossed the membranes and with the help of capping agents such as sulfate polysaccharidespolymers undergo multivalent bond interactions with virus glycoproteins ie hemagglutinin ha bachmaier balakrishna such surface proteins often act as a prime inducer of neutralizing antibodies as observed by boulware and chaturvedi and shrivastava where antigenic determinants enable nucleocapsid to take entry into the host cells as reported for in vitro sarscov inhibition of virus replication and the fusion between the viral and host cell endosomal membrane are often facilitated by some emerging nanobased technologies ie silver nanorod array surface enhanced raman spectroscopy sers substrate interferometric biosensor immunoassay chauhan chhikara choudhary the development of such biomolecular detectors enable the molecular binding of virus ps to target specific antigen phase antibodies coated waveguide which help in the fast and reliable detection of viral infections kirchdoerfer and ward cojocaru das considering the high transmissibility r0 ‰ and low to moderate pathogenicity of sarscov2 compared to middle east respiratory syndrome coronavirus merscov and sarscov the quest for clinical development of suitable antiviral nanotherapeutics is need of table a summary of biocompatible nanomaterials and antiviral nanopharmaceuticals commonly used for biomedical drug delivery action as virucidal agents souce of data weiss udugama letko jamshidi gao dong nanocarriers aunps immobilized aunps tio2 nps modified tio2nps agnps engineered agnps sinps mesoporous si fullerenes modified fullerenes fenps engineered fenps acidbasic functionalized nanotube metal functionalized nanotube carbon dots graphene oxide go polystyrene nps virucidal action immunizationviral detection viral detection inactivation of virus by photolysis virus inhibitioninactivation viral entry inhibition viral replication deformation virus detection viral entry inhibition virus destabilization inhibition of virus entry host pathogen interaction viral detectionremoval virus inactivation immunization viral inhibition viral entry hindrance mucosal vaccine development target specific cell bhk21 helacdltr vero cells c636 balbc mice na mdck vero cells human rhabdomyosarcoma vero cells hek293t na supt1 na na ncih292 grass carp pk15 marc145 vero cells na virus types hin1 h3n2 hiv1 h3n2 dengue virus h5n1 h3n2 h5n1 h1n1 h7n3 feline calicivirus influenza papilloma virus hsv1 bacteriophage λ hiv1 wild and resistant type zika virus bacteriophage ms2 h5n2 h3n2 reovirus rsv pseudorabies virus porcine epidemic diarrhea virus hiv1 mode of antiviral activity binding with peroxidasemimic enzymes and viral gp120 antibody mediated inhibition viral capsid protein interaction viral surface protein interaction inhibit cd4based binding viral envelop rupture cell mediated immunenucleic acid inhibition hinder viral attachment viral capsidenvelop attachment and interaction impairing viral polyproteinhinder gag processing viral envelopprotein binding phosphatidylserine inhibit viral tropism photoactivated mediated viral inhibition destabilization vp7dna mediated inhibition type i interferon production inhibited negative single layered sharp edged p interaction with virus viral gp120 antigen binding and mannoselectin specific inhibition action immune system inhibition cd8 t cells inhibition antibodydependent cellmediated phagocytosiscytotoxicity viral transcriptase inhibition gene silencing action viral cell entry inhibition peroxidase inhibition viral entry inhibition coagulation results from virus surface protein interaction magnb mediated enzymeatic signaling inhibition cell cycle inhibition at g2m phase glycosaminoglycan binding affinity and in vitro replication inhibition chitosan coated nps peptide coated nps protein coated nps amide coated nps nanoliposomes nanomicelle aunpscarbon nanotubes polymeric micelle aunpsagnps aufenpscarbon nanotubes nanolipid carriers dendrimer na balbc strain balbc mice neuro 2a cell lines na apc49 huh75 na male wistar rats cell mdck na vk2e6e7 vero cells helfs rabies virus influenza a virus influenza a virus hiv na hepatitis c virus h3n2 hiv h1n1 h3n2 norovirus h1n1 papilloma virus hsv12 immunization nanoparticulate vaccine influenza vaccine antiviral therapy drug delivery immunomodulator antiviral activity and bioavailable vaccines viral detection by colorimetric assay oral bioavailable drugs viral inhibition and drug delivery action viral dna detection nontoxic viral inhibition mrna vaccine na not applicable aunpsgold nanops agnpssilver nanops fenpsiron nanops sinpssilica nanops tio2 titanium nanops gographene oxides environmentalresearch19120201101192 0ccome under anic nanocarrier systems based on the their function as therapeutic agents encapsulated chemical attachment adsorbed or dissolved grein neogi gacem fig demonstrates the comparative size range of nanops whereas for hybrid nanobased systems the molecular composition and target specific action of individual engineered nanoparticulate systems play a vital role in drug delivery actions table the mode of action of such nanobased therapeutic agents can be categorised based on the permeability of vasculature passive targeting for malignancy or attachment of bioactive ligands to the selective nanotherapeutics kirchdoerfer and ward gao gadade and pekamwar role of metallic nanops as in vivo and in vitro drug discharging agents s mukherjee the hour rosenberg nalla dung though it is noteworthy to mention that r0 is not data specific to the covid19 disease rather it depends on the place and the behavior of the population it is associated with social isolation hygienic habits among others and the variation must occur in different ways viceconte and petrosillo li the drug development for antiviral treatment of sarscov2 depends on several factors such as pharmacokinetics drug properties collateral acutechronic side effects and spike proteins s arrangement of coronaviruses virus properties these can act as therapeutic targets to prevent the fusion of the virus to host cell via binding with host cell receptors trimeric scaffolds such as nsp10 a viral transcription factor used along with secondary antigens in vaccines for lung and lower respiratory tract infections these are essential to nucleate and stabilize pseudosubdomains of protein and peptide antigens which are also responsible for cell infection and polycistronic expression that may help in the nanobased therapeutic vaccine development dong grein etman peginterferon α2a2b pegasys ifnα2b show excellent drug delivery actions against hepatitis virus c whereas cationiccrosslinked nanops biodegradable polymers such as mpolyethylene glycol mpeg€“pla clpm are found suitable for hepatitis b and c virus infections synthetic and natural polymeric nanops pnps are widely used since the last decade for the effective control of pathogenic viruses based on their individual biochemical properties immuno biocompatibility biodistribution factors table low to zero toxicity profile high stability against proteinase degradation improved safety efficacy profiles and good internalization properties make such nanozymes effective against chronic infectious diseases and also help in exerting their cytoprotective actions against cytopathic effect polymeric nanops in hybridized forms have found their way in biomedical sectors as therapeutics and diagnostics of human adenovirus influenza virus and hiv therefore the widespread application of hybridized nanoformulation systems have been documented by kerry and kim where functionalized dnazyme along with cellular peptides encoding viral envelopes help in the knockdown of hsv2 hvc and influenza a viruses for the first time the purpose of the present systematic review lays out the framework for a developing a critical understanding of selfassembling metal nanops targeting a variety of fusion proteins for vaccine development b the spatial geometry three fold symmetry axis and radial distributions that drive the rapid antigen processing and render virucidal activity c building up a deep insight for biomarkers research in both prophylactic and therapeutic approaches d the critical assessment of nanops as therapeutics pathways biodegradation this review also provides a guide map for the regulation of metabolic enzymes on selected nanopharmaceuticals through which the multigenerational effects can be evaluated this publication is designed to provide vital information on biocompatible nanocarriers active vs passive targeted drug delivery action of nanomedicines and critically analyze the possible hybrid nanobased therapy for sarscov2 inhibition our current review also highlights the stateofart molecular fingerprinting techniques of virus identification through advanced biosensing methods which critically explore integral surveillance and monitoring of novel viral genotypes mode of action and biomedical applications of biocompatible nanocarriers the presence of multiple surface binding sites in vivo clinical interaction with the targeted sites composition based multiple interactions shape luminescence and large surfacevolume ratio of inanic nanops render their multifarious biomedical applications ignatov dong the virucidal activity of silver nanops agnps allows its wider application in the annihilation or amelioration of several viral infections such as poliovirus type1 coxsackievirus b3 influenza a virus etc the mode of action of such nanops can be described either inhibition of cd4dependent cellular bindingpathogenesis or by covalent linking with sulfhydryl group virion surface hill neogi elsheekh the viral internalization can also be prevented by merging nanops with viral genomecore protein that hinders viral replicationattachment release of viral core into the cytoplasm and governing conformational changes in viruscoreceptor association letko hu this type of viral replication inhibitions are quite effective in curing of dsrna viruses infectious hsv2 and bursal disease viruses iravani jamshidi on the other hand gold nanops aunps may also exert their antiviral efficacy through hindering of gp120 fusion to cd4 that are surrounded by capping agents encapsulated aunps table hybridized and charged aunps render virucidal mechanism after associating with nanobased formulation and mimicking peroxidase enzymatic reaction where inhibited viral entry is facilitated by blocking of transcription within the host cell isida kumar 2020a 2020b 2020c kang on the other hand stabilization of magnetic nanops is often carried out by biocompatible polymers which helps in the translation of magneto responsive nanoparticulate systems in clinical diagnostics bio imaging bioresonanceimaging and cell separation the use of superparamagnetic iron nanops γfe2o3fe2o3fe3o4 is not directly involved in the therapeutics but their indirect application may inhibit viral multiplication even at postentry cellular level as seen for zika virus h5n2 and hcv zhou 2020ab dong photothermal nanotherapy high refractive index photocatalytic activities and high solubility properties of titanium nanops tinps enable them to find their wide antiviral application for bacteriophages and h3n2 viruses grein kar the clinical application of functionalized silica nanops sinps drives the diverse antiviral therapeutics or diagnostics application of nanocarriers in inhibition of hiv hepatitis b virus and other recombinant viruses kim nucleic acid hybridization and fluorescent based virus viral protein detection methods are getting popular based on the antiviral efficacy of sinps though their meso to nanoporous biocompatible surfaces allow hostpathogen interaction the emerging demand for novel antiviral nanotherapeutics triggers the researchers to ponder about immunomodulation and immunization of host cells these are essential to regulate premature drug release or inhibition of viral entry through in vivo biodistribution kalantarzadeh kerry the different forms of nanomaterials that are mostly used as antiviral agents depend largely on the pathway of drug delivery system which provide versatile forms of nanobased carriers starting from complex anic hybrid nanosystems to simple inanic metallic composites nanocapsules nanocages and nanospheres are categorised under inanic nanop based systems though nanocapsules can sometimes through and associated drug metabolism environmentalresearch19120201101193 0cs mukherjee fig schematic of the size range of nanops commonly applied in clinical practice as drug delivery agents for gene and drug delivery system quantum dots qd a type of nanosized crystals have excellent nanobased sensing which allow them to be used as antiviral therapeutics or in vitro diagnosis of virulent pathogens where strong chemical interactionsbonding render their biochemical conjugation with therapeutic molecules kostarelos kumar et al 2020a 2020b 2020c and zhou 2020ab have shown that different metallic composition pb cu ga zn hg based qd showed target specific actions against hiv1 human tlymphotropic virus1 after their binding with nh2 receptorbiotin acceptor some researchers have shown that nanoformulations based qds crossed the bbb in vitro model along with target dna and saquinavir antiviral agent which have been widely utilized as highly active antiretroviral therapy kumar 2020a 2020b 2020c lembo evaluation of anic and hybrid nanops as therapeutics and in drug delivery action anic nanopsnanocarriers onp play an important role in the drug delivery action if the therapeutic compounds are of large sized molecules €“ nm encapsulation of such nanoagents through environmentalresearch19120201101194 0cs mukherjee specific designstructure render offtarget toxicity which is required for target specific action lopez gacem slow release kinetics and specific target site chemical synthesis have direct influence on onps therapeutic competence kumar 2020a 2020b 2020c li polymeric nanops pnps have the clinical potential to carry the target drugs to its core or can coordinate with target molecules on its planar surface mainardes and diedrich nasrollahzadeh biocorona formation ease of biodegradation strong mechanicalthermal properties of carbonbased nanocarriers nanorods nanodots etc render their antiviral activities against respiratory syncytial virus rsv hiv1 ebola virus etc table in spite of having broad spectrum activities against antiviral infections the ratelimiting phase of these carbon materials limits their further biomedical applications nikaeen and nguyen have suggested that drug conjugated nanops having excellent antiviral activities can help in the development of an influenza vaccine with matrix protein m2e and hemagglutinin haamine functionalized gelatin surface coating though more research is required to validate the immunity and protection of cellular genes for such impeccable protein vaccines against infectious virulent pathogens like sarscov2 with the advancement of medical sciences the emergence of multifunctional nanobased lipid nanocarriers opens a new door in the field of novel antiviral nanotherapeutics table read and risitano have demonstrated that podophyllotoxin stearic acidglycol loaded lipid nanocarriers are useful in maintaining in vitro slow drug release nontoxic viral inhibition and hemocompatibility of vk2e6e7 hela cells such solidlipid nanops and other nanolipid carriers can fig a and b schematic of the internalization of nanodrugs through the plasma membrane and targeted drug release a and transcytosis of nanodrugs through cell barriers b nanoparticulate drug carriers through the cellular and mechanistic establishment crossed the membranes bloodbrain barrier and bloodtestis barrier and with the help of capping agents such as sulfate polysaccharidespolymers undergo multivalent bond interactions with virus glycoproteins ie hemagglutinin ha environmentalresearch19120201101195 0cs mukherjee become the exceptional antiviral drug discharging agents for infectious viral pathogens ie hpv hiv and hepatitis c virus nú˜nezdelgado prather hyperbranched monodispersed easily biodegradable anic dendrimers have gained importance for more than a decade in the field of nanomedicine because they act as targeted carriers for biological systems the antiviral activity of dendrimers is still under scientific investigations which may be facilitated through conjugated drug delivery mechanisms patil palestino the efficacy of such nanoparticulated dendrimer system can be observed for the inhibition of hsv1influenza virus where antibody mediated responsecd8 t cell activation and regulation of gene expressions are achieved through small rnas inactivation shang sportelli niosomes the nonionic surfactant based liposome alike anic nanovesicle are getting popular in the advanced biomedical sciences considering their nonimmunogenicity and stable optical properties which make them a suitable carrier of both hydrolipophilic drug molecules excellent bioavailability and controlled release of specific drugs at the targeted sites make such nanocarriers ideal antiviral agents hsv virus where nanoniosome was loaded with suitable antiviral drug acyclovir s´anchezl´opez et al improved drug delivery mechanism and suitable drug release kinetics may allow such nanocarriers to be used in infectious virus diseases nanomicelles fig supramolecular globular micelles exhibit colloidal stability and super encapsulation potential which help such polymeric micelles to show antiviral activity in vitro as observed from curcumin loaded bioavailable nanoformulations hepatitis c virus sivasankarapillai some researchers used graphene oxides conjugated adnps against infectious sars and bursal viruses where the drug resistance event after such antiviral efficacy was mediated by selenium nanops senps andor amantadine am arrangement te velthuis kumar 2020a 2020b 2020c diagnosis or detection of such virulent pathogens have been documented by viceconte and petrosillo and vazquezmunoz and lopez through thiolstabilized gold cluster or enteroviruses labeling with cysteine molecule a brief overview about antiviral nanomedicines rsv virus tremiliosi udugama the size and zeta potential of silver nanops can exert inhibition effects on different human parainfluenza three virus strains or on their replication event the nanocolloidal system of vivagel® is immensely used for the control of zika virus infection tkm130803 is widely used in the treatment of ebola virus utilizing the concept of rnaibased therapy for the lipidbased nanosystem it is well doccumented that for human norovirus treatment the employment of goldcopper sulfide coreshell capsid protein binding results into excellent virucidal activity ziaie on the other hand nanotrap ps are quite often used in the inhibition of infection of target cells by capturing viral rnaviral proteins ie influenza virus treatment zhou 2020ab zhang intrinsic in vivo instability poor immunogenicity and toxicity multiple therapeutic and prophylactic approaches can be overcome by nanovaccinology where cellular and humoral immune response drive the faster uptake of mucosagut associated lymphoid tissue slow controlled release of antigens is facilitated by surface modifications of nanovaccines with antibodiescarbohydrates which results in the target specific immune response by different immune cells additionally their small size and prolonged shelf life help in the faster recognition of the hostreceptor immune system ie hepatitis a virus hav and influenza virus where epaxalexapal is used with immuno targeting agents yu yang and wang nonresponsive immune systems high dose administration coldchain transport of parenteral vaccines limit their widespread application in drug therapy particularly for mucosally administered vaccines the chitosannanop embedded system might be useful for therapeutic proteins or antigens having negative charges which makes its wider application in vaccination against hbv virus through gene delivery systems table the utilization of mouse model employing humoral and mucosal immune responses helped in the liposomebased vaccine development in case of hepaxen used for hepatitis a c and e which further utilized the recombinant surface antigen as a prophylactic vaccine waris wang 2020a 2020b the usages of inflexal v and influvac as standard virosomal vaccine against influenza virus are getting quite popular considering its active biocompatible and immunogenicity wu weiss these types of licensed subunit nanovaccines are found quite useful for older infants and middleaged group people in terms of nanosafety issue as they mimic natural infections as seen in table a cysteineguanine rich oligonucleotide combination with extracellular m2egold conjugates renders molecular protection for pr8h1n1 influenza which was further activated by thiolgold interactions zhang zhou 2020ab from the discussion provided here it is clear that most of the research has been done for influenza virus vaccination but some scanty literature also report several nanomedicines including nanovaccines are under clinical trial or at least in the stage of commercialization for the cure of infectious viral diseases table in general the use of drugs for antiviral therapy is usually employed to target different life cycles of virulent pathogens ie hiv ebola virus or hsv1 valdiglesias and laffon alnrsv01 is a commonly used lipid nanop drug for lower tract respiratory disease which targets the nucleocapsid €œn€ gene of table commercial nanomedicines or under clinical trial for the antiviral therapytreatment souce of data neogi letko dong kalantarzadeh kang nanomedicines influvac plus tkmhbv cervisil doravirine dermavir inflexal v epaxal pegasys geovax novavax fluquit curevac peglntron vivagel a pepreclinical evaluation uceunder clinical evaluation hav hepatitis a virus hiv human immunodeficiency virus hbv hepatitis b virus hpv human papillomavirus hcv hepatitis c virus hsv herpes simplex virus mode of action presence of neuraminidase and hemagglutinin rnai therapeutics gene silencing reverse trancriptase inhibitor nonnucleoside dna immunogen with hiv specific t cell precursor antigens specific on speherical carriers surface natural process mimics peroxidases pegylation control stability of protein ankara€”virus alike drug therapy clinical stage antiviral nanobiotechnology gene silencing mrna technique pegylation control stability of protein dendrimer with sulphonic acid group interaction disease indication influenza hbv hpv hiv hiv influenza hav hbv hcv sarscov2 sarscov2 influenza sarscov2 hcv hsv hiv biomedical application virosome vaccine solidlipid nanop sirna therapeutic nanoparticulate formulation therapeutic vaccine liposome vaccine liposome vaccine pegylated interferon antiviral therapy nanoparticulate therapeutics sirna therapeutic infections virus vaccine pegylated interferon dendrimer yearstage of development uce preclinical evaluation ucea uce pe pe pea pe uce environmentalresearch19120201101196 0cs mukherjee vaccine research for rotanoroebola virus hpv rsv and others dung das metabolic pathway of nanotherapeutics and their limitations in clinical practice pulmonary and hepatotoxicity studies are required to build a safety profile of such nanodelivery therapeutic or diagnostic agents nanobased approach for sarscov2 infection inhibition nanops uptake cellular process in nanotherapeutics are governed by their physicochemical properties along with cellular membrane characteristics which may have direct influence on the rate of administered drug dosages and structure of engineered nanops it is hypothesized that nanops with optimum diameter of ‰ nm and high surface charge density are quite effective in crossing the cellular membranes for hivderived tat cell penetrating peptides weiss waris immunoliposomes and other carbon based nanotubesnanocarriers play an important role in the activation of the complement pathway of host immune systems to deregulate in vitro utilization of nps neogi kumar 2020a 2020b 2020c antibodies that are specifically targeted at polyethylene glycol pegmacrogol polymers and peglike nanostructures can show independent therapeutic efficacy based on their individual immunotoxicology and risk assessment strategies letko read experimental findings wang 2020a 2020b hill with nanobased therapeutic agents reveal the urgent requirement of more rigorous scientific investigations to prove their clinical efficiency in reversing the drug resistance event ie h1n1 virus through seam biodegradation process of nanotherapeutics has gained special attention considering uniform biodistribution kinetics and sustained drug release which are essential for improved drug design process distribution metabolism absorption excretion are important pharmacokinetic features which rate of biochemical features are directly governed by hydrophobichydrophilic profile and tacticity of the nano based formulations at in vitro level patil exocytosis process plays a very important role in the clearance of the foreign nanops out of the cell depending on administered nanocarriers it was hypothesized that ps with nm diameter can Answer:

Colon_Cancer

" introduction it is well known that immune system is highly specific to protect the body against various environmental pathogens the concept of conventional vaccination has overcome the pandemic situation of several infectious diseases outbreak area covered the recent idea of immunization through oral route edible vaccine is vital alternatives over conventional vaccines edible vaccines are composed of antigenic protein introduced into the plant cells which induce these altered plants to produce the encoded protein edible vaccine has no way of forming infection and safety is assured as it only composed of antigenic protein and is devoid of pathogenic genes edible vaccines have significant role in stimulating mucosal immunity as they come in contact with digestive tract lining they are safe costeffective easytoadminister and have reduced manufacturing cost preproofhence have a dramatic impact on health care in developing countries expert opinion the edible vaccine might be the solution for the potential hazard associated with the parenteral vaccines in this review we discuss the detailed study of pros cons mechanism of immune stimulation various outbreaks that might be controlled by edible vaccines with the possible future research and applied application of edible vaccine keywords edible vaccine outbreak transformation mucosal immunity adjuvants conventional vaccine probiotic 0c preproof introduction the immune system is a dynamic structure in our body that protects us from various pathogens immune system continuous tracking of molecules which circulate within the body to detect substances which negatively affect our health once the foreign body pathogens are identified the immune system attacks to neutralize them with the help of antibodies1 human immune cells are extremely complex and quickly adjusted to overcome every day™s challenges literarily the immune system can be described as a complex collection of cells tissues ans and process working together to prevent disease the immune system targets microanism like a virus bacteria pathogen parasitic worm toxins allergens and even sometimes own cell that show unusual characteristics as microanism rapidly evolves in a very short period the immune system has to be prepared to handle massive diversity of antigens which are commonly identified as an agent that activates immune system2 generally each molecule may be an antigen but researchers have found that carbohydrate and proteins offer the best response whereas lipids and nucleic acids are poor antigens the preproofsensitivity and the specificity of the immune system should be taken in to account for the developments of highly specific chemical and cellular tools as it is known that immune system is complex but well anized as a result this destroys or kills the invading pathogens ensuring longterm protection against pathogens and immunological memory for the body that immediately reacts to subsequent encounters with the same antigen3sometimes body immune system is unable to combat with these pathogens or microanisms may be due to the resistance causes in the microbe over the long period in which they modified their internal external structure modify receptors that present on their surface or due to the new strain that does not attack before to the human body to overcome this problem scientists develops vaccines usually contains whole microbe either killed or as a live form microbe small parts a protein molecule or toxins that mimic the diseasecausing pathogen these vaccine elements are used to activate the body's immune responses to identify kill or prevent further attacks of pathogens such as viruses bacteria fungi or other foreign bodies4 in both infectious and noninfectious disease vaccines play a crucial role to prevent it nowadays conventional approaches have successfully developed a vaccine against many infectious diseases particularly by attenuating the pathogen inactivation of microbes or by subunits of microbes still the effectiveness stability cost and safety of these current conventional vaccines remain important considerations in the production of vaccine delivery storage and availability of vaccine therefore it is important to create a new vaccine that is more costeffective and safer for the benefits of people relative to the current vaccine hence 0c preproofa new alternative approach comes into the picture nowadays that is œedible vaccine refer to the use of edible plantvegetable parts or probiotics5 live microanisms as a vaccine which is taken orally as whole or parts of plantvegetable the concept of œedible vaccine first given by œcharles arntzen in as per the new research the probiotics and plant may be genetically modified with the help of gene transfer or transformation and plant virus that contains the human pathogen proteins for the development of an edible vaccine against various lifethreatening disease such as cholera chickenpox aids malaria foot and mouth disease fmd hepatitis b and c etc some of these diseases require booster vaccination or multiple antigens to promote and retain protective immunity the plants can express more than one transgene which allows delivery of multiple antigens for repeated inoculation8 mostly the oral vaccine is associated with degradation of proteins in the stomach due to the action of the gastric enzyme and low ph and gut which restricts immune response but due to the rigid protective plant cell wall these proteins get protection from the stomach acids and enzymes as human enzymes are incapable of breaking down glycosidic bonds of plant cell wall carbohydrates9but subsequently release proteins at the gut lumen due to the action of microbial enzymes which break the plant cell wall bioencapsulation lactobacillus species preproofare considered intrinsically resistant to acid and the survival rate of probiotic lactobacilli increased in acidic condition when given with glucose10the resistant of probiotic and by encapsulation of antigensin plants stomach acid or at low ph will provide extra future prospects and tools to over come the challenges that will be faced by edible vaccines regarding its degradation in the stomach due to gastric enzyme now fda has approved plant cells for costeffective production of protein drugs pds in large scale current good manufacturing practice cgmp hydroponic growth facilities11 in freezedry lyophilized plant cells proteindrugs pds are more stable at ambient temperature for a longer period and maintain their folding and efficacy11 outbreak of diseases an outbreak occurs when several cases of a particular disease increase more than expected in a specific region over a specific period in the last three decades numbers of highly transmissible or pathogenic infectious disease like zika ebola monkeypox sars measles polio cholera diphtheria cases have increased in many parts of the globe12 this trend further continues with zoonotic spillover events expected to continue as a result of population expansion or overcrowding of cities unhygienic condition pollution and migration to uninhabited areas and effect of global warming on vector distribution due to the presence of these factors it decreases the ability of the individual to combat these diseases in recent 0c preproofyear's demand for pharmaceutical products vaccine increase over the world to treat these diseases however some counties are unable to manufacture these vaccines due to poor infrastructure or buy these vaccines at higher prices in these countries the edible vaccine may be a better option to combat diseases as edible vaccines are cheap and stable for a longer time table criteria for the selection of plant or plant part for the development of an edible vaccine edible vaccines are the form of medication taken as food by the humans and animals to boost their immunity thus the criteria needed in selecting plants for the production of edible vaccine must meet specific requirements the far most important criteria of selecting plant is its life cycle13 it shouldn™t be longer as if it takes long time for maturation then their production and maintenance cost increase the next criteria are the choice of plant for antigen incorporation which should not contain any biomolecule that will interact with it next many plants have been identified and studied for the edible vaccine which was transformed to express antigen for rotavirus gastroenteritis cholera autoimmune diseases and rabies moreover several experiments have used vegetable potato but potatoes may not be the ideal choice for edible vaccines since frying or boiling will degrade certain antigenic proteins certain foods such as bananas tomato carrots peanuts corn and tobacco have a more promising potential as edible vaccines as it can be eaten raw not only because they are commonly available but since genetic engineering is efficiently developed these kinds of vegetable plants the following plant list contains edible vaccines previously studied in animals and which are required to be approved in both human and animal use potato preproofimportant one is the stability of the antigen under high temperature as all vegetable or plant parts are not eaten raw eg potato rice pea sometime they are boiled or fried at higher temperature plant and plant parts that can be consumed raw are preferable for human immunization however banana might be the ideal source of edible vaccines because they are consumed raw even by infants and are a major crop in many developing counties8 the one disadvantage of both banana and tomato is the low protein content of the fruit which might limit the amount of antigen that can be expressed13 in contrast seeds of the plants such as maize and soybean have higher protein content and have been reported to express very high level of foreign proteins edible vaccine from the plant source 0c preproofmason et al was the first person who conducts the vaccinebased assay produced in potato solanum tuberosum to fight against ltb stain produced by e coli in mice14 in that same year in rats and human volunteers in the same year the effectiveness of the antigens produced from potatoes solanum tuberosum towards the nontoxic subunit of vibrio cholera endotoxin and the norwalk virus capsid pathogen was identified in rats and human volunteers1215thanavalas'group proposed in that potatoes could play a role in human hepatitis b as an oral reinforcement since injectable vaccine cause redness swelling or itching at the site of administration also the edible vaccine for the animal has now been developed to replace the injectable vaccine for animal protection12 tomatotobacco model species for the edible vaccine1 cherry tomatillos tomato solanum lycopersicum an appropriate candidate for vaccine development for coronavirus that causes a highly acute respiratory syndrome sars for the development of recombinant sarscoronavirus cov vaccine sspike protein sprotein and its truncated fragment are considered as the best choice912 the genome of tomato and tobacco is incorporated with nterminal fragment of sarscov protein s1 used to develop the safe effective and inexpensive vaccine when these plantbased vaccines for sars give to mice shows significant increase level of sarscovspecific iga after oral ingestion of tomato expressing s1 protein whereas tobaccoderived s1protein indicate the presence of sarscovspecific igg detect by elisa analysis and western blot16tobacco is not an edible plant but play a major role in the development of the vaccine as it is used as a proofofconcept preprooffor hbsag gene of hepatitis b lines of transgenic cherry tomatillos have been grownthe expression of genes was seen through the whole plant but it was maximum in the fresh leaves weight of ngg and with fresh fruit weight of ngg12 lettuce lactuca sativa express the bsubunit of the thermolabile protein of e coli responsible for both human and animal enteric disease show the possibility of this vegetable as an edible vaccine in the typical swine fear hog pest virus glycoprotein e2 was expressed by lettuce in poland the transgenic lettuce that shows effect against hepatitis b virus is in the development stage17 soybean in the study e coli bacteria bsubunit of thermolabile toxin expression was performed in the endoplasmic reticulum er of soybean glycine max which yielded a total antigen level 0c preproofof up to of the total soybean seed protein without any problem during drying for further processing moreover when this protein is given orally to rats leads to a rise in systemic igg and iga1819 algae chlamydomonas reinhardtii green algae has been used as a tool to achieve a large number of proteins specific to both animal and humans for therapeutic purpose1819the use of algae for the production of vaccines is optimistic as algae have a very high growth rate the entire system can be used as a raw material for the development of edible vaccines besides to facilitate the already rapidly growing algae can be cultivated in bioreactors c reinhardtii contains one chloroplast which facilitates the stability of the desired antigens in the algal line notably the effectiveness of algal vaccines after lyophilization is unchanged which might based on the expression of the capsid protein norwalk virus the transgenic plant was developed protein deposition in the unripened fruit with a lower accumulation in red fruit was reported up to of soluble protein expression in seeds allowed the storage of antigenic peptides thus creating a plant with a high yield of proteins with an average protein content of about which would preclude intensive purification procedure by pharmaceutical industries1220in addition to the expression of hemagglutinin protein h a pa against rinder pest virus rpv pea plants were used the total soluble protein level of expression in leaves was observed to be determined by western blot even more preproofpromote global delivery of edible algae vaccine12 pea studies are also required to improve the expression of a protein in transgenic peas banana in banana plants hbsag expression was reported with four distinct cassettes pher phb pefeher and pefehbs at the different level expression of hbsag were studied with pcr reverse transcription pcr and southern hybridization method expression levels reached a height of ngg in the plant and the antigen was found in banana leaves however because of the long period required the shrub needs to grow the use of this vaccine was denied21 papaya in a papaya carica papaya vaccine was developed to counter cysticercosis caused by taenia solium by expressing synthetic peptides in transgenic papaya clones vaccine was tested in rats with an immunogenic response of per cent in vaccinated animals these 0c preproofedible vaccines may offer good relief both in humans and in pigs which are the two major carriers of the disease22“ carrot in an experiment carrot along with a thaliana was utilized to develop an edible vaccine for surface hiv antigen expression and in the study it was reported that rats showed more positive effect compared to those nontreated animals25 carrot daucus carota has a positive effect in the treatment of hiv not only because carrots are nutritious and tasty but because of carrot main chemical constituent carotenoids which on consumption by rats increases monocytes lymphocytes and other immune defence thus people with a weakened immune system might benefit from the use of this potential edible antihiv vaccine26 the efficacy of this antihiv vaccine must be confirmed by a clinical trial in it has been reported that the ureb subunit of helicobacter pylori was used in transgenic carrots as a potential vaccine transgenic carrot expressing the b subunit from e coli thermolabile toxininduced iga and igg production and occurred at the intestinal and systemic level in the rat2728 rice preproofa research in found that transgenic rice oryza sativa plants expressing the b subunit of e coli induces significant number of antibodies to this subunit in the same year an immune response was found to be caused in chicken by transgenic rice that is a result of the vp2 antigenic protein from infectious bursitis in pcr and southern blot analysis confirmed the functional expression of hbsag in rice seeds2930 in addition transgenic rice was developed in in parallel to express the subunit b of the e coli thermolabile toxin used to convert plant cells using bioballistic approach pcr verified the expression india and china both are the world two biggest rice producer and have the capability to export these modified rice vaccine plant all over the globe31 selected gene encoding antigen genes from a pathogenic anism bacteria viruses or parasites that have already been identified and for which antibodies are easily accessible can be controlled in a twofold fashion in one scenario the whole structural gene is inserted inside the functional elements of the ' to ' plant transformation vector allowing the transcription and aggregation of the coding sequence in the plant32 for the second scenario the epitope is identified within the antigen dna fragment encoding may be used to create genes through fusion with coated protein gene with the plant virus eg cmv or tmv then the recombinant virus is then introduced into suitable healthy plants which produce several new plants 0c preprooftable method for the transformation of genedna into selected plants essentially there are two types of plant transformation methods but many other approaches have also been utilized to transformation vectorplasmid carrier system agrobacterium tumefacien mediated gene transfer agrobacterium a soil bacterium naturally occurring has been used to transfer a small fragment of dna into the plant genome and is called transformation5051 in this method the appropriate recombinant dna is inserted into the tregion of disarmed tiplasmid of agrobacterium tumefaciens plant pathogens which is cocultured with the plant cells andor the tissue that will be transformed the insertion of the exogenous genes and the infection of such a plant tissue into sufficiently modified agrobacterium tdna cell contributed to studies of the stable gene incorporation in the genome of the plant as well as transgenic protein production32 however this technique is sluggish and yield are lower but the application of this transformation is first limited to tobacco plant and too few other plant species which extend to most preproofvegetable species including leguminose and graminae microprojectile bombardment biolistic method this is a sophisticated method based on the microprojectile bombardment the selected dna sequence is precipitated on microparticles of metals eg tungsten gold and bombarded by a particle gun at an accelerated rate toward selected plant tissue5253 these metallic microparticles penetrate the cell walls and the exogenous dna is emitted into the cell where it is integrated into the nuclear genome through a process known for the photosynthetic role of cytoplasmic anelles called chloroplast comprising chlorophyll particle gun shoots adequately charged metallic particles with selected and processed dna which penetrate chloroplast and merge with its genome32 transformation of the chloroplast is an effective alternative to nuclear transformation5455 pds in chloroplast are more stable when plant cells lyophilized and when preserved at ambient temperature therefore the freezedrying method improves pds concentration and prevents bacterial contamination11 electroporation in this method dna is introduced into the cells to which the electrical pulses of high voltage are released which are intended to create transitory pores in the 0c preproofplasmalemma it requires the extra effort to weaken the cell wall as it serves as an efficient barrier to the entrance of dna into the cell cytoplasm32 mechanisms of action of edible vaccines for mucosal and peripheral immune figure response mucosal and peripheral immunity a critical issue for oral vaccination immune response to the vaccine is affected by the route of immunization the form of antigen and the active content of vaccine mediate specific tissue tropism there is now substantial evidence supporting the existence of at least two immune systems a peripheral immune system and a mucosal immune system20 these systems operate separately and simultaneously in most species including human protective immunity acquired during recuperation is usually referred to as systemic immunity but the fact is that it might be dominated by an incomplete form of immunity dictated by a specific pathogen as the systemic immunity might be a combination of mucosal and peripheral immunity lymphocyte traffic patterns regulated by selective expression of adhesion proteins in peripheral or mucosal lymphatic tissues affects the outcome of an immune response for example the same antigen may produce qualitatively different immune responses in lymph nodes spleen or peyer's patches the antigens in the lymph are presented over the fixed antigenpresenting cells in lymph nodes results in peripheral immunity characterized by the appearance of specific igg in the blood the antigen in the blood is presented in strategic preprooftissue interface in the spleen this also results in peripheral immunity however the microenvironment of the spleen is somewhat more complicated as it also accommodates circulating antigenpresenting cells and immunoreactive t and b cells from other tissues committed to either peripheral or mucosal immunity triggers of antigen in the lumens of enteric ans presented on payer's patches commitment to mucosal immunity characterized by the release of specific iga into the secretions2056 the mucosal surfaces are a popular site for delivering therapeutic small molecules due to the ease of administration and speed of uptake across the large surface areas efficacy of the mucosal route of immunization is largely based on the fact that mucous membranes constitute the largest immunogenic an of the body this interface is endowed with the wellanized lymphatic structure called malt mucosaassociated lymphoid tissue which constitute t and b cells innate and adaptive arms of the immune system oral vaccines stimulate the generation of immunity in gutassociated lymphoid tissue galt which includes lymph nodes payer's patches in which lymphocytes are the major component 0c preproof are b cells while are t cells and isolated lymphoid follicles in the gastrointestinal tract git a significant hurdle impacting protein delivery to the git that the antigens are rapidly degraded within the harsh environment of the digestive tract is the remarkable challenge for vaccine development it will also be important to consider the characteristics of the git in which several factors including proteolytic enzymes acidic ph bile salts and limited permeability that may hinder the induction of a protective immune response20 mucosal immunity system described above have a clear image that induction of mucosal immune response starts with the recognition of an antigen by specialized cells called mcells located in the mucosal membranes of lymphoid tissues such as peyer™s patches within the small intestine57 then the apc internalize and process the antigen as soon mcell channel antigens into the underlying tissues causing activation of cd4cells57 which leads to the pathogen this whole process is elicited in figure the antigen bioencapsulation by the plant cell which avoids degradation and conformational alterations and the enhancement of mcells uptake of the conjugation of the vaccine antigen with specific ligands will overcome the challenge faced by the conventional vaccine for mucosal immunity stimulation458 second generation edible vaccine figure the secondgeneration edible vaccines are multiplecomponent protective vaccines against multiple pathogens which can produce more than one antigenic protein by crossing two cell lines containing different antigens in the same plant the adjuvant is coexpressible with the same antigen a trivalent edible vaccine against cholera enterotoxigenic escherichia germinal center development bcells maturation and class switching to iga through cd40cd40 ligand interaction and cytokine secretion the antigenic epitopes present on apc then activate bcells with the help of tcells12 due to the expression of chemokine hormone receptors like cxcr5 or cxcr10 the bcells migrate to the mesenteric lymph nodes where they mature into plasma cells and finally migrate to the mucosal membrane and differentiate into plasma cells causing secretion of dimeric and polymeric iga12 on passing through the mucosal epithelial layer toward the lumen the iga molecules complex with membranebound secretary components to form secretary iga siga transported into the lumen the siga interacts with specific antigenic epitopes and neutralize the invading preprooffigure 0c preproofcolietec and rotavirus are the examples that could effectively initiate an immune response59 factor affecting the efficacy of an edible vaccine antigen loaded in the specific plant tissue is the principle of edible vaccines thus the efficacy and the potency of the vaccines are significantly affected by the nature of œadjuvants œadjuvants are the biomolecules lectins saponins that do not exhibit immunogenic response but potentiate the immune response when coadministered with an antigen adjuvants can improve immune and potentiate responses by acting as a depot to guide antigens to relevant sites protect them from degradation control release and activate apcs20 immunogenically inert biodegradable adjuvants like lipids proteins starch polysaccharides or polyesters act as the delivery vehicles for efficient availability at antigen presenting cells apcs there are major two methods to associate antigen and particles ie encapsulated by the particle by entrapment and linked to the surface by chemical conjugation or physical adsorption the choice of carrier particles is critical to maximizing the bioavailability of its complex with antigens encapsulated antigens are afforded protection against extracellular proteases at the time of transport to the target immune responsive sites20 preproofthe erosion of the carrier particles leads to the exposure of antigens to apcs including dendritic cells dcs macrophages and monocytes thus a fine balancing act is needed to avoid overprotection of the encapsulated antigen inhibiting the release of the therapeutic or premature release if protection is inadequate the above case leads to the reduced bioavailability that weakened the host immunity alternatively the conjugation or linked to the surface by chemical bonding or physical adsorption have efficient therapeutic action as it can facilitate delivery too specific immune responsive sites or cells the structures like liposomes virus like particles vlps virosomes and immunostimulatory complexes iscoms are well recognized by apcs because they have characteristics including size shape and surface properties that are similar to viral and bacterial pathogens that the immune system has evolved to attack20 beyond the factors regarding adjuvants and vehicles for the efficacy of an edible vaccine these are some factors that significantly affect the efficacy of edible vaccines \uf0b7 antigen selection \uf0b7 efficacy in the model system \uf0b7 choice of plant species \uf0b7 delivery and dosing frequency 0c preproof\uf0b7 release patterncontrolled and sustained \uf0b7 public perceptions and attitudes to genetic modification the advantage over the conventional vaccine figure due to the use of the oral route the administration of the edible vaccine is less complicated than the conventional methods that are given through im sc and intradermal thus it removes the needs of trained medical personnel and decreases the risk of infection as there is no need for the sterilization of premises and the manufacturing areas \uf0b7 such oral immunization will become a practical key strategy for effective disease prevention in lowincome countries in general \uf0b7 processing purification sterilization packaging or delivery does not require rigorous structure in edible vaccines minimizing longterm costs in relation to conventional vaccines32 \uf0b7 maintenance and distribution of edible vaccine are easier than the conventional vaccines as it enabling the preservation without the constant cold chain storage \uf0b7 improved storage possibilities for edible vaccines become possible as transgenic plant seeds have lesser moisture content heat stable and can quickly be dried6032 \uf0b7 a plant containing therapeutically active edible vaccine protein is free of toxins pathogens and do not have the risk of proteins to reform into the infectious anism8 \uf0b7 improved compliance is particularly related to children's who refuse to take injections preproof\uf0b7 most specifically in this case the immunity is activated on the mucosal surfaces of the gi including those which are the first line of defence on the mouth mucosal of the vaccine immunity \uf0b7 sophisticated equipment and machinery are not required in edible vaccines since they can easily be cultivated on rich soils and are costeffective relative to fermenters where the recombinant cell is cultured in a controlled manner32 limitation beyond the several advantages and the convenience over the conventional vaccines edible vaccines have certain challenges that have to be overcome for efficient and pure beneficial edible vaccination some of them are pointed below 0c preproof\uf0b7 uncertainty in calculating the appropriate oral dosage which might take multiple rounds for a patient to get the effective therapeutic action and raises the final expense of its application32 \uf0b7 the concentration of peptide or protein of edible vaccine varies from generationtogeneration planttoplant and fruitto fruit32 \uf0b7 patientrelated factor such as patient age and weight also affected the dose to be administered \uf0b7 repeated intakes of these antigenbearing plants which stimulate the immune system might over stimulate the immune system itse

cancer7536

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " introduction it is well known that immune system is highly specific to protect the body against various environmental pathogens the concept of conventional vaccination has overcome the pandemic situation of several infectious diseases outbreak area covered the recent idea of immunization through oral route edible vaccine is vital alternatives over conventional vaccines edible vaccines are composed of antigenic protein introduced into the plant cells which induce these altered plants to produce the encoded protein edible vaccine has no way of forming infection and safety is assured as it only composed of antigenic protein and is devoid of pathogenic genes edible vaccines have significant role in stimulating mucosal immunity as they come in contact with digestive tract lining they are safe costeffective easytoadminister and have reduced manufacturing cost preproofhence have a dramatic impact on health care in developing countries expert opinion the edible vaccine might be the solution for the potential hazard associated with the parenteral vaccines in this review we discuss the detailed study of pros cons mechanism of immune stimulation various outbreaks that might be controlled by edible vaccines with the possible future research and applied application of edible vaccine keywords edible vaccine outbreak transformation mucosal immunity adjuvants conventional vaccine probiotic 0c preproof introduction the immune system is a dynamic structure in our body that protects us from various pathogens immune system continuous tracking of molecules which circulate within the body to detect substances which negatively affect our health once the foreign body pathogens are identified the immune system attacks to neutralize them with the help of antibodies1 human immune cells are extremely complex and quickly adjusted to overcome every day™s challenges literarily the immune system can be described as a complex collection of cells tissues ans and process working together to prevent disease the immune system targets microanism like a virus bacteria pathogen parasitic worm toxins allergens and even sometimes own cell that show unusual characteristics as microanism rapidly evolves in a very short period the immune system has to be prepared to handle massive diversity of antigens which are commonly identified as an agent that activates immune system2 generally each molecule may be an antigen but researchers have found that carbohydrate and proteins offer the best response whereas lipids and nucleic acids are poor antigens the preproofsensitivity and the specificity of the immune system should be taken in to account for the developments of highly specific chemical and cellular tools as it is known that immune system is complex but well anized as a result this destroys or kills the invading pathogens ensuring longterm protection against pathogens and immunological memory for the body that immediately reacts to subsequent encounters with the same antigen3sometimes body immune system is unable to combat with these pathogens or microanisms may be due to the resistance causes in the microbe over the long period in which they modified their internal external structure modify receptors that present on their surface or due to the new strain that does not attack before to the human body to overcome this problem scientists develops vaccines usually contains whole microbe either killed or as a live form microbe small parts a protein molecule or toxins that mimic the diseasecausing pathogen these vaccine elements are used to activate the body's immune responses to identify kill or prevent further attacks of pathogens such as viruses bacteria fungi or other foreign bodies4 in both infectious and noninfectious disease vaccines play a crucial role to prevent it nowadays conventional approaches have successfully developed a vaccine against many infectious diseases particularly by attenuating the pathogen inactivation of microbes or by subunits of microbes still the effectiveness stability cost and safety of these current conventional vaccines remain important considerations in the production of vaccine delivery storage and availability of vaccine therefore it is important to create a new vaccine that is more costeffective and safer for the benefits of people relative to the current vaccine hence 0c preproofa new alternative approach comes into the picture nowadays that is œedible vaccine refer to the use of edible plantvegetable parts or probiotics5 live microanisms as a vaccine which is taken orally as whole or parts of plantvegetable the concept of œedible vaccine first given by œcharles arntzen in as per the new research the probiotics and plant may be genetically modified with the help of gene transfer or transformation and plant virus that contains the human pathogen proteins for the development of an edible vaccine against various lifethreatening disease such as cholera chickenpox aids malaria foot and mouth disease fmd hepatitis b and c etc some of these diseases require booster vaccination or multiple antigens to promote and retain protective immunity the plants can express more than one transgene which allows delivery of multiple antigens for repeated inoculation8 mostly the oral vaccine is associated with degradation of proteins in the stomach due to the action of the gastric enzyme and low ph and gut which restricts immune response but due to the rigid protective plant cell wall these proteins get protection from the stomach acids and enzymes as human enzymes are incapable of breaking down glycosidic bonds of plant cell wall carbohydrates9but subsequently release proteins at the gut lumen due to the action of microbial enzymes which break the plant cell wall bioencapsulation lactobacillus species preproofare considered intrinsically resistant to acid and the survival rate of probiotic lactobacilli increased in acidic condition when given with glucose10the resistant of probiotic and by encapsulation of antigensin plants stomach acid or at low ph will provide extra future prospects and tools to over come the challenges that will be faced by edible vaccines regarding its degradation in the stomach due to gastric enzyme now fda has approved plant cells for costeffective production of protein drugs pds in large scale current good manufacturing practice cgmp hydroponic growth facilities11 in freezedry lyophilized plant cells proteindrugs pds are more stable at ambient temperature for a longer period and maintain their folding and efficacy11 outbreak of diseases an outbreak occurs when several cases of a particular disease increase more than expected in a specific region over a specific period in the last three decades numbers of highly transmissible or pathogenic infectious disease like zika ebola monkeypox sars measles polio cholera diphtheria cases have increased in many parts of the globe12 this trend further continues with zoonotic spillover events expected to continue as a result of population expansion or overcrowding of cities unhygienic condition pollution and migration to uninhabited areas and effect of global warming on vector distribution due to the presence of these factors it decreases the ability of the individual to combat these diseases in recent 0c preproofyear's demand for pharmaceutical products vaccine increase over the world to treat these diseases however some counties are unable to manufacture these vaccines due to poor infrastructure or buy these vaccines at higher prices in these countries the edible vaccine may be a better option to combat diseases as edible vaccines are cheap and stable for a longer time table criteria for the selection of plant or plant part for the development of an edible vaccine edible vaccines are the form of medication taken as food by the humans and animals to boost their immunity thus the criteria needed in selecting plants for the production of edible vaccine must meet specific requirements the far most important criteria of selecting plant is its life cycle13 it shouldn™t be longer as if it takes long time for maturation then their production and maintenance cost increase the next criteria are the choice of plant for antigen incorporation which should not contain any biomolecule that will interact with it next many plants have been identified and studied for the edible vaccine which was transformed to express antigen for rotavirus gastroenteritis cholera autoimmune diseases and rabies moreover several experiments have used vegetable potato but potatoes may not be the ideal choice for edible vaccines since frying or boiling will degrade certain antigenic proteins certain foods such as bananas tomato carrots peanuts corn and tobacco have a more promising potential as edible vaccines as it can be eaten raw not only because they are commonly available but since genetic engineering is efficiently developed these kinds of vegetable plants the following plant list contains edible vaccines previously studied in animals and which are required to be approved in both human and animal use potato preproofimportant one is the stability of the antigen under high temperature as all vegetable or plant parts are not eaten raw eg potato rice pea sometime they are boiled or fried at higher temperature plant and plant parts that can be consumed raw are preferable for human immunization however banana might be the ideal source of edible vaccines because they are consumed raw even by infants and are a major crop in many developing counties8 the one disadvantage of both banana and tomato is the low protein content of the fruit which might limit the amount of antigen that can be expressed13 in contrast seeds of the plants such as maize and soybean have higher protein content and have been reported to express very high level of foreign proteins edible vaccine from the plant source 0c preproofmason et al was the first person who conducts the vaccinebased assay produced in potato solanum tuberosum to fight against ltb stain produced by e coli in mice14 in that same year in rats and human volunteers in the same year the effectiveness of the antigens produced from potatoes solanum tuberosum towards the nontoxic subunit of vibrio cholera endotoxin and the norwalk virus capsid pathogen was identified in rats and human volunteers1215thanavalas'group proposed in that potatoes could play a role in human hepatitis b as an oral reinforcement since injectable vaccine cause redness swelling or itching at the site of administration also the edible vaccine for the animal has now been developed to replace the injectable vaccine for animal protection12 tomatotobacco model species for the edible vaccine1 cherry tomatillos tomato solanum lycopersicum an appropriate candidate for vaccine development for coronavirus that causes a highly acute respiratory syndrome sars for the development of recombinant sarscoronavirus cov vaccine sspike protein sprotein and its truncated fragment are considered as the best choice912 the genome of tomato and tobacco is incorporated with nterminal fragment of sarscov protein s1 used to develop the safe effective and inexpensive vaccine when these plantbased vaccines for sars give to mice shows significant increase level of sarscovspecific iga after oral ingestion of tomato expressing s1 protein whereas tobaccoderived s1protein indicate the presence of sarscovspecific igg detect by elisa analysis and western blot16tobacco is not an edible plant but play a major role in the development of the vaccine as it is used as a proofofconcept preprooffor hbsag gene of hepatitis b lines of transgenic cherry tomatillos have been grownthe expression of genes was seen through the whole plant but it was maximum in the fresh leaves weight of ngg and with fresh fruit weight of ngg12 lettuce lactuca sativa express the bsubunit of the thermolabile protein of e coli responsible for both human and animal enteric disease show the possibility of this vegetable as an edible vaccine in the typical swine fear hog pest virus glycoprotein e2 was expressed by lettuce in poland the transgenic lettuce that shows effect against hepatitis b virus is in the development stage17 soybean in the study e coli bacteria bsubunit of thermolabile toxin expression was performed in the endoplasmic reticulum er of soybean glycine max which yielded a total antigen level 0c preproofof up to of the total soybean seed protein without any problem during drying for further processing moreover when this protein is given orally to rats leads to a rise in systemic igg and iga1819 algae chlamydomonas reinhardtii green algae has been used as a tool to achieve a large number of proteins specific to both animal and humans for therapeutic purpose1819the use of algae for the production of vaccines is optimistic as algae have a very high growth rate the entire system can be used as a raw material for the development of edible vaccines besides to facilitate the already rapidly growing algae can be cultivated in bioreactors c reinhardtii contains one chloroplast which facilitates the stability of the desired antigens in the algal line notably the effectiveness of algal vaccines after lyophilization is unchanged which might based on the expression of the capsid protein norwalk virus the transgenic plant was developed protein deposition in the unripened fruit with a lower accumulation in red fruit was reported up to of soluble protein expression in seeds allowed the storage of antigenic peptides thus creating a plant with a high yield of proteins with an average protein content of about which would preclude intensive purification procedure by pharmaceutical industries1220in addition to the expression of hemagglutinin protein h a pa against rinder pest virus rpv pea plants were used the total soluble protein level of expression in leaves was observed to be determined by western blot even more preproofpromote global delivery of edible algae vaccine12 pea studies are also required to improve the expression of a protein in transgenic peas banana in banana plants hbsag expression was reported with four distinct cassettes pher phb pefeher and pefehbs at the different level expression of hbsag were studied with pcr reverse transcription pcr and southern hybridization method expression levels reached a height of ngg in the plant and the antigen was found in banana leaves however because of the long period required the shrub needs to grow the use of this vaccine was denied21 papaya in a papaya carica papaya vaccine was developed to counter cysticercosis caused by taenia solium by expressing synthetic peptides in transgenic papaya clones vaccine was tested in rats with an immunogenic response of per cent in vaccinated animals these 0c preproofedible vaccines may offer good relief both in humans and in pigs which are the two major carriers of the disease22“ carrot in an experiment carrot along with a thaliana was utilized to develop an edible vaccine for surface hiv antigen expression and in the study it was reported that rats showed more positive effect compared to those nontreated animals25 carrot daucus carota has a positive effect in the treatment of hiv not only because carrots are nutritious and tasty but because of carrot main chemical constituent carotenoids which on consumption by rats increases monocytes lymphocytes and other immune defence thus people with a weakened immune system might benefit from the use of this potential edible antihiv vaccine26 the efficacy of this antihiv vaccine must be confirmed by a clinical trial in it has been reported that the ureb subunit of helicobacter pylori was used in transgenic carrots as a potential vaccine transgenic carrot expressing the b subunit from e coli thermolabile toxininduced iga and igg production and occurred at the intestinal and systemic level in the rat2728 rice preproofa research in found that transgenic rice oryza sativa plants expressing the b subunit of e coli induces significant number of antibodies to this subunit in the same year an immune response was found to be caused in chicken by transgenic rice that is a result of the vp2 antigenic protein from infectious bursitis in pcr and southern blot analysis confirmed the functional expression of hbsag in rice seeds2930 in addition transgenic rice was developed in in parallel to express the subunit b of the e coli thermolabile toxin used to convert plant cells using bioballistic approach pcr verified the expression india and china both are the world two biggest rice producer and have the capability to export these modified rice vaccine plant all over the globe31 selected gene encoding antigen genes from a pathogenic anism bacteria viruses or parasites that have already been identified and for which antibodies are easily accessible can be controlled in a twofold fashion in one scenario the whole structural gene is inserted inside the functional elements of the ' to ' plant transformation vector allowing the transcription and aggregation of the coding sequence in the plant32 for the second scenario the epitope is identified within the antigen dna fragment encoding may be used to create genes through fusion with coated protein gene with the plant virus eg cmv or tmv then the recombinant virus is then introduced into suitable healthy plants which produce several new plants 0c preprooftable method for the transformation of genedna into selected plants essentially there are two types of plant transformation methods but many other approaches have also been utilized to transformation vectorplasmid carrier system agrobacterium tumefacien mediated gene transfer agrobacterium a soil bacterium naturally occurring has been used to transfer a small fragment of dna into the plant genome and is called transformation5051 in this method the appropriate recombinant dna is inserted into the tregion of disarmed tiplasmid of agrobacterium tumefaciens plant pathogens which is cocultured with the plant cells andor the tissue that will be transformed the insertion of the exogenous genes and the infection of such a plant tissue into sufficiently modified agrobacterium tdna cell contributed to studies of the stable gene incorporation in the genome of the plant as well as transgenic protein production32 however this technique is sluggish and yield are lower but the application of this transformation is first limited to tobacco plant and too few other plant species which extend to most preproofvegetable species including leguminose and graminae microprojectile bombardment biolistic method this is a sophisticated method based on the microprojectile bombardment the selected dna sequence is precipitated on microparticles of metals eg tungsten gold and bombarded by a particle gun at an accelerated rate toward selected plant tissue5253 these metallic microparticles penetrate the cell walls and the exogenous dna is emitted into the cell where it is integrated into the nuclear genome through a process known for the photosynthetic role of cytoplasmic anelles called chloroplast comprising chlorophyll particle gun shoots adequately charged metallic particles with selected and processed dna which penetrate chloroplast and merge with its genome32 transformation of the chloroplast is an effective alternative to nuclear transformation5455 pds in chloroplast are more stable when plant cells lyophilized and when preserved at ambient temperature therefore the freezedrying method improves pds concentration and prevents bacterial contamination11 electroporation in this method dna is introduced into the cells to which the electrical pulses of high voltage are released which are intended to create transitory pores in the 0c preproofplasmalemma it requires the extra effort to weaken the cell wall as it serves as an efficient barrier to the entrance of dna into the cell cytoplasm32 mechanisms of action of edible vaccines for mucosal and peripheral immune figure response mucosal and peripheral immunity a critical issue for oral vaccination immune response to the vaccine is affected by the route of immunization the form of antigen and the active content of vaccine mediate specific tissue tropism there is now substantial evidence supporting the existence of at least two immune systems a peripheral immune system and a mucosal immune system20 these systems operate separately and simultaneously in most species including human protective immunity acquired during recuperation is usually referred to as systemic immunity but the fact is that it might be dominated by an incomplete form of immunity dictated by a specific pathogen as the systemic immunity might be a combination of mucosal and peripheral immunity lymphocyte traffic patterns regulated by selective expression of adhesion proteins in peripheral or mucosal lymphatic tissues affects the outcome of an immune response for example the same antigen may produce qualitatively different immune responses in lymph nodes spleen or peyer's patches the antigens in the lymph are presented over the fixed antigenpresenting cells in lymph nodes results in peripheral immunity characterized by the appearance of specific igg in the blood the antigen in the blood is presented in strategic preprooftissue interface in the spleen this also results in peripheral immunity however the microenvironment of the spleen is somewhat more complicated as it also accommodates circulating antigenpresenting cells and immunoreactive t and b cells from other tissues committed to either peripheral or mucosal immunity triggers of antigen in the lumens of enteric ans presented on payer's patches commitment to mucosal immunity characterized by the release of specific iga into the secretions2056 the mucosal surfaces are a popular site for delivering therapeutic small molecules due to the ease of administration and speed of uptake across the large surface areas efficacy of the mucosal route of immunization is largely based on the fact that mucous membranes constitute the largest immunogenic an of the body this interface is endowed with the wellanized lymphatic structure called malt mucosaassociated lymphoid tissue which constitute t and b cells innate and adaptive arms of the immune system oral vaccines stimulate the generation of immunity in gutassociated lymphoid tissue galt which includes lymph nodes payer's patches in which lymphocytes are the major component 0c preproof are b cells while are t cells and isolated lymphoid follicles in the gastrointestinal tract git a significant hurdle impacting protein delivery to the git that the antigens are rapidly degraded within the harsh environment of the digestive tract is the remarkable challenge for vaccine development it will also be important to consider the characteristics of the git in which several factors including proteolytic enzymes acidic ph bile salts and limited permeability that may hinder the induction of a protective immune response20 mucosal immunity system described above have a clear image that induction of mucosal immune response starts with the recognition of an antigen by specialized cells called mcells located in the mucosal membranes of lymphoid tissues such as peyer™s patches within the small intestine57 then the apc internalize and process the antigen as soon mcell channel antigens into the underlying tissues causing activation of cd4cells57 which leads to the pathogen this whole process is elicited in figure the antigen bioencapsulation by the plant cell which avoids degradation and conformational alterations and the enhancement of mcells uptake of the conjugation of the vaccine antigen with specific ligands will overcome the challenge faced by the conventional vaccine for mucosal immunity stimulation458 second generation edible vaccine figure the secondgeneration edible vaccines are multiplecomponent protective vaccines against multiple pathogens which can produce more than one antigenic protein by crossing two cell lines containing different antigens in the same plant the adjuvant is coexpressible with the same antigen a trivalent edible vaccine against cholera enterotoxigenic escherichia germinal center development bcells maturation and class switching to iga through cd40cd40 ligand interaction and cytokine secretion the antigenic epitopes present on apc then activate bcells with the help of tcells12 due to the expression of chemokine hormone receptors like cxcr5 or cxcr10 the bcells migrate to the mesenteric lymph nodes where they mature into plasma cells and finally migrate to the mucosal membrane and differentiate into plasma cells causing secretion of dimeric and polymeric iga12 on passing through the mucosal epithelial layer toward the lumen the iga molecules complex with membranebound secretary components to form secretary iga siga transported into the lumen the siga interacts with specific antigenic epitopes and neutralize the invading preprooffigure 0c preproofcolietec and rotavirus are the examples that could effectively initiate an immune response59 factor affecting the efficacy of an edible vaccine antigen loaded in the specific plant tissue is the principle of edible vaccines thus the efficacy and the potency of the vaccines are significantly affected by the nature of œadjuvants œadjuvants are the biomolecules lectins saponins that do not exhibit immunogenic response but potentiate the immune response when coadministered with an antigen adjuvants can improve immune and potentiate responses by acting as a depot to guide antigens to relevant sites protect them from degradation control release and activate apcs20 immunogenically inert biodegradable adjuvants like lipids proteins starch polysaccharides or polyesters act as the delivery vehicles for efficient availability at antigen presenting cells apcs there are major two methods to associate antigen and particles ie encapsulated by the particle by entrapment and linked to the surface by chemical conjugation or physical adsorption the choice of carrier particles is critical to maximizing the bioavailability of its complex with antigens encapsulated antigens are afforded protection against extracellular proteases at the time of transport to the target immune responsive sites20 preproofthe erosion of the carrier particles leads to the exposure of antigens to apcs including dendritic cells dcs macrophages and monocytes thus a fine balancing act is needed to avoid overprotection of the encapsulated antigen inhibiting the release of the therapeutic or premature release if protection is inadequate the above case leads to the reduced bioavailability that weakened the host immunity alternatively the conjugation or linked to the surface by chemical bonding or physical adsorption have efficient therapeutic action as it can facilitate delivery too specific immune responsive sites or cells the structures like liposomes virus like particles vlps virosomes and immunostimulatory complexes iscoms are well recognized by apcs because they have characteristics including size shape and surface properties that are similar to viral and bacterial pathogens that the immune system has evolved to attack20 beyond the factors regarding adjuvants and vehicles for the efficacy of an edible vaccine these are some factors that significantly affect the efficacy of edible vaccines \uf0b7 antigen selection \uf0b7 efficacy in the model system \uf0b7 choice of plant species \uf0b7 delivery and dosing frequency 0c preproof\uf0b7 release patterncontrolled and sustained \uf0b7 public perceptions and attitudes to genetic modification the advantage over the conventional vaccine figure due to the use of the oral route the administration of the edible vaccine is less complicated than the conventional methods that are given through im sc and intradermal thus it removes the needs of trained medical personnel and decreases the risk of infection as there is no need for the sterilization of premises and the manufacturing areas \uf0b7 such oral immunization will become a practical key strategy for effective disease prevention in lowincome countries in general \uf0b7 processing purification sterilization packaging or delivery does not require rigorous structure in edible vaccines minimizing longterm costs in relation to conventional vaccines32 \uf0b7 maintenance and distribution of edible vaccine are easier than the conventional vaccines as it enabling the preservation without the constant cold chain storage \uf0b7 improved storage possibilities for edible vaccines become possible as transgenic plant seeds have lesser moisture content heat stable and can quickly be dried6032 \uf0b7 a plant containing therapeutically active edible vaccine protein is free of toxins pathogens and do not have the risk of proteins to reform into the infectious anism8 \uf0b7 improved compliance is particularly related to children's who refuse to take injections preproof\uf0b7 most specifically in this case the immunity is activated on the mucosal surfaces of the gi including those which are the first line of defence on the mouth mucosal of the vaccine immunity \uf0b7 sophisticated equipment and machinery are not required in edible vaccines since they can easily be cultivated on rich soils and are costeffective relative to fermenters where the recombinant cell is cultured in a controlled manner32 limitation beyond the several advantages and the convenience over the conventional vaccines edible vaccines have certain challenges that have to be overcome for efficient and pure beneficial edible vaccination some of them are pointed below 0c preproof\uf0b7 uncertainty in calculating the appropriate oral dosage which might take multiple rounds for a patient to get the effective therapeutic action and raises the final expense of its application32 \uf0b7 the concentration of peptide or protein of edible vaccine varies from generationtogeneration planttoplant and fruitto fruit32 \uf0b7 patientrelated factor such as patient age and weight also affected the dose to be administered \uf0b7 repeated intakes of these antigenbearing plants which stimulate the immune system might over stimulate the immune system itse Answer:

Colon_Cancer

pediatric obesity remains a public health burden and continues to increasein prevalence the gut microbiota plays a causal role in obesity and is a promising therapeutic target specifically the microbial production of shortchain fatty acids scfa fromthe fermentation of otherwise indigestible dietary carbohydrates may protect against pediatric obesity and metabolic syndrome still it has not been demonstrated that therapies involving microbiotatargeting carbohydrates known as prebiotics will enhance gutbacterial scfa production in children and adolescents with obesity age to yearsold here we used an in vitro system to examine the scfa production by fecal microbiota from children with obesity when exposed to five different commercially availableoverthecounter otc prebiotic supplements we found microbiota from all patientsactively metabolized most prebiotics still supplements varied in their acidogenic potential significant interdonor variation also existed in scfa production which 16s rrna sequencing supported as being associated with differences in the host microbiota composition last we found that neither fecal scfa concentration microbiota scfa productioncapacity nor markers of obesity positively correlated with one another together thesein vitro findings suggest the hypothesis that otc prebiotic supplements may be unequalin their ability to stimulate scfa production in children and adolescents with obesityand that the most acidogenic prebiotic may differ across individualsimportance pediatric obesity remains a major public health problem in the unitedstates where of children and adolescents are obese and rates of pediatric œsevereobesity are increasing children and adolescents with obesity face higher health risksand noninvasive therapies for pediatric obesity often have limited success the humangut microbiome has been implicated in adult obesity and microbiotadirected therapiescan aid weight loss in adults with obesity however less is known about the microbiome in pediatric obesity and microbiotadirected therapies are understudied in children and adolescents our research has two important findings i dietary prebiotics fiber result in the microbiota from adolescents with obesity producing more scfa andii the effectiveness of each prebiotic is donor dependent together these findings suggest that prebiotic supplements could help children and adolescents with obesity butthat these therapies may not be œone size fits allkeywords fermentation microbiome pediatric obesity prebiotics shortchain fattyacidscitation holmes zc silverman jd dressmanhk wei z dallow ep armstrong sc seed pcrawls jf david la shortchain fatty acidproduction by gut microbiota from childrenwith obesity differs according to prebioticchoice and bacterial community compositionmbio 11e0091420 101128mbio0091420invited editor thomas mitchell schmidtuniversity of michigan”ann arboreditor jose c clemente icahn school ofmedicine at mount sinaicopyright holmes this is anopenaccess distributed under the termsof the creative commons attribution international licenseaddress correspondence to lawrence a davidlawrencedaviddukeedu present address justin d silverman collegeof information science and technologypennsylvania state university state collegepennsylvania usa institute for computationaland data science pennsylvania stateuniversity state college pennsylvania usaand department of medicine pennsylvaniastate university hershey pennsylvania usareceived april accepted july published august julyaugust volume issue e0091420®mbioasm 0cholmes ®approximately of children in the united states have obesity and the prevalence continues to increase among all ages and populations the prevalence ofpediatric obesity is even higher in hispanic and african american populations in theunited states where rates of severe obesity continue to increase children withobesity have an increased risk of adverse health events and incur higher health carecosts “ despite the severity of the pediatric obesity epidemic current commontreatment strategies centered around lifestyle changes including behavioral dietaryand exercise interventions often fail or have limited success the high prevalence ofpediatric obesity coupled with the low success rate of common interventions highlights the need for more efficacious safe strategies to lower the body mass index bmiin children and adolescentsthe human gut microbiome has emerged as a promising therapeutic target in pediatricobesity over the past decade differences in gut microbial community composition andmetabolic activity between obese and lean individuals have been observed “ causallinks have also been established fecal transplantation can transfer the obesity phenotypefrom obese donors to lean recipients and recapitulate some key metabolic changes inhuman obesity multiple mechanisms for this link have been proposedincludingincreased energy harvest by obese microbiota activation of enteroendocrine signalingpathways by shortchain fatty acids scfas “ modulation of glucose and energyhomeostasis through bile acid signaling and increased local and systemic ‚ammation caused by a variety of microbial metabolites recent attention in obesity research has been specifically drawn to the role ofmicrobially derived scfas scfas”primarily acetate propionate and butyrate”areproduced by enteric microbes as end products of anaerobic fermentation of undigested microbially accessible dietary carbohydrates and serve a variety of importantroles in the gut of particular interest is the scfa butyrate which serves as the primarynutrient source for colonocytes and functions as a histone deacetylase inhibitor through its inhibition of nf 242cb signaling in colonocytes butyrate contributesto barrier integrity maintenance and reduces levels of intestinal ‚ammation markers“ acetate propionate and butyrate also each activate gproteincoupled receptors gprs that modulate key metabolic hormones including peptide yy pyy andglp1 consistent with these mechanistic findings mouse studies have shownthat supplementation with acetate propionate butyrate or some mixture of these canprotect against weight gain improve insulin sensitivity and reduce obesityassociated‚ammation “ given the experimental evidence for scfa supplementationhaving an antiobesogenic effect in a murine system maintaining high levels of scfasduring a weight loss treatment may improve results if increasing scfa levels is a potential approach to promote weight loss in childrenprebiotic supplementation may provide an effective and lowrisk adjunctive therapyprebiotics are dietary carbohydrates that are indigestible by humanproduced enzymesand thus survive transit to the lower gastrointestinal gi tract once in the colonprebiotics serve as carbon sources for bacterial fermentation which in turn yield scfasas metabolic end products multiple types of prebiotics eg fructooligosaccharides [fos] and inulintype fructans have been tested in children with obesityranging from ages to years old in select cases these treatments have beenassociated with smaller increases in bmi and fat mass and reductions in bodyweight zscores body fat and trunk fat still other prebiotic trials in children whoare overweight have reported no significant beneficial effects interpreting the mixed outcomes of prior prebiotic clinical trials in pediatric obesitythough is complicated by several challenges first in vivo studies in pediatric obesity todate have each used only one prebiotic supplement due to the logistical constraints ofclinical trials “ trials employing testing only a single type of supplement hinderthe ability to generalize s regarding the efficacy of prebiotics and also makeit challenging to determine whether some prebiotics are inherently more acidogenicthan others second in vivo trials in healthy adults have shown substantial interindividual variation in the single prebiotic effects on stool scfa concentration julyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®variation in the primary and secondary outcomes could be due to differences inmicrobial scfa production or differences in host physiology such as scfa absorptionpotential third while scfa concentrations have been shown to be altered in childrenwho are overweight or obese changes in fecal scfas during dietary interventionhave not been measured in past in vivo studies in pediatric populations if prebioticsmediate their effects through scfas directly tracking scfas could helpdetermine treatment success fourth in vivo studies in adults especially those withobesity may be confounded by the concurrence of chronic disease and the medications a person may be taking to treat chronic diseasein this study we have taken an in vitro approach to address the limitations of priorhuman studies an in vitro approach facilitates more direct comparisons of differentprebiotic supplements the higher throughput of in vitro experiments allows widervariety of prebiotics to be tested and the effects of these supplements can be testedon identical microbiota samples rather than over time within subjects which isconfounded by microbiota drift over time as well as inconsistencies in dietarycomposition taking an in vitro approach to studying the effects of prebiotics on gutmicrobiota allows a more direct investigation of microbial scfa production since wecan study the effects of prebiotic supplementation independent of the effects of hostabsorption using a preclinical in vitro fermentation model and samples fromadolescents with obesity who have not developed longterm complications we pursued three specific lines of inquiry i whether different types of prebiotics lead todifferences in scfa production by gut microbiota from adolescents with obesity iiwhether the effects of prebiotics are shaped by interindividual differences in gutmicrobiota structure and iii whether fecal scfa production is likely to be associatedwith protection from obesityresultsscfa production capacity to measure scfa production by gut microbiota weadapted the in vitro approach of edwards this method was specificallydesigned to study fermentation of starch in the human lower gi tract and has sincebeen used to measure metabolite production from human stool samples when exposed to prebiotic fiber “ in brief we homogenized previously frozen feces inreduced phosphatebuffered saline pbs ph «¾ to create a fecal slurry with afinal concentration of gliter fig these fecal slurries were then supplied witheach of five prebiotic carbon sources as well as a carbonfree control and allowed toferment at °c in anaerobic conditions for h to approximate colonic transit time after the incubation period the concentrations of scfas in the samples weremeasured by gas chromatography to control for differences in overall cell viability orstool slurry nutrient content between donors we corrected measurements of scfaconcentration by dividing the treatment scfa concentration by the control scfaconcentrationto validate our assay we ran a series of experiments using feces from validationsample sets we verified that our controlcorrected scfa production data were not‚uenced by bacterial abundance p «½ 2433 «½ spearman correlation seefig s1 in the supplemental material absolute not relativized to control scfaconcentrations are supplied in the supplement see fig s2 and s3 since our fermentation experiments used previously frozen fecal samples we verified that total scfaproduction was strongly correlated between fresh samples and twice freezethawedsamples p ¬ 2433«½ spearman correlation see fig s4a since we elected tonot provide our fermentation reactions with nutrients in excess of what was containedin the fecal slurries we verified that there existed strong correlation in total scfaproduction between pbsgrown and colonic mediumgrown cultures both whensupplied with dextrin and inulin dextrin p «½ 2433«½ inulin p «½ 2433«½ spearman correlations see fig s5 we found that total scfa production over controlwas positively correlated with the ph of starting fecal slurries p «½ 2433 «½ spearman correlation fig 2a a weaker correlation may exist between scfa producjulyaugust volume issue e0091420mbioasm 0cholmes ®fig overview of in vitro fermentation methodstion and the final ph of the fermentation vessels p «½ 2433 «½ spearmancorrelation fig 2bwe subsequently applied our assay to fecal microbiota from a cohort of children male female one unknown ranging in age from to years old average age years tanner stages to and a body mass index bmi of to averagebmi see table s1 in the supplemental material one patient provided samplesused in all analyses but was lost to followup before providing clinical metadata thiscohort was a subset of a cohort of patients enrolled in the pediatric obesity microbiomefig relationship between in vitro scfa production and ph a in vitro total scfa production over control is positively correlated with the phof starting fecal slurries p «½ 2433 «½ spearman correlation b relationship between scfa production and the final ph of fermentationvessels p «½ 2433 «½ spearman correlationjulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®fig in vitro scfa production by prebiotic a donor b and individually c in a twoway anova of the effects of œdonor and œprebiotic on œscfaconcentrationcontrol œdonor œprebiotic and their interaction were all statistically significant p ¬ p ¬ and p ¬ respectively shownis the total scfa concentration of an in vitro culture after h of anaerobic incubation divided by the scfa concentration of the corresponding prebioticfreecontrol culture for each of five prebiotic growth conditions across donors black dots gray diamonds are means and gray bars are standard deviationsabsolute scfa concentrations are depicted in fig s3and metabolism study we found all individuals demonstrated a net gain ofscfas relative to the controlin at least one prebiotic treatment which led us toconclude that all tested cultures were viable and metabolically active fig donor and prebiotic both impact scfa production in vitro we next tested thehypothesis that different prebiotics equally promote the production of scfas byjulyaugust volume issue e0091420mbioasm 0cholmes ®fig eighteen genera were found to be credibly associated with scfa production in at least one of our five prebiotic growth conditions shownare the mean lambda values and and credible intervals for all genera credibly associated with at least one prebioticgrowth condition plotted on centered logratio clr coordinates red centers denote associations with credible intervals that do not cover lambda represents the strength of the effect of each covariate on each taxa a lambda value of reflects a unit fold change in scfaconcentration over control as being associated with a unit fold change in the clrtransformed relative abundance of the genusperforming statistical analysis of scfa production as a function of the prebiotic typeand individual identity our analysis revealed heterogeneity in the efficacy of prebioticsupplements twoway analysis of variance [anova] p ¬ see table s2 fig 3aranging from inulin which resulted in a mean fold change in total scfas togalactooligosaccharides gos which resulted in mean fold change in total scfasfrequently only two or three of the five tested prebiotics resulted in increased totalscfa production within an individual our statistical testing also revealed consistentpatterns between individuals™ gut microbiota in terms of scfa production twowayanova p ¬ see table s2 fig 3b with mean fold changes in scfas over controlranging from to within individuals the average fold change in scfa concentration in the prebiotic treatments often appeared to be driven by a few stronglyacidogenic prebiotics last our analysis indicated a significant interaction betweenidentity twoway anova p ¬ see table s2prebiotic type and individualfig 3c because our statistical analysis considered technical replicates as separateexperimental conditions this result suggests the presence of consistent prebioticindividual responses across in vitro assay replicate runs”not whether such interactionsare consistent within an individual over timescfa production in vitro predicts the abundance of bacteria in the startingculture if interindividual differences in gut microbiota mediated responses to prebiotictreatment we would expect that specific bacterial taxa which varied between individuals could also be associated with scfa production to evaluate this hypothesis weused the r package stray to create a bayesian multinomial logistic normal linearregression pibble model that tested for correlations between in vitro scfa productionin response to each prebiotic and 16s rrna community composition of patient stoolused in the fermentations at the genus level this analysis revealed that scfa production from prebiotics was correlated with the relative abundances of differentbacterial genera credible interval not covering fig of the generapositively associated with scfa production are known or likely fiber degraders“ one akkermansia is often observed to increase in abundance after prebiotictreatment and one methanobrevibacter an archaeon hydrogenotrophic methanojulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesitytable associations between microbial genera and scfa production on five different prebiotic substratesassociation with scfa productiondextrin«¹«¹«¹«¹«¹«¹«¹«¹«¹genusakkermansiaruminococcus_2coprostanoligenes_groupparabacteroidesbutyricimonasmethanobrevibactertyzzerella_4tyzzerella_3lachnospiraceae_nk4b4lactobacilluscoprococcus_1collinsellalachnospiraceae_fcs020xosgosfosinulin«¹«¹«¹“«¹fiber degrader statussupporterdegraderno evidencedegraderassociatedsupporterdegraderdegraderdegraderdegraderdegraderno evidencedegrader®referencegen is known to increase the efficiency of carbohydrate metabolism by the microbiota table most genera identified by stray were associated with scfa production ina limited set of prebiotic treatments one genus lactobacillus is positively associatedwith scfa production on xylooligosaccharides xos but was negatively associatedwith scfa production on gos overall the presence of specific associations betweenbacterial taxa and different prebiotics supports a model where different individuals varyin their levels of prebiotic degrading gut bacteriametrics of obesity do not appear to correlate with scfa production capacity ofstool finally we tested the hypothesis that in vitro scfa production would beassociated with obesityrelated phenotypes we compared clinical metadata fromindividuals which included bmi insulin and hba1c with average total scfa production across prebiotics and found no significant correlations in our population spearman correlation table fecal microbial scfa production capacity may not be directlyassociated with obesity though because rates of host scfa uptake likely vary and thisvariance may ‚uence host intestinal physiology “ indeed in support of theidea that scfa absorption rate which was not measured in this study shape metabolichomeostasis and host health we observed a negative association between fecal scfaconcentrations and in vitro scfa production across the range of tested prebioticsfig furthermore if scfa absorption efficiencies varied by individual residual fecalscfa concentrations may not directly reflect the complete effect of bacterial metabolism on obesity consistent with this notion no significant relationships were apparentbetween concentrations of scfa in patient stool and clinical markers of obesitymeasured at enrollment including bmi insulin levels and hba1c table althoughthis may also be explained by uncontrolled patient parametersdiscussionin this study we found that the microbiota of all tested adolescents with obesityincreased total scfa production when exposed in vitro to at least one prebiotic bothdonor and prebiotic were significant factors in determining scfa production in vitro aswas their interaction our modeling revealed distinct associations between specificmicrobial taxa and scfa production on different prebiotics we interpret this result astable neither average scfa production in vitro nor fecal scfa concentrationcorrelated with metrics of obesity measured in individuals at the time of enrollmentabmiparameteravg net scfa productionfecal scfa concentrationap and 2433 values were determined from spearman correlations 2433““pinsulinp 2433hba1cp 2433“julyaugust volume issue e0091420mbioasm 0cholmes ®fig spearman correlations between in vitro scfa production and scfa concentration of the starting fecal inoculum scfaproduction is the average of technical replicates with the linear regression line plottedsuggesting that the associated bacteria play a role in the fiber fermenting capacity ofthe community we observed no correlations between either stool scfa concentrationsor in vitro acidogenic capacity of communities and any metrics of obesity table we have recapitulated previous findings that both donor and prebiotic areimportant in determining the scfa production from in vitro prebiotic supplementation and we found that not all prebiotics appear equally acidogenic since our in vitro system removes the host as a potential source of variationour data support a gut microbial role for interdonor variation in fecal scfaproduction in addition the strength of the interaction between donor and prebiotic strongly suggests that prebiotics are not œone size fits all rather inconsistentresults from prior studies of prebiotics in pediatric obesity may be dueto variation in the scfa production capacity of individuals™ gut microbiota acrossthe tested prebiotics future therapeutic efforts involving prebiotics in patients withobesity may benefit from stratified or personalized treatments nutritional therapiesthat are personalized to individuals™ microbiota are already in development murine and in vitro studies show that increased signaling through gpcrs mediatedby acetate propionate and butyrate increases satiety and insulin sensitivity whiledecreasing adipogenesis yet we did not observe associations betweenfecal scfa levels and metrics of obesity the effects of scfa on obesity may be maskedby uncontrolled patient factors such as differences in caloric intake and variation inindividual nutrient harvest and utilization in order to observe the effects of scfa onobesity it would be necessary to control for these variable physiological and lifestyleparameters which we did not attempt these patient factors may also have ‚uencedour inability to observe an association between acidogenic capacity of microbiota andfecal scfa concentrations however this may also be explained by the potentialjulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®uncoupling of fecal scfa production and fecal scfa concentration in vitro increasedluminal concentrations of butyrate have been shown to upregulate the sodiumcoupled monocarboxylate transporter slc5a8 and the addition of physiologicalmixtures of scfa has been shown to upregulate the monocarboxylate transporterslc16a1 both of which uptake acetate propionate and butyrate from the lumensince gut epithelia have the capacity to absorb up to of scfa before excretion increased host scfa uptake triggered by increased gut bacterial production couldtherefore lead to constant or even decreased fecal scfa concentrations this complexrelationship could explain the absence of positive correlations we observed betweenstool scfa levels and the acidogenic capacity of gut microbiota it may be necessary todelve further upstream of fecal scfa concentration by measuring proxies for host scfauptakes such as the expression of scfa transporters slc5a8 and slc16a1 and scfareceptors gpr43 gpr41 and gpr109a the primary limitations of this study involve constraints common to in vitro culturestudies first many factors affecting bacterial scfa production in vivo are difficult toreplicate in vitro including the availability of nutrients such as nitrogen the startingconcentration of scfas the redox state of the environment and the efficiency ofcrossfeeding interactions different metabolic results between prebiotics mayhave occurred if we provided alternative cometabolites or nutrients in addition to thetested prebiotics we chose our culture conditions namely a mediumfree approachthat does not add any nutrients beyond what is present in the stool in an effort toavoid inducing artificial selective conditions within our cultures prior experimentaldigestion studies have shown that prebiotic response patterns can be recapitulatedacross various culture conditions indeed we found strong correlation in scfaproduction between cultures grown with our mediumfree approach and those grownin a more conventional medium containing added nitrogen vitamins minerals andacetate further this approach allowed us to minimize the ‚uence of the host onmeasurements of microbiota production of scfa we did observe shifts in communitycomposition during the h fermentations fig s6 however we remained able to findstatistical associations between scfa production capacity and prefermentation community composition a second set of limitations in this study involves our reliance onpatient collection of stoolinterdonor variation in prebiotic response could haveoriginated in technical variation between how patients exposed stool to aerobicconditions or how they froze their samples which in turn could have affectedthe fraction of viable microbial cells in stool samples still we found a significantcorrelation between in vitro total scfa production from fresh stool and stool that hadbeen frozen and thawed twice variation in donor prebiotic response could also havebiological origins due to physiological differences between people eg efficiency offood digestion consistency of stool [] or differences in diet which can lead tovariation in stool microbial load and nutrient content rather than control for amyriad of different sources of variation whose origins we did not measure we chosethe straightforward approach of standardizing donor samples by employing a consistent concentration of stool slurry [wtvol] stool in pbs in our experimentsfuture work to address these limitations could test multiple stool samples persubject to confirm whether the observed variation in prebiotic response is durablebetween individuals over time future studies could also examine the correlationbetween the metabolic effects of prebiotic supplementation in vitro and in vivo usingrandomized human trials that couple human prebiotic supplementation in vivo measurement of scfa production and in vitro tests of microbiota metabolic activity itwould also be useful for such studies to explore the impact of prebiotic supplementation on host physiology both in vitro and in vivo specifically the effects of prebioticsupplementation on colonic epithelial barrier integrity scfa receptor gpr41 gpr43and gpr109a expression and scfa transporter mct1 and smct1 expression couldprovide greater insight into the health impacts of prebiotic supplementation as well asexplain why fecal scfa concentrations may not mirror the metabolic capacity of gutmicrobiotajulyaugust volume issue e0091420mbioasm 0c®holmes materials and methodscohort stool was collected from human donors under a protocol approved by the duke healthinstitutional review board duke health irb pro00074547 for a prospective longitudinal cohort studyand biorepository participants whose samples were used in this study were treatmentseeking adolescents with obesity who were newly enrolled in a multidisciplinary weight management program allsubjects received familybased intensive lifestyle modification based on clinical necessity some participants also were placed on a lowcarbohydrate diet medications to facilitate weight loss or underwentweight loss surgery see table s3 due to the low number of patients assigned to each treatment armwe did not attempt to base any analyses on patient treatment plan patients were to years old witha bmi ±– 95th percentile none had antibiotic use in the month prior to enrollment used medicationsknown to interfere with the intestinal microbiome or had other significant medical problems stoolsamples used in this study were from enrollment 3month 45month and 6month followup visits seetable s3 the clinical metadata used for correlations was collected at enrollment months and months the metadata collected nearest to the stool sample collection date was used in our analysesstool collection patients collected intact stool samples in the clinic or at home using a plastic stoolcollection container fisher scientific and were asked to immediately store this container intheir home freezer patients then returned the sample by either bringing it to the study team orscheduling a home pickup within h of stooling stool was transported frozen in an insulated containerwith an ice pack upon receipt in the lab samples were placed on dry ice until transferred to a “ °cfreezer for longterm storage all patient samples were frozen at “ °c within h of stooling range h to h median h except for one which was stored h after stooling the timebetween stooling and freezing at “ °c did not have a significant effect on average scfa productionp «½ 2433 «½ «º pearson correlation stool samples for analysis were processed by removingcontainers from “ °c storage and thawing on ice in a biological safety cabinet until soft enough toaliquot thawed containers of stool were opened to atmosphere for a maximum of min while sampleswere aliquoted after primary aliquoting the remaining stool was transferred to an anaerobic chambercoy laboratory products hydrogen co2 nitrogen and further portioned into ¬ƒ2galiquots for this study these aliquots were then stored as solid stool pellets at “ °c until used for thisstudyin vitro fermentation see fig for an overview of in vitro fermentation methods aliquotedstool was thawed at room temperature in an anaerobic chamber once thawed stool was weighedand placed into a polyethylene filter bag with 033mm pore size whirlpak b01385 and ml ofanaerobic «» pbs was added for each gram of stool resulting in a wtvol fecal slurry similarto previous studies during our validation experiments a medium designed tosimulate colonic contents was used in place of «» pbs to create stool slurries the filter bag wasthen closed and placed into a stomacher seward stomacher where the contents were homogenized on the medium speed setting for s the liquid fraction was removed from the downstreamside of the filter membrane and the solid fraction was discarded a 1ml aliquot of this liquid fractionwas removed for analysis of the scfa concentration to determine the scfa concentration of thestarting stool sample during our validation experiments two separate 1ml aliquots of this liquidfraction were removed one was used to estimate relative bacteria abundance of starting fecalslurries using total extracted dna concentration as has been previously published and theremaining aliquot was used to determine the ph of the starting fecal slurry using a handheld phmeter elite ph spear thermo fisher scientific the remaining liquid fraction was incubated induplicate across six different treatments either supplemented with inulin now foods inulin powderpart fructooligosaccharides fos cargill part galactooligosaccharides gosbimuno powder xylooligosaccharides xos bionutrition prebiotic with llifeoligo part wheatdextrin benefiber original or unsupplemented for each reaction ml of fecal slurry wasplaced in one well of a 24well cell culture plate each well was then delivered ml of wtvolprebiotic solution in «» pbs or ml of «» pbs without prebiotic during our validation experimentsprebiotics were dissolved in colonic medium instead of «» pbs the resulting fermentationconditions were therefore fecal slurry with prebiotic wtvol a fecal slurry was selectedbecause its fermentative capacity has been previously demonstrated to be insensitive to smallvariations in concentration and is feasible to work with using this method a finalconcentration of prebiotic in the context of a fecal slurry i

cancer7537

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: pediatric obesity remains a public health burden and continues to increasein prevalence the gut microbiota plays a causal role in obesity and is a promising therapeutic target specifically the microbial production of shortchain fatty acids scfa fromthe fermentation of otherwise indigestible dietary carbohydrates may protect against pediatric obesity and metabolic syndrome still it has not been demonstrated that therapies involving microbiotatargeting carbohydrates known as prebiotics will enhance gutbacterial scfa production in children and adolescents with obesity age to yearsold here we used an in vitro system to examine the scfa production by fecal microbiota from children with obesity when exposed to five different commercially availableoverthecounter otc prebiotic supplements we found microbiota from all patientsactively metabolized most prebiotics still supplements varied in their acidogenic potential significant interdonor variation also existed in scfa production which 16s rrna sequencing supported as being associated with differences in the host microbiota composition last we found that neither fecal scfa concentration microbiota scfa productioncapacity nor markers of obesity positively correlated with one another together thesein vitro findings suggest the hypothesis that otc prebiotic supplements may be unequalin their ability to stimulate scfa production in children and adolescents with obesityand that the most acidogenic prebiotic may differ across individualsimportance pediatric obesity remains a major public health problem in the unitedstates where of children and adolescents are obese and rates of pediatric œsevereobesity are increasing children and adolescents with obesity face higher health risksand noninvasive therapies for pediatric obesity often have limited success the humangut microbiome has been implicated in adult obesity and microbiotadirected therapiescan aid weight loss in adults with obesity however less is known about the microbiome in pediatric obesity and microbiotadirected therapies are understudied in children and adolescents our research has two important findings i dietary prebiotics fiber result in the microbiota from adolescents with obesity producing more scfa andii the effectiveness of each prebiotic is donor dependent together these findings suggest that prebiotic supplements could help children and adolescents with obesity butthat these therapies may not be œone size fits allkeywords fermentation microbiome pediatric obesity prebiotics shortchain fattyacidscitation holmes zc silverman jd dressmanhk wei z dallow ep armstrong sc seed pcrawls jf david la shortchain fatty acidproduction by gut microbiota from childrenwith obesity differs according to prebioticchoice and bacterial community compositionmbio 11e0091420 101128mbio0091420invited editor thomas mitchell schmidtuniversity of michigan”ann arboreditor jose c clemente icahn school ofmedicine at mount sinaicopyright holmes this is anopenaccess distributed under the termsof the creative commons attribution international licenseaddress correspondence to lawrence a davidlawrencedaviddukeedu present address justin d silverman collegeof information science and technologypennsylvania state university state collegepennsylvania usa institute for computationaland data science pennsylvania stateuniversity state college pennsylvania usaand department of medicine pennsylvaniastate university hershey pennsylvania usareceived april accepted july published august julyaugust volume issue e0091420®mbioasm 0cholmes ®approximately of children in the united states have obesity and the prevalence continues to increase among all ages and populations the prevalence ofpediatric obesity is even higher in hispanic and african american populations in theunited states where rates of severe obesity continue to increase children withobesity have an increased risk of adverse health events and incur higher health carecosts “ despite the severity of the pediatric obesity epidemic current commontreatment strategies centered around lifestyle changes including behavioral dietaryand exercise interventions often fail or have limited success the high prevalence ofpediatric obesity coupled with the low success rate of common interventions highlights the need for more efficacious safe strategies to lower the body mass index bmiin children and adolescentsthe human gut microbiome has emerged as a promising therapeutic target in pediatricobesity over the past decade differences in gut microbial community composition andmetabolic activity between obese and lean individuals have been observed “ causallinks have also been established fecal transplantation can transfer the obesity phenotypefrom obese donors to lean recipients and recapitulate some key metabolic changes inhuman obesity multiple mechanisms for this link have been proposedincludingincreased energy harvest by obese microbiota activation of enteroendocrine signalingpathways by shortchain fatty acids scfas “ modulation of glucose and energyhomeostasis through bile acid signaling and increased local and systemic ‚ammation caused by a variety of microbial metabolites recent attention in obesity research has been specifically drawn to the role ofmicrobially derived scfas scfas”primarily acetate propionate and butyrate”areproduced by enteric microbes as end products of anaerobic fermentation of undigested microbially accessible dietary carbohydrates and serve a variety of importantroles in the gut of particular interest is the scfa butyrate which serves as the primarynutrient source for colonocytes and functions as a histone deacetylase inhibitor through its inhibition of nf 242cb signaling in colonocytes butyrate contributesto barrier integrity maintenance and reduces levels of intestinal ‚ammation markers“ acetate propionate and butyrate also each activate gproteincoupled receptors gprs that modulate key metabolic hormones including peptide yy pyy andglp1 consistent with these mechanistic findings mouse studies have shownthat supplementation with acetate propionate butyrate or some mixture of these canprotect against weight gain improve insulin sensitivity and reduce obesityassociated‚ammation “ given the experimental evidence for scfa supplementationhaving an antiobesogenic effect in a murine system maintaining high levels of scfasduring a weight loss treatment may improve results if increasing scfa levels is a potential approach to promote weight loss in childrenprebiotic supplementation may provide an effective and lowrisk adjunctive therapyprebiotics are dietary carbohydrates that are indigestible by humanproduced enzymesand thus survive transit to the lower gastrointestinal gi tract once in the colonprebiotics serve as carbon sources for bacterial fermentation which in turn yield scfasas metabolic end products multiple types of prebiotics eg fructooligosaccharides [fos] and inulintype fructans have been tested in children with obesityranging from ages to years old in select cases these treatments have beenassociated with smaller increases in bmi and fat mass and reductions in bodyweight zscores body fat and trunk fat still other prebiotic trials in children whoare overweight have reported no significant beneficial effects interpreting the mixed outcomes of prior prebiotic clinical trials in pediatric obesitythough is complicated by several challenges first in vivo studies in pediatric obesity todate have each used only one prebiotic supplement due to the logistical constraints ofclinical trials “ trials employing testing only a single type of supplement hinderthe ability to generalize s regarding the efficacy of prebiotics and also makeit challenging to determine whether some prebiotics are inherently more acidogenicthan others second in vivo trials in healthy adults have shown substantial interindividual variation in the single prebiotic effects on stool scfa concentration julyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®variation in the primary and secondary outcomes could be due to differences inmicrobial scfa production or differences in host physiology such as scfa absorptionpotential third while scfa concentrations have been shown to be altered in childrenwho are overweight or obese changes in fecal scfas during dietary interventionhave not been measured in past in vivo studies in pediatric populations if prebioticsmediate their effects through scfas directly tracking scfas could helpdetermine treatment success fourth in vivo studies in adults especially those withobesity may be confounded by the concurrence of chronic disease and the medications a person may be taking to treat chronic diseasein this study we have taken an in vitro approach to address the limitations of priorhuman studies an in vitro approach facilitates more direct comparisons of differentprebiotic supplements the higher throughput of in vitro experiments allows widervariety of prebiotics to be tested and the effects of these supplements can be testedon identical microbiota samples rather than over time within subjects which isconfounded by microbiota drift over time as well as inconsistencies in dietarycomposition taking an in vitro approach to studying the effects of prebiotics on gutmicrobiota allows a more direct investigation of microbial scfa production since wecan study the effects of prebiotic supplementation independent of the effects of hostabsorption using a preclinical in vitro fermentation model and samples fromadolescents with obesity who have not developed longterm complications we pursued three specific lines of inquiry i whether different types of prebiotics lead todifferences in scfa production by gut microbiota from adolescents with obesity iiwhether the effects of prebiotics are shaped by interindividual differences in gutmicrobiota structure and iii whether fecal scfa production is likely to be associatedwith protection from obesityresultsscfa production capacity to measure scfa production by gut microbiota weadapted the in vitro approach of edwards this method was specificallydesigned to study fermentation of starch in the human lower gi tract and has sincebeen used to measure metabolite production from human stool samples when exposed to prebiotic fiber “ in brief we homogenized previously frozen feces inreduced phosphatebuffered saline pbs ph «¾ to create a fecal slurry with afinal concentration of gliter fig these fecal slurries were then supplied witheach of five prebiotic carbon sources as well as a carbonfree control and allowed toferment at °c in anaerobic conditions for h to approximate colonic transit time after the incubation period the concentrations of scfas in the samples weremeasured by gas chromatography to control for differences in overall cell viability orstool slurry nutrient content between donors we corrected measurements of scfaconcentration by dividing the treatment scfa concentration by the control scfaconcentrationto validate our assay we ran a series of experiments using feces from validationsample sets we verified that our controlcorrected scfa production data were not‚uenced by bacterial abundance p «½ 2433 «½ spearman correlation seefig s1 in the supplemental material absolute not relativized to control scfaconcentrations are supplied in the supplement see fig s2 and s3 since our fermentation experiments used previously frozen fecal samples we verified that total scfaproduction was strongly correlated between fresh samples and twice freezethawedsamples p ¬ 2433«½ spearman correlation see fig s4a since we elected tonot provide our fermentation reactions with nutrients in excess of what was containedin the fecal slurries we verified that there existed strong correlation in total scfaproduction between pbsgrown and colonic mediumgrown cultures both whensupplied with dextrin and inulin dextrin p «½ 2433«½ inulin p «½ 2433«½ spearman correlations see fig s5 we found that total scfa production over controlwas positively correlated with the ph of starting fecal slurries p «½ 2433 «½ spearman correlation fig 2a a weaker correlation may exist between scfa producjulyaugust volume issue e0091420mbioasm 0cholmes ®fig overview of in vitro fermentation methodstion and the final ph of the fermentation vessels p «½ 2433 «½ spearmancorrelation fig 2bwe subsequently applied our assay to fecal microbiota from a cohort of children male female one unknown ranging in age from to years old average age years tanner stages to and a body mass index bmi of to averagebmi see table s1 in the supplemental material one patient provided samplesused in all analyses but was lost to followup before providing clinical metadata thiscohort was a subset of a cohort of patients enrolled in the pediatric obesity microbiomefig relationship between in vitro scfa production and ph a in vitro total scfa production over control is positively correlated with the phof starting fecal slurries p «½ 2433 «½ spearman correlation b relationship between scfa production and the final ph of fermentationvessels p «½ 2433 «½ spearman correlationjulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®fig in vitro scfa production by prebiotic a donor b and individually c in a twoway anova of the effects of œdonor and œprebiotic on œscfaconcentrationcontrol œdonor œprebiotic and their interaction were all statistically significant p ¬ p ¬ and p ¬ respectively shownis the total scfa concentration of an in vitro culture after h of anaerobic incubation divided by the scfa concentration of the corresponding prebioticfreecontrol culture for each of five prebiotic growth conditions across donors black dots gray diamonds are means and gray bars are standard deviationsabsolute scfa concentrations are depicted in fig s3and metabolism study we found all individuals demonstrated a net gain ofscfas relative to the controlin at least one prebiotic treatment which led us toconclude that all tested cultures were viable and metabolically active fig donor and prebiotic both impact scfa production in vitro we next tested thehypothesis that different prebiotics equally promote the production of scfas byjulyaugust volume issue e0091420mbioasm 0cholmes ®fig eighteen genera were found to be credibly associated with scfa production in at least one of our five prebiotic growth conditions shownare the mean lambda values and and credible intervals for all genera credibly associated with at least one prebioticgrowth condition plotted on centered logratio clr coordinates red centers denote associations with credible intervals that do not cover lambda represents the strength of the effect of each covariate on each taxa a lambda value of reflects a unit fold change in scfaconcentration over control as being associated with a unit fold change in the clrtransformed relative abundance of the genusperforming statistical analysis of scfa production as a function of the prebiotic typeand individual identity our analysis revealed heterogeneity in the efficacy of prebioticsupplements twoway analysis of variance [anova] p ¬ see table s2 fig 3aranging from inulin which resulted in a mean fold change in total scfas togalactooligosaccharides gos which resulted in mean fold change in total scfasfrequently only two or three of the five tested prebiotics resulted in increased totalscfa production within an individual our statistical testing also revealed consistentpatterns between individuals™ gut microbiota in terms of scfa production twowayanova p ¬ see table s2 fig 3b with mean fold changes in scfas over controlranging from to within individuals the average fold change in scfa concentration in the prebiotic treatments often appeared to be driven by a few stronglyacidogenic prebiotics last our analysis indicated a significant interaction betweenidentity twoway anova p ¬ see table s2prebiotic type and individualfig 3c because our statistical analysis considered technical replicates as separateexperimental conditions this result suggests the presence of consistent prebioticindividual responses across in vitro assay replicate runs”not whether such interactionsare consistent within an individual over timescfa production in vitro predicts the abundance of bacteria in the startingculture if interindividual differences in gut microbiota mediated responses to prebiotictreatment we would expect that specific bacterial taxa which varied between individuals could also be associated with scfa production to evaluate this hypothesis weused the r package stray to create a bayesian multinomial logistic normal linearregression pibble model that tested for correlations between in vitro scfa productionin response to each prebiotic and 16s rrna community composition of patient stoolused in the fermentations at the genus level this analysis revealed that scfa production from prebiotics was correlated with the relative abundances of differentbacterial genera credible interval not covering fig of the generapositively associated with scfa production are known or likely fiber degraders“ one akkermansia is often observed to increase in abundance after prebiotictreatment and one methanobrevibacter an archaeon hydrogenotrophic methanojulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesitytable associations between microbial genera and scfa production on five different prebiotic substratesassociation with scfa productiondextrin«¹«¹«¹«¹«¹«¹«¹«¹«¹genusakkermansiaruminococcus_2coprostanoligenes_groupparabacteroidesbutyricimonasmethanobrevibactertyzzerella_4tyzzerella_3lachnospiraceae_nk4b4lactobacilluscoprococcus_1collinsellalachnospiraceae_fcs020xosgosfosinulin«¹«¹«¹“«¹fiber degrader statussupporterdegraderno evidencedegraderassociatedsupporterdegraderdegraderdegraderdegraderdegraderno evidencedegrader®referencegen is known to increase the efficiency of carbohydrate metabolism by the microbiota table most genera identified by stray were associated with scfa production ina limited set of prebiotic treatments one genus lactobacillus is positively associatedwith scfa production on xylooligosaccharides xos but was negatively associatedwith scfa production on gos overall the presence of specific associations betweenbacterial taxa and different prebiotics supports a model where different individuals varyin their levels of prebiotic degrading gut bacteriametrics of obesity do not appear to correlate with scfa production capacity ofstool finally we tested the hypothesis that in vitro scfa production would beassociated with obesityrelated phenotypes we compared clinical metadata fromindividuals which included bmi insulin and hba1c with average total scfa production across prebiotics and found no significant correlations in our population spearman correlation table fecal microbial scfa production capacity may not be directlyassociated with obesity though because rates of host scfa uptake likely vary and thisvariance may ‚uence host intestinal physiology “ indeed in support of theidea that scfa absorption rate which was not measured in this study shape metabolichomeostasis and host health we observed a negative association between fecal scfaconcentrations and in vitro scfa production across the range of tested prebioticsfig furthermore if scfa absorption efficiencies varied by individual residual fecalscfa concentrations may not directly reflect the complete effect of bacterial metabolism on obesity consistent with this notion no significant relationships were apparentbetween concentrations of scfa in patient stool and clinical markers of obesitymeasured at enrollment including bmi insulin levels and hba1c table althoughthis may also be explained by uncontrolled patient parametersdiscussionin this study we found that the microbiota of all tested adolescents with obesityincreased total scfa production when exposed in vitro to at least one prebiotic bothdonor and prebiotic were significant factors in determining scfa production in vitro aswas their interaction our modeling revealed distinct associations between specificmicrobial taxa and scfa production on different prebiotics we interpret this result astable neither average scfa production in vitro nor fecal scfa concentrationcorrelated with metrics of obesity measured in individuals at the time of enrollmentabmiparameteravg net scfa productionfecal scfa concentrationap and 2433 values were determined from spearman correlations 2433““pinsulinp 2433hba1cp 2433“julyaugust volume issue e0091420mbioasm 0cholmes ®fig spearman correlations between in vitro scfa production and scfa concentration of the starting fecal inoculum scfaproduction is the average of technical replicates with the linear regression line plottedsuggesting that the associated bacteria play a role in the fiber fermenting capacity ofthe community we observed no correlations between either stool scfa concentrationsor in vitro acidogenic capacity of communities and any metrics of obesity table we have recapitulated previous findings that both donor and prebiotic areimportant in determining the scfa production from in vitro prebiotic supplementation and we found that not all prebiotics appear equally acidogenic since our in vitro system removes the host as a potential source of variationour data support a gut microbial role for interdonor variation in fecal scfaproduction in addition the strength of the interaction between donor and prebiotic strongly suggests that prebiotics are not œone size fits all rather inconsistentresults from prior studies of prebiotics in pediatric obesity may be dueto variation in the scfa production capacity of individuals™ gut microbiota acrossthe tested prebiotics future therapeutic efforts involving prebiotics in patients withobesity may benefit from stratified or personalized treatments nutritional therapiesthat are personalized to individuals™ microbiota are already in development murine and in vitro studies show that increased signaling through gpcrs mediatedby acetate propionate and butyrate increases satiety and insulin sensitivity whiledecreasing adipogenesis yet we did not observe associations betweenfecal scfa levels and metrics of obesity the effects of scfa on obesity may be maskedby uncontrolled patient factors such as differences in caloric intake and variation inindividual nutrient harvest and utilization in order to observe the effects of scfa onobesity it would be necessary to control for these variable physiological and lifestyleparameters which we did not attempt these patient factors may also have ‚uencedour inability to observe an association between acidogenic capacity of microbiota andfecal scfa concentrations however this may also be explained by the potentialjulyaugust volume issue e0091420mbioasm 0cgut microbial scfa production in pediatric obesity®uncoupling of fecal scfa production and fecal scfa concentration in vitro increasedluminal concentrations of butyrate have been shown to upregulate the sodiumcoupled monocarboxylate transporter slc5a8 and the addition of physiologicalmixtures of scfa has been shown to upregulate the monocarboxylate transporterslc16a1 both of which uptake acetate propionate and butyrate from the lumensince gut epithelia have the capacity to absorb up to of scfa before excretion increased host scfa uptake triggered by increased gut bacterial production couldtherefore lead to constant or even decreased fecal scfa concentrations this complexrelationship could explain the absence of positive correlations we observed betweenstool scfa levels and the acidogenic capacity of gut microbiota it may be necessary todelve further upstream of fecal scfa concentration by measuring proxies for host scfauptakes such as the expression of scfa transporters slc5a8 and slc16a1 and scfareceptors gpr43 gpr41 and gpr109a the primary limitations of this study involve constraints common to in vitro culturestudies first many factors affecting bacterial scfa production in vivo are difficult toreplicate in vitro including the availability of nutrients such as nitrogen the startingconcentration of scfas the redox state of the environment and the efficiency ofcrossfeeding interactions different metabolic results between prebiotics mayhave occurred if we provided alternative cometabolites or nutrients in addition to thetested prebiotics we chose our culture conditions namely a mediumfree approachthat does not add any nutrients beyond what is present in the stool in an effort toavoid inducing artificial selective conditions within our cultures prior experimentaldigestion studies have shown that prebiotic response patterns can be recapitulatedacross various culture conditions indeed we found strong correlation in scfaproduction between cultures grown with our mediumfree approach and those grownin a more conventional medium containing added nitrogen vitamins minerals andacetate further this approach allowed us to minimize the ‚uence of the host onmeasurements of microbiota production of scfa we did observe shifts in communitycomposition during the h fermentations fig s6 however we remained able to findstatistical associations between scfa production capacity and prefermentation community composition a second set of limitations in this study involves our reliance onpatient collection of stoolinterdonor variation in prebiotic response could haveoriginated in technical variation between how patients exposed stool to aerobicconditions or how they froze their samples which in turn could have affectedthe fraction of viable microbial cells in stool samples still we found a significantcorrelation between in vitro total scfa production from fresh stool and stool that hadbeen frozen and thawed twice variation in donor prebiotic response could also havebiological origins due to physiological differences between people eg efficiency offood digestion consistency of stool [] or differences in diet which can lead tovariation in stool microbial load and nutrient content rather than control for amyriad of different sources of variation whose origins we did not measure we chosethe straightforward approach of standardizing donor samples by employing a consistent concentration of stool slurry [wtvol] stool in pbs in our experimentsfuture work to address these limitations could test multiple stool samples persubject to confirm whether the observed variation in prebiotic response is durablebetween individuals over time future studies could also examine the correlationbetween the metabolic effects of prebiotic supplementation in vitro and in vivo usingrandomized human trials that couple human prebiotic supplementation in vivo measurement of scfa production and in vitro tests of microbiota metabolic activity itwould also be useful for such studies to explore the impact of prebiotic supplementation on host physiology both in vitro and in vivo specifically the effects of prebioticsupplementation on colonic epithelial barrier integrity scfa receptor gpr41 gpr43and gpr109a expression and scfa transporter mct1 and smct1 expression couldprovide greater insight into the health impacts of prebiotic supplementation as well asexplain why fecal scfa concentrations may not mirror the metabolic capacity of gutmicrobiotajulyaugust volume issue e0091420mbioasm 0c®holmes materials and methodscohort stool was collected from human donors under a protocol approved by the duke healthinstitutional review board duke health irb pro00074547 for a prospective longitudinal cohort studyand biorepository participants whose samples were used in this study were treatmentseeking adolescents with obesity who were newly enrolled in a multidisciplinary weight management program allsubjects received familybased intensive lifestyle modification based on clinical necessity some participants also were placed on a lowcarbohydrate diet medications to facilitate weight loss or underwentweight loss surgery see table s3 due to the low number of patients assigned to each treatment armwe did not attempt to base any analyses on patient treatment plan patients were to years old witha bmi ±– 95th percentile none had antibiotic use in the month prior to enrollment used medicationsknown to interfere with the intestinal microbiome or had other significant medical problems stoolsamples used in this study were from enrollment 3month 45month and 6month followup visits seetable s3 the clinical metadata used for correlations was collected at enrollment months and months the metadata collected nearest to the stool sample collection date was used in our analysesstool collection patients collected intact stool samples in the clinic or at home using a plastic stoolcollection container fisher scientific and were asked to immediately store this container intheir home freezer patients then returned the sample by either bringing it to the study team orscheduling a home pickup within h of stooling stool was transported frozen in an insulated containerwith an ice pack upon receipt in the lab samples were placed on dry ice until transferred to a “ °cfreezer for longterm storage all patient samples were frozen at “ °c within h of stooling range h to h median h except for one which was stored h after stooling the timebetween stooling and freezing at “ °c did not have a significant effect on average scfa productionp «½ 2433 «½ «º pearson correlation stool samples for analysis were processed by removingcontainers from “ °c storage and thawing on ice in a biological safety cabinet until soft enough toaliquot thawed containers of stool were opened to atmosphere for a maximum of min while sampleswere aliquoted after primary aliquoting the remaining stool was transferred to an anaerobic chambercoy laboratory products hydrogen co2 nitrogen and further portioned into ¬ƒ2galiquots for this study these aliquots were then stored as solid stool pellets at “ °c until used for thisstudyin vitro fermentation see fig for an overview of in vitro fermentation methods aliquotedstool was thawed at room temperature in an anaerobic chamber once thawed stool was weighedand placed into a polyethylene filter bag with 033mm pore size whirlpak b01385 and ml ofanaerobic «» pbs was added for each gram of stool resulting in a wtvol fecal slurry similarto previous studies during our validation experiments a medium designed tosimulate colonic contents was used in place of «» pbs to create stool slurries the filter bag wasthen closed and placed into a stomacher seward stomacher where the contents were homogenized on the medium speed setting for s the liquid fraction was removed from the downstreamside of the filter membrane and the solid fraction was discarded a 1ml aliquot of this liquid fractionwas removed for analysis of the scfa concentration to determine the scfa concentration of thestarting stool sample during our validation experiments two separate 1ml aliquots of this liquidfraction were removed one was used to estimate relative bacteria abundance of starting fecalslurries using total extracted dna concentration as has been previously published and theremaining aliquot was used to determine the ph of the starting fecal slurry using a handheld phmeter elite ph spear thermo fisher scientific the remaining liquid fraction was incubated induplicate across six different treatments either supplemented with inulin now foods inulin powderpart fructooligosaccharides fos cargill part galactooligosaccharides gosbimuno powder xylooligosaccharides xos bionutrition prebiotic with llifeoligo part wheatdextrin benefiber original or unsupplemented for each reaction ml of fecal slurry wasplaced in one well of a 24well cell culture plate each well was then delivered ml of wtvolprebiotic solution in «» pbs or ml of «» pbs without prebiotic during our validation experimentsprebiotics were dissolved in colonic medium instead of «» pbs the resulting fermentationconditions were therefore fecal slurry with prebiotic wtvol a fecal slurry was selectedbecause its fermentative capacity has been previously demonstrated to be insensitive to smallvariations in concentration and is feasible to work with using this method a finalconcentration of prebiotic in the context of a fecal slurry i Answer:

Colon_Cancer

continuously increasing with development of the economy and the environment [“] the prognosis for hcc patients remains extremely poor although significant progress has been achieved strategies for early diagnosis are urgently needed because the majority of patients with hcc are diagnosed in very late stages however the molecular mechanism of hcc has not been clearly defined circular rnas circrnas are a new class of rna molecules that have functions as regulators of parental gene transcription in alternative splicing and as mirna sponges through use of rna deep sequencing gtechnology numerous circrnas have been identified as the predominant regulatory elements in diseases moreover accumulating evidence shows that circrnas play pivotal roles in many diseases in particular abnormally expressed circrnas are involved in tumor progression including cell proliferation migration and invasion [“] in addition some research indicates that circrnas level are closely correlated wit specific phenotypes and tumorigenesis in hcc [“] nevertheless the research concerning circrnas is frankly in its infancy which greatly hinders the application of circrnas as biomarkers for diagnosis of hcc in clinicsrelated research shows that circrnas possess great potential to be used for diagnosis of hcc recent studies have found that hsa_circ_0067934 plays oncogenic roles by accelerating cell proliferation and metastasis in glioblastoma gbm circsmarca5 was significantly elevated and thereby suppressed cell apoptosis and arrested cell cycle in prostate cancer in addition previous studies have shown that downregulation of hsa_circ_0005986 facilitated cell proliferation by promoting the g0g1 to s phase transition in hcc similarly alteration in expression of circrnas correlated with development and metastasis of malignant tumors these data suggest that circrnas may be of greater benefit in clinical diagnosis of hcc however reliable circrna biomarkers for hcc are still lacking therefore this review synthetically integrates available data on the role of circrna in hcc progression and attempts to provide crucial clues for investigating the molecular mechanism regarding hccoverview of circrnacircrnas are a category of singlestranded closedcircle molecules which take part in multifaceted biological regulation recently research has verified that the majority of circrnas are synthesized by backspliced exons and that others are formed from intron intergenic and untranslated regions utr therefore biogenesis of circrnas can be divided into eicirnas exonintron circrnas ecircrnas circular exonic rnas and cirnas circular intronic rnas meanwhile over circrnas have been identified and this type of transcript has been considered a new form of gene expression generally the structure of the transcription is inverted and the order of genomic exons is altered and these exons are spliced over time the biological functions of circrnas gradually have been recognized including roles in embryonic development maintainenance of homeostasis and promotion of tumor progression figure properties of circrnascircrnas recently have attracted great attention related to their pathological role in disease development compared with linear rnas circrnas have special properties including biological roles and clinical use circrnas are mainly enriched in certain body fluids comprising blood saliva and urine they are covalently closed loop structures degradation of most rna is highly dependent on rna exonuclease or rnase hence circrnas remain highly stable based on their high resistance to enzyme degradation moreover studies have shown that expression of circrnas is tissuespecific and correlated with different phases of development and they exhibit different expression patterns at different developmental stages roles of circrnasaccumulating evidence shows that circrnas play a crucial role in the pathogenesis of diseases as a result of their complex biological functions generally the molecular functions of circrnas mainly include being sponges of mirna acting as rnabinding proteins performing alternative splicing of premrnas regulating transcription and translation and potentially encoding proteins these properties are described in detail belowsponges of mirnathe different types of circrnas have different mirna binding sites some circrnas negatively regulate mirnas by absorbing and specifically binding to mirnas then decreasing mirna activity and elevating expression of mirnarelated target genes researchers have shown that cirs7 inhibits mir7 function and positively mediates mir7 target genes acting as a molecular sponge in addition functional analyses have indicated that circrnas constitute an entire molecular regulatory network which specifically regulates degradation of mirnas as mirna sponges this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238322indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832premrna™e1e2abcbasepairinge3dguriche4™ecrichpretrnarna bindingproteinsrbp™™™™™™™™gnicilpskcablariat splicingaecircrnaelcirnacirnatrnatricrnafigure1 biogenesis of circular rnas a lariatdriven circularization the ™ hydroxyl of the upstream exon reacts with the ™ phosphate of the downstream exon to form a covalent linkage then producing a lariat including exons and introns the ™ hydroxyl of the ™ intron interacts with the ™ phosphate of the ™intron to form an ecircrna following an interaction between the ™ hydroxyl of the ™ exon and the ™ phosphate of the ™ exon b rnabinding protein rbpdriven circularization rbps accelerate interaction of the downstream intron and upstream intron thereby promoting formation of ecircrna c basepairingdriven circularization the downstream introns and upstream introns are paired depends on inverserepeatingcomplementary sequences formation of ecircrnaeicirna was derived from the introns are removedretained d biosynthesis of cirna formation of cirnas mainly based on a 7nt gurich element and an 11nt crich element to escape debranching and exonucleolytic degradation e formation of tricrna trna splicing enzymes divide pretrna into two parts tricrnas are generated by a ™“™ phosphodiester bond and the other part generates trnascircrnasbinding proteinsrna binding proteins rbps are a broad class of proteins involved in gene transcription translation and interaction studies suggest that distribution of rbps is widespread in many tissue types furthermore rbps participate in development of disorders by regulating posttranscriptional regulation of rnas rbps assemble ribonucleoprotein complexes to bind rna sequences thereby affecting the function of the target rnas previous research has shown that circrnas serve as protein decoys to harbor binding sites of specific proteins and block protein activity circfoxo3 induces cell cycle arrest resulting in defective cdk2 gene function by binding to p21 and cdk2 moreover circrna ciacgas binds to cgas protein and suppresses enzymatic activity of cgas thereby preventing cgas from recognizing selfdna e9238323indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832circrnas regulate alternative splicing transcription and translationcellular localization of most circrnas is cytoplasmic which is the basis for the biological function of mirna and protein decoys several studies have suggested that circrnas participate in rna splicing assembly and biosynthesis recently research has shown that circrnas may play pivotal roles in regulating alternative splicing transcription and translation in addition the exon of the splicing factor may form a circrna by affecting formation of linear rna eicirnas interact with the u1 small nuclear ribonucleoproteinsnrnp thereby regulating parental gene transcription by binding to rna polymerase ii interestingly translation of circrnas is mediated by ires and n6methyladenosine m6a and translation efficiency of circrna is regulated by the level of m6a modification moreover circfbxw7 effectively inhibits glioma proliferation and cell cycle progression by antagonizing usp28induced cmyc stabilization potential to encode proteinscircrnas are implicated in numerous physiological processes and pathogenesis of diseases strong evidence indicates that circrnas can encode proteins by mimicking dna rolling circle amplification related studies indicate that circrna circppp1r12a plays a key molecular role by encoding a functional protein circppp1r12a73aa which promotes proliferation migration and invasion of colon cancer circanril interacts with pescadillo zebrafish homolog pes1 to mediate ribosome biogenesis and prerrna processing in vascular macrophages and smooth muscle cells these studies have significantly increased the knowledge base about the biological functions of circrnascircrnas in diseasescircrnas are involved in processes that lead to development of various disorders such as neuronal and cardiovascular diseases and cancers circrnas participate in regulating gene transcription and protein expression and are indirectly and directly associated with time and regionspecific variations as mentioned previously abnormal expression of circrnas is implicated in neurological disorders atherosclerosis and ribosomal rna maturation reportedly are regulated by circanril simultaneously some studies have suggested that circrnas upregulation significantly affects sprouting and proliferation of vascular endothelial cells and elicits vascular dysfunction recently several experiments have implicated circrnas in pathogenesis of cancer via activation of a series of cascade reactions however the underlying mechanism for the effect of circrnas in initiation and progression of tumors has not been fully clarified to date related studies have revealed that certain circrnas are highly expressed in tumor tissues and overexpression of circrnas promotes tumor proliferation and deterioration an investigation revealed that hsa_circ_002059 was downregulated in gastric cancer while hsa_circ_0004018 was upregulated in hcc meanwhile tumorspecific circrnas candidates were screened in lung adenocarcinoma tissue by microarrays and circrnas were identified downregulated and upregulated of the circrnas hsa_circ_0013958 clearly was positive correlated with lymph node metastasis and tnm stage these findings indicate that circrnas have important roles in tumor progression and may have potential for broad applicatoins in medicine scienceoverview of hcchcc is one of the most prevalent tumors worldwide with diagnoses and approximately deaths annually epidemiological survey data indicate that morbidity and mortality from hcc are gradually increasing risk factors for hcc include diabetes mellitus obesity smoking alcohol consumption older age male sex chronic hbv liver cirrhosis and chronic hepatitis c virus hcv the primary risk factors include liver cirrhosis viral hepatitis alcohol intake and obesity worldwide approximately hcc patients are infected with hepatitis b virus hbv or hcv in addition alcohol abuse is a crucial factor for onset of hcc [“] obesity hypertension and diabetes are closely linked with development of hcc but specific correlations remain unknown moreover regular screening has been widely applied for early detection and to ensure effective treatment of hcc most commonly good results have been achieved with regular screening with ultrasonography blood alphafetoprotein content testing mri and ct generally surgical resection and chemotherapy are mainstays of therapy in patients with hcc yet some tumors cannot be fully removed which results in tumor growth invasion and metastasis local and systemic metastases are the main reasons for the unsatisfactory prognosis in patients with hcc therefore more effective therapeutic approaches need to be developedroles of circrnas in hccnumerous studies have documented the important role that circrnas play in tumorigenesis metastasis and invasion research has shown that circrnas are localized in the nucleus and interfere with transcription and promote alternative this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238324indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832hsa_circ_0001649circzkscan1circitchwntbetacatenincircmto1mir9p21hsa_circ_00059836mir1295phmgb1 ragenfκbmir7hsa_circ_101368hsa_circ_001569cdr1ashsa_circ_0000673figure the function of circrnas in hcc carcinogenesis this graph demonstrates the role of circrnas in hcc carcinogenesis including positive and negative effects respectivelytable brief summary of circrnas as biomarkers for hccnamediseaseconclusiondoicirs7hsa_circ_0003570hsa_circ_0005075hepatocellular carcinomahepatocellular carcinomahepatocellular carcinomacirs7 was one of the independent factors and may be a promising biomarker for hepatic mvi and a novel therapy target for restraining mvi101007s0043201622567hsa_circ_0003570 expression levels were associated with hcc clinicopathological characteristics101002jcla22239hsa_circ_0005075 promotes proliferation migration and invasiveness of hcc via mir431 regulation101016jbiopha201801150splicing circpvt1 is overexpressed in gastric cancer tissues compared with nontumor tissues and circpvt1 acts as an oncogene to mediate expression of mir4975p however studies concerning the role of circrnas in development and progression of hcc remain in their infancytumor inhibitioncurrently circrnas are considered promising diagnostic biomarkers and ideal therapeutic targets for hcc studies have revealed that circitch inhibits tumor proliferation by suppressing the wntbetacatenin pathway expression of circitch has been positively correlated with good survival outcome in patients with hcc analysis of the circrnas expression profile in human hcc tissues showed that circmto1 was markedly decreased in hcc tissues and that expression of circmto1 was positively correlated with survival rate circmto1 reportedly inhibits hcc progress by sponging mir9 and thereby increasing p21 expression meanwhile overexpression of hsa_circ_0001649 negatively affects invasion and proliferation and promotes apoptosis of hcc cells downregulation of zkscan1 and circzkscan1 enhances cell proliferation and promotes progression of hcc tumor promotionin patients with hcc cdr1was more abundant in tumor specimens than in adjacent normal tissues cdr1as effectively suppresses the invasion and proliferation of hcc cells by targeting mir7 some reports have shown that hsa_circ_0000673 is significantly upregulated in hcc tissues and hsa_circ_0000673 downregulation markedly inhibits proliferation and invasion of hcc cells in vitro meanwhile a positive correlation was found between circ_001569 expression level and tumor size advanced tnm stages and unfavorable prognosis in patients with hcc circrna101368 was abundantly expressed in hcc tissue which correlated with poorer prognosis in addition circrna101368 inhibited cell migration by reducing protein levels in nfkb rage and hmgb1 figure e9238325indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832biomarkerconclusionsprevious studies have shown that circrnas are closely related to development of tumors clinicopathological features in patients with hcc are correlated to with levels of expression of cirs7 and its targeted mrnas global circrna expression profile analysis showed that hsa_circ_0005075 exhibited significant differences in tumor tissue versus adjacent tissues in patients with hcc expression of hsa_circ_0005075 also was related to tumor proliferation and metastasis therefore an increasing number of circrnas have been identified as diagnostic markers as summarized in table given the high incidence and mortality fo hcc worldwide it is one of the most serious diseases threatening human health increasing attention is being paid due to this serious situation evidence is increasing to support the close association between circrnas progression of hcc circrnas may play an important role in the occurrence and development of tumors however the molecular mechanism underlying the relationship between circrnas and hcc has not been fully elucidated therefore indepth research is needed on the potential regulatory relationships and to uncover regulatory patterns between circrnas and hcc so that new diagnostic markers for hcc can be developedreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries cancer j clin “ feng rm zong yn cao sm xu rh current cancer situation in china good or bad news from the global cancer statistics cancer commun lond jemal a bray f center mm global cancer statistics cancer j clin “ li r jiang j shi h circrna a rising star in gastric cancer cell mol life sci “ salzman j gawad c wang pl circular rnas are the predominant transcript isoform from hundreds of human genes in diverse cell types plos one e30733 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hsa_circrna_102958 may serve as a diagnostic marker for gastric cancer cancer biomark “ li p chen s chen h using circular rna as a novel type of biomarker in the screening of gastric cancer clin chim acta “ xin j zhang xy sun dk upregulated circular rna hsa_circ_0067934 contributes to glioblastoma progression through activating pi3kakt pathway eur rev med pharmacol sci “ kong z wan x zhang y androgenresponsive circular rna circsmarca5 is upregulated and promotes cell proliferation in prostate cancer biochem biophys res commun “ fu l chen q yao t hsa_circ_0005986 inhibits carcinogenesis by acting as a mir1295p sponge and is used as a novel biomarker for hepatocellular carcinoma oncotarget “ zhu x wang x wei s hsa_circ_0013958 a circular rna and potential novel biomarker for lung adenocarcinoma febs j “ zhang q wang w zhou q roles of circrnas in the tumour microenvironment mol cancer qu z jiang c wu j ding y exosomes as potent regulators of hcc malignancy and potential biotools in clinical application int j clin exp med “ memczak s jens m elefsinioti a circular rnas are a large class of animal rnas with regulatory potency nature “ cocquerelle c mascrez b hetuin d bailleul b missplicing yields circular rna molecules faseb j “ zhao x cai y xu j circular rnas biogenesis mechanism and function in human cancers int j mol sci qu s yang x li x circular rna a new star of noncoding rnas cancer lett “ bahn jh zhang q li f the landscape of microrna piwiinteracting rna and circular rna in human saliva clin chem “ hsu mt cocaprados m electron microscopic evidence for the circular form of rna in the cytoplasm of eukaryotic cells nature “ yu x odenthal m fries jw exosomes as mirna carriers formationfunctionfuture int j mol sci hanan m soreq h kadener s circrnas in the brain rna biol “ constantin l circular rnas and neuronal development adv exp med biol “ van rossum d verheijen bm pasterkamp rj circular rnas novel regulators of neuronal development front mol neurosci ebert ms sharp pa microrna sponges progress and possibilities rna “ ebert ms neilson jr sharp pa microrna sponges competitive inhibitors of small rnas in mammalian cells nat methods “ hansen tb jensen ti clausen bh natural rna circles function as efficient microrna sponges nature “ hsiao ky lin yc gupta sk noncoding effects of circular rna ccdc66 promote colon cancer growth and metastasis cancer res “ janga sc mittal n construction structure and dynamics of posttranscriptional regulatory network directed by rnabinding proteins adv exp med biol “ du ww yang w liu e foxo3 circular rna retards cell cycle progression via forming ternary complexes with p21 and cdk2 nucleic acids res “ xia p wang s ye b a circular rna protects dormant hematopoietic stem cells from dna sensor cgasmediated exhaustion immunity “701e7 li z huang c bao c exonintron circular rnas regulate transcription in the nucleus nat struct mol biol “this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238326indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832 yang y fan x mao m extensive translation of circular rnas driven by n6methyladenosine cell res “ yang y gao x zhang m novel role of fbxw7 circular rna in repressing glioma tumorigenesis j natl cancer inst “ abe n hiroshima m maruyama h rolling circle amplification in a prokaryotic translation system using small circular rna angew chem int ed engl “ zheng x chen l zhou y a novel protein encoded by a circular rna circppp1r12a promotes tumor pathogenesis and metastasis of colon cancer via hippo“yap signaling mol cancer holdt lm stahringer a sass k circular noncoding rna anril modulates ribosomal rna maturation and atherosclerosis in humans nat commun gokul s rajanikant gk circular rnas in brain physiology and disease adv exp med biol “ idda ml munk r abdelmohsen k gorospe m noncoding rnas in alzheimer™s disease wiley interdiscip rev rna luo q chen y long noncoding rnas and alzheimer™s disease clin interv aging “ li cy ma l yu b circular rna hsa_circ_0003575 regulates oxldl induced vascular endothelial cells proliferation and angiogenesis biomed pharmacother “ chen j cui l yuan j circular rna wdr77 target fgf2 to regulate vascular smooth muscle cells proliferation and migration by sponging mir biochem biophys res commun “ “ kristensen ls hansen tb veno mt kjems j circular rnas in cancer opportunities and challenges in the field oncogene “ fu l yao t chen q screening differential circular rna expression profiles reveals hsa_circ_0004018 is associated with hepatocellular carcinoma oncotarget “ massarweh nn elserag hb epidemiology of hepatocellular carcinoma and intrahepatic cholangiocarcinoma cancer control salem r gilbertsen m butt z increased quality of life among hepatocellular carcinoma patients treated with radioembolization compared with chemoembolization clin gastroenterol hepatol “65e1 ozer ed suna n boyacioglu as management of hepatocellular carcinoma prevention surveillance diagnosis and staging exp clin transplant 15suppl “ lou w liu j ding b identification of potential mirnamrna regulatory network contributing to pathogenesis of hbvrelated hcc j transl med yang t hu ly li zl liver resection for hepatocellular carcinoma in nonalcoholic fatty liver disease a multicenter propensity matching analysis with hbvhcc j gastrointest surg “ nishibatake km minami t tateishi r impact of directacting antivirals on early recurrence of hcvrelated hcc comparison with interferonbased therapy j hepatol “ toyoda h kumada t tada t the impact of hcv eradication by directacting antivirals on the transition of precancerous hepatic nodules to hcc a prospective observational study liver int “ zhao j o™neil m vittal a prmt1dependent macrophage il6 production is required for alcoholinduced hcc progression gene expr “ vandenbulcke h moreno c colle i alcohol intake increases the risk of hcc in hepatitis c virusrelated compensated cirrhosis a prospective study j hepatol “ fabris c toniutto p falleti e mthfr c677t polymorphism and risk of hcc in patients with liver cirrhosis role of male gender and alcohol consumption alcohol clin exp res “ vernon g baranova a younossi zm systematic review the epidemiology and natural history of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in adults aliment pharmacol ther “ bruix j reig m sherman m evidencebased diagnosis staging and treatment of patients with hepatocellular carcinoma gastroenterology “ zhang bh yang bh tang zy randomized controlled trial of screening for hepatocellular carcinoma j cancer res clin oncol “ verduci l ferraiuolo m sacconi a the oncogenic role of circpvt1 in head and neck squamous cell carcinoma is mediated through the mutant p53yaptead transcriptioncompetent complex genome biol yu j xu qg wang zg circular rna csmarca5 inhibits growth and metastasis in hepatocellular carcinoma j hepatol “ wang m yu f li p circular rnas characteristics function and clinical significance in hepatocellular carcinoma cancers basel guo w zhang j zhang d et al polymorphisms and expression pattern of circular rna circitch contributes to the carcinogenesis of hepatocellular carcinoma oncotarget “ han d li j wang h circular rna circmto1 acts as the sponge of microrna9 to suppress hepatocellular carcinoma progression hepatology “ qin m liu g huo x hsa_circ_0001649 a circular rna and potential novel biomarker for hepatocellular carcinoma cancer biomark “ yao z luo j hu k zkscan1 gene and its related circular rna circzkscan1 both inhibit hepatocellular carcinoma cell growth migration and invasion but through different signaling pathways mol oncol “ xu l zhang m zheng x the circular rna cirs7 cdr1as acts as a risk factor of hepatic microvascular invasion in hepatocellular carcinoma j cancer res clin oncol “ yu l gong x sun l the circular rna cdr1as act as an oncogene in hepatocellular carcinoma through targeting mir7 expression plos one e0158347 jiang w wen d gong l circular rna hsa_circ_0000673 promotes hepatocellular carcinoma malignance by decreasing mir7673p targeting set biochem biophys res commun “ liu h xue l song c overexpression of circular rna circ_001569 indicates poor prognosis in hepatocellular carcinoma and promotes cell growth and metastasis by sponging mir4115p and mir4325p biochem biophys res commun “ li s gu h huang y circular rna 101368mir200a axis modulates the migration of hepatocellular carcinoma through hmgb1rage signaling cell cycle “ “ shang x li g liu h comprehensive circular rna profiling reveals that hsa_circ_0005075 a new circular rna biomarker is involved in hepatocellular carcinoma development medicine baltimore e3811 yao t chen q shao z circular rna as a new biomarker for hepatocellular carcinoma metastasis j clin lab anal e22572e9238327indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0c'

cancer7538

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: continuously increasing with development of the economy and the environment [“] the prognosis for hcc patients remains extremely poor although significant progress has been achieved strategies for early diagnosis are urgently needed because the majority of patients with hcc are diagnosed in very late stages however the molecular mechanism of hcc has not been clearly defined circular rnas circrnas are a new class of rna molecules that have functions as regulators of parental gene transcription in alternative splicing and as mirna sponges through use of rna deep sequencing gtechnology numerous circrnas have been identified as the predominant regulatory elements in diseases moreover accumulating evidence shows that circrnas play pivotal roles in many diseases in particular abnormally expressed circrnas are involved in tumor progression including cell proliferation migration and invasion [“] in addition some research indicates that circrnas level are closely correlated wit specific phenotypes and tumorigenesis in hcc [“] nevertheless the research concerning circrnas is frankly in its infancy which greatly hinders the application of circrnas as biomarkers for diagnosis of hcc in clinicsrelated research shows that circrnas possess great potential to be used for diagnosis of hcc recent studies have found that hsa_circ_0067934 plays oncogenic roles by accelerating cell proliferation and metastasis in glioblastoma gbm circsmarca5 was significantly elevated and thereby suppressed cell apoptosis and arrested cell cycle in prostate cancer in addition previous studies have shown that downregulation of hsa_circ_0005986 facilitated cell proliferation by promoting the g0g1 to s phase transition in hcc similarly alteration in expression of circrnas correlated with development and metastasis of malignant tumors these data suggest that circrnas may be of greater benefit in clinical diagnosis of hcc however reliable circrna biomarkers for hcc are still lacking therefore this review synthetically integrates available data on the role of circrna in hcc progression and attempts to provide crucial clues for investigating the molecular mechanism regarding hccoverview of circrnacircrnas are a category of singlestranded closedcircle molecules which take part in multifaceted biological regulation recently research has verified that the majority of circrnas are synthesized by backspliced exons and that others are formed from intron intergenic and untranslated regions utr therefore biogenesis of circrnas can be divided into eicirnas exonintron circrnas ecircrnas circular exonic rnas and cirnas circular intronic rnas meanwhile over circrnas have been identified and this type of transcript has been considered a new form of gene expression generally the structure of the transcription is inverted and the order of genomic exons is altered and these exons are spliced over time the biological functions of circrnas gradually have been recognized including roles in embryonic development maintainenance of homeostasis and promotion of tumor progression figure properties of circrnascircrnas recently have attracted great attention related to their pathological role in disease development compared with linear rnas circrnas have special properties including biological roles and clinical use circrnas are mainly enriched in certain body fluids comprising blood saliva and urine they are covalently closed loop structures degradation of most rna is highly dependent on rna exonuclease or rnase hence circrnas remain highly stable based on their high resistance to enzyme degradation moreover studies have shown that expression of circrnas is tissuespecific and correlated with different phases of development and they exhibit different expression patterns at different developmental stages roles of circrnasaccumulating evidence shows that circrnas play a crucial role in the pathogenesis of diseases as a result of their complex biological functions generally the molecular functions of circrnas mainly include being sponges of mirna acting as rnabinding proteins performing alternative splicing of premrnas regulating transcription and translation and potentially encoding proteins these properties are described in detail belowsponges of mirnathe different types of circrnas have different mirna binding sites some circrnas negatively regulate mirnas by absorbing and specifically binding to mirnas then decreasing mirna activity and elevating expression of mirnarelated target genes researchers have shown that cirs7 inhibits mir7 function and positively mediates mir7 target genes acting as a molecular sponge in addition functional analyses have indicated that circrnas constitute an entire molecular regulatory network which specifically regulates degradation of mirnas as mirna sponges this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238322indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832premrna™e1e2abcbasepairinge3dguriche4™ecrichpretrnarna bindingproteinsrbp™™™™™™™™gnicilpskcablariat splicingaecircrnaelcirnacirnatrnatricrnafigure1 biogenesis of circular rnas a lariatdriven circularization the ™ hydroxyl of the upstream exon reacts with the ™ phosphate of the downstream exon to form a covalent linkage then producing a lariat including exons and introns the ™ hydroxyl of the ™ intron interacts with the ™ phosphate of the ™intron to form an ecircrna following an interaction between the ™ hydroxyl of the ™ exon and the ™ phosphate of the ™ exon b rnabinding protein rbpdriven circularization rbps accelerate interaction of the downstream intron and upstream intron thereby promoting formation of ecircrna c basepairingdriven circularization the downstream introns and upstream introns are paired depends on inverserepeatingcomplementary sequences formation of ecircrnaeicirna was derived from the introns are removedretained d biosynthesis of cirna formation of cirnas mainly based on a 7nt gurich element and an 11nt crich element to escape debranching and exonucleolytic degradation e formation of tricrna trna splicing enzymes divide pretrna into two parts tricrnas are generated by a ™“™ phosphodiester bond and the other part generates trnascircrnasbinding proteinsrna binding proteins rbps are a broad class of proteins involved in gene transcription translation and interaction studies suggest that distribution of rbps is widespread in many tissue types furthermore rbps participate in development of disorders by regulating posttranscriptional regulation of rnas rbps assemble ribonucleoprotein complexes to bind rna sequences thereby affecting the function of the target rnas previous research has shown that circrnas serve as protein decoys to harbor binding sites of specific proteins and block protein activity circfoxo3 induces cell cycle arrest resulting in defective cdk2 gene function by binding to p21 and cdk2 moreover circrna ciacgas binds to cgas protein and suppresses enzymatic activity of cgas thereby preventing cgas from recognizing selfdna e9238323indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832circrnas regulate alternative splicing transcription and translationcellular localization of most circrnas is cytoplasmic which is the basis for the biological function of mirna and protein decoys several studies have suggested that circrnas participate in rna splicing assembly and biosynthesis recently research has shown that circrnas may play pivotal roles in regulating alternative splicing transcription and translation in addition the exon of the splicing factor may form a circrna by affecting formation of linear rna eicirnas interact with the u1 small nuclear ribonucleoproteinsnrnp thereby regulating parental gene transcription by binding to rna polymerase ii interestingly translation of circrnas is mediated by ires and n6methyladenosine m6a and translation efficiency of circrna is regulated by the level of m6a modification moreover circfbxw7 effectively inhibits glioma proliferation and cell cycle progression by antagonizing usp28induced cmyc stabilization potential to encode proteinscircrnas are implicated in numerous physiological processes and pathogenesis of diseases strong evidence indicates that circrnas can encode proteins by mimicking dna rolling circle amplification related studies indicate that circrna circppp1r12a plays a key molecular role by encoding a functional protein circppp1r12a73aa which promotes proliferation migration and invasion of colon cancer circanril interacts with pescadillo zebrafish homolog pes1 to mediate ribosome biogenesis and prerrna processing in vascular macrophages and smooth muscle cells these studies have significantly increased the knowledge base about the biological functions of circrnascircrnas in diseasescircrnas are involved in processes that lead to development of various disorders such as neuronal and cardiovascular diseases and cancers circrnas participate in regulating gene transcription and protein expression and are indirectly and directly associated with time and regionspecific variations as mentioned previously abnormal expression of circrnas is implicated in neurological disorders atherosclerosis and ribosomal rna maturation reportedly are regulated by circanril simultaneously some studies have suggested that circrnas upregulation significantly affects sprouting and proliferation of vascular endothelial cells and elicits vascular dysfunction recently several experiments have implicated circrnas in pathogenesis of cancer via activation of a series of cascade reactions however the underlying mechanism for the effect of circrnas in initiation and progression of tumors has not been fully clarified to date related studies have revealed that certain circrnas are highly expressed in tumor tissues and overexpression of circrnas promotes tumor proliferation and deterioration an investigation revealed that hsa_circ_002059 was downregulated in gastric cancer while hsa_circ_0004018 was upregulated in hcc meanwhile tumorspecific circrnas candidates were screened in lung adenocarcinoma tissue by microarrays and circrnas were identified downregulated and upregulated of the circrnas hsa_circ_0013958 clearly was positive correlated with lymph node metastasis and tnm stage these findings indicate that circrnas have important roles in tumor progression and may have potential for broad applicatoins in medicine scienceoverview of hcchcc is one of the most prevalent tumors worldwide with diagnoses and approximately deaths annually epidemiological survey data indicate that morbidity and mortality from hcc are gradually increasing risk factors for hcc include diabetes mellitus obesity smoking alcohol consumption older age male sex chronic hbv liver cirrhosis and chronic hepatitis c virus hcv the primary risk factors include liver cirrhosis viral hepatitis alcohol intake and obesity worldwide approximately hcc patients are infected with hepatitis b virus hbv or hcv in addition alcohol abuse is a crucial factor for onset of hcc [“] obesity hypertension and diabetes are closely linked with development of hcc but specific correlations remain unknown moreover regular screening has been widely applied for early detection and to ensure effective treatment of hcc most commonly good results have been achieved with regular screening with ultrasonography blood alphafetoprotein content testing mri and ct generally surgical resection and chemotherapy are mainstays of therapy in patients with hcc yet some tumors cannot be fully removed which results in tumor growth invasion and metastasis local and systemic metastases are the main reasons for the unsatisfactory prognosis in patients with hcc therefore more effective therapeutic approaches need to be developedroles of circrnas in hccnumerous studies have documented the important role that circrnas play in tumorigenesis metastasis and invasion research has shown that circrnas are localized in the nucleus and interfere with transcription and promote alternative this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238324indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832hsa_circ_0001649circzkscan1circitchwntbetacatenincircmto1mir9p21hsa_circ_00059836mir1295phmgb1 ragenfκbmir7hsa_circ_101368hsa_circ_001569cdr1ashsa_circ_0000673figure the function of circrnas in hcc carcinogenesis this graph demonstrates the role of circrnas in hcc carcinogenesis including positive and negative effects respectivelytable brief summary of circrnas as biomarkers for hccnamediseaseconclusiondoicirs7hsa_circ_0003570hsa_circ_0005075hepatocellular carcinomahepatocellular carcinomahepatocellular carcinomacirs7 was one of the independent factors and may be a promising biomarker for hepatic mvi and a novel therapy target for restraining mvi101007s0043201622567hsa_circ_0003570 expression levels were associated with hcc clinicopathological characteristics101002jcla22239hsa_circ_0005075 promotes proliferation migration and invasiveness of hcc via mir431 regulation101016jbiopha201801150splicing circpvt1 is overexpressed in gastric cancer tissues compared with nontumor tissues and circpvt1 acts as an oncogene to mediate expression of mir4975p however studies concerning the role of circrnas in development and progression of hcc remain in their infancytumor inhibitioncurrently circrnas are considered promising diagnostic biomarkers and ideal therapeutic targets for hcc studies have revealed that circitch inhibits tumor proliferation by suppressing the wntbetacatenin pathway expression of circitch has been positively correlated with good survival outcome in patients with hcc analysis of the circrnas expression profile in human hcc tissues showed that circmto1 was markedly decreased in hcc tissues and that expression of circmto1 was positively correlated with survival rate circmto1 reportedly inhibits hcc progress by sponging mir9 and thereby increasing p21 expression meanwhile overexpression of hsa_circ_0001649 negatively affects invasion and proliferation and promotes apoptosis of hcc cells downregulation of zkscan1 and circzkscan1 enhances cell proliferation and promotes progression of hcc tumor promotionin patients with hcc cdr1was more abundant in tumor specimens than in adjacent normal tissues cdr1as effectively suppresses the invasion and proliferation of hcc cells by targeting mir7 some reports have shown that hsa_circ_0000673 is significantly upregulated in hcc tissues and hsa_circ_0000673 downregulation markedly inhibits proliferation and invasion of hcc cells in vitro meanwhile a positive correlation was found between circ_001569 expression level and tumor size advanced tnm stages and unfavorable prognosis in patients with hcc circrna101368 was abundantly expressed in hcc tissue which correlated with poorer prognosis in addition circrna101368 inhibited cell migration by reducing protein levels in nfkb rage and hmgb1 figure e9238325indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832biomarkerconclusionsprevious studies have shown that circrnas are closely related to development of tumors clinicopathological features in patients with hcc are correlated to with levels of expression of cirs7 and its targeted mrnas global circrna expression profile analysis showed that hsa_circ_0005075 exhibited significant differences in tumor tissue versus adjacent tissues in patients with hcc expression of hsa_circ_0005075 also was related to tumor proliferation and metastasis therefore an increasing number of circrnas have been identified as diagnostic markers as summarized in table given the high incidence and mortality fo hcc worldwide it is one of the most serious diseases threatening human health increasing attention is being paid due to this serious situation evidence is increasing to support the close association between circrnas progression of hcc circrnas may play an important role in the occurrence and development of tumors however the molecular mechanism underlying the relationship between circrnas and hcc has not been fully elucidated therefore indepth research is needed on the potential regulatory relationships and to uncover regulatory patterns between circrnas and hcc so that new diagnostic markers for hcc can be developedreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries cancer j clin “ feng rm zong yn cao sm xu rh current cancer situation in china good or bad news from the global cancer statistics cancer commun lond jemal a bray f center mm global cancer statistics cancer j clin “ li r jiang j shi h circrna a rising star in gastric cancer cell mol life sci “ salzman j gawad c wang pl circular rnas are the predominant transcript isoform from hundreds of human genes in diverse cell types plos one e30733 lukiw wj circular rna circrna in alzheimer™s disease ad front genet liu y yang y wang z insights into the regulatory role of circrna in angiogenesis and clinical implications atherosclerosis “ zhao y alexandrov pn jaber v lukiw wj deficiency in the ubiquitin conjugating enzyme ube2a in alzheimer™s disease ad is linked to deficits in a natural circular mirna7 sponge circrna cirs7 genes basel shen f liu p xu z circrna_001569 promotes cell proliferation through absorbing mir“ in gastric cancer j biochem “ song t xu a zhang z circrna hsa_circrna_101996 increases cervical cancer proliferation and invasion through activating tpx2 expression by restraining mir8075 j cell physiol “ min l wang h zeng y circrna_104916 regulates migration apoptosis and epithelial“mesenchymal transition in colon cancer cells front biosci landmark ed “ verduci l strano s yarden y blandino g the circrnamicrorna code emerging implications for cancer diagnosis and treatment mol oncol “ wei j wei w xu h circular rna hsa_circrna_102958 may serve as a diagnostic marker for gastric cancer cancer biomark “ li p chen s chen h using circular rna as a novel type of biomarker in the screening of gastric cancer clin chim acta “ xin j zhang xy sun dk upregulated circular rna hsa_circ_0067934 contributes to glioblastoma progression through activating pi3kakt pathway eur rev med pharmacol sci “ kong z wan x zhang y androgenresponsive circular rna circsmarca5 is upregulated and promotes cell proliferation in prostate cancer biochem biophys res commun “ fu l chen q yao t hsa_circ_0005986 inhibits carcinogenesis by acting as a mir1295p sponge and is used as a novel biomarker for hepatocellular carcinoma oncotarget “ zhu x wang x wei s hsa_circ_0013958 a circular rna and potential novel biomarker for lung adenocarcinoma febs j “ zhang q wang w zhou q roles of circrnas in the tumour microenvironment mol cancer qu z jiang c wu j ding y exosomes as potent regulators of hcc malignancy and potential biotools in clinical application int j clin exp med “ memczak s jens m elefsinioti a circular rnas are a large class of animal rnas with regulatory potency nature “ cocquerelle c mascrez b hetuin d bailleul b missplicing yields circular rna molecules faseb j “ zhao x cai y xu j circular rnas biogenesis mechanism and function in human cancers int j mol sci qu s yang x li x circular rna a new star of noncoding rnas cancer lett “ bahn jh zhang q li f the landscape of microrna piwiinteracting rna and circular rna in human saliva clin chem “ hsu mt cocaprados m electron microscopic evidence for the circular form of rna in the cytoplasm of eukaryotic cells nature “ yu x odenthal m fries jw exosomes as mirna carriers formationfunctionfuture int j mol sci hanan m soreq h kadener s circrnas in the brain rna biol “ constantin l circular rnas and neuronal development adv exp med biol “ van rossum d verheijen bm pasterkamp rj circular rnas novel regulators of neuronal development front mol neurosci ebert ms sharp pa microrna sponges progress and possibilities rna “ ebert ms neilson jr sharp pa microrna sponges competitive inhibitors of small rnas in mammalian cells nat methods “ hansen tb jensen ti clausen bh natural rna circles function as efficient microrna sponges nature “ hsiao ky lin yc gupta sk noncoding effects of circular rna ccdc66 promote colon cancer growth and metastasis cancer res “ janga sc mittal n construction structure and dynamics of posttranscriptional regulatory network directed by rnabinding proteins adv exp med biol “ du ww yang w liu e foxo3 circular rna retards cell cycle progression via forming ternary complexes with p21 and cdk2 nucleic acids res “ xia p wang s ye b a circular rna protects dormant hematopoietic stem cells from dna sensor cgasmediated exhaustion immunity “701e7 li z huang c bao c exonintron circular rnas regulate transcription in the nucleus nat struct mol biol “this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238326indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832 yang y fan x mao m extensive translation of circular rnas driven by n6methyladenosine cell res “ yang y gao x zhang m novel role of fbxw7 circular rna in repressing glioma tumorigenesis j natl cancer inst “ abe n hiroshima m maruyama h rolling circle amplification in a prokaryotic translation system using small circular rna angew chem int ed engl “ zheng x chen l zhou y a novel protein encoded by a circular rna circppp1r12a promotes tumor pathogenesis and metastasis of colon cancer via hippo“yap signaling mol cancer holdt lm stahringer a sass k circular noncoding rna anril modulates ribosomal rna maturation and atherosclerosis in humans nat commun gokul s rajanikant gk circular rnas in brain physiology and disease adv exp med biol “ idda ml munk r abdelmohsen k gorospe m noncoding rnas in alzheimer™s disease wiley interdiscip rev rna luo q chen y long noncoding rnas and alzheimer™s disease clin interv aging “ li cy ma l yu b circular rna hsa_circ_0003575 regulates oxldl induced vascular endothelial cells proliferation and angiogenesis biomed pharmacother “ chen j cui l yuan j circular rna wdr77 target fgf2 to regulate vascular smooth muscle cells proliferation and migration by sponging mir biochem biophys res commun “ “ kristensen ls hansen tb veno mt kjems j circular rnas in cancer opportunities and challenges in the field oncogene “ fu l yao t chen q screening differential circular rna expression profiles reveals hsa_circ_0004018 is associated with hepatocellular carcinoma oncotarget “ massarweh nn elserag hb epidemiology of hepatocellular carcinoma and intrahepatic cholangiocarcinoma cancer control salem r gilbertsen m butt z increased quality of life among hepatocellular carcinoma patients treated with radioembolization compared with chemoembolization clin gastroenterol hepatol “65e1 ozer ed suna n boyacioglu as management of hepatocellular carcinoma prevention surveillance diagnosis and staging exp clin transplant 15suppl “ lou w liu j ding b identification of potential mirnamrna regulatory network contributing to pathogenesis of hbvrelated hcc j transl med yang t hu ly li zl liver resection for hepatocellular carcinoma in nonalcoholic fatty liver disease a multicenter propensity matching analysis with hbvhcc j gastrointest surg “ nishibatake km minami t tateishi r impact of directacting antivirals on early recurrence of hcvrelated hcc comparison with interferonbased therapy j hepatol “ toyoda h kumada t tada t the impact of hcv eradication by directacting antivirals on the transition of precancerous hepatic nodules to hcc a prospective observational study liver int “ zhao j o™neil m vittal a prmt1dependent macrophage il6 production is required for alcoholinduced hcc progression gene expr “ vandenbulcke h moreno c colle i alcohol intake increases the risk of hcc in hepatitis c virusrelated compensated cirrhosis a prospective study j hepatol “ fabris c toniutto p falleti e mthfr c677t polymorphism and risk of hcc in patients with liver cirrhosis role of male gender and alcohol consumption alcohol clin exp res “ vernon g baranova a younossi zm systematic review the epidemiology and natural history of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in adults aliment pharmacol ther “ bruix j reig m sherman m evidencebased diagnosis staging and treatment of patients with hepatocellular carcinoma gastroenterology “ zhang bh yang bh tang zy randomized controlled trial of screening for hepatocellular carcinoma j cancer res clin oncol “ verduci l ferraiuolo m sacconi a the oncogenic role of circpvt1 in head and neck squamous cell carcinoma is mediated through the mutant p53yaptead transcriptioncompetent complex genome biol yu j xu qg wang zg circular rna csmarca5 inhibits growth and metastasis in hepatocellular carcinoma j hepatol “ wang m yu f li p circular rnas characteristics function and clinical significance in hepatocellular carcinoma cancers basel guo w zhang j zhang d et al polymorphisms and expression pattern of circular rna circitch contributes to the carcinogenesis of hepatocellular carcinoma oncotarget “ han d li j wang h circular rna circmto1 acts as the sponge of microrna9 to suppress hepatocellular carcinoma progression hepatology “ qin m liu g huo x hsa_circ_0001649 a circular rna and potential novel biomarker for hepatocellular carcinoma cancer biomark “ yao z luo j hu k zkscan1 gene and its related circular rna circzkscan1 both inhibit hepatocellular carcinoma cell growth migration and invasion but through different signaling pathways mol oncol “ xu l zhang m zheng x the circular rna cirs7 cdr1as acts as a risk factor of hepatic microvascular invasion in hepatocellular carcinoma j cancer res clin oncol “ yu l gong x sun l the circular rna cdr1as act as an oncogene in hepatocellular carcinoma through targeting mir7 expression plos one e0158347 jiang w wen d gong l circular rna hsa_circ_0000673 promotes hepatocellular carcinoma malignance by decreasing mir7673p targeting set biochem biophys res commun “ liu h xue l song c overexpression of circular rna circ_001569 indicates poor prognosis in hepatocellular carcinoma and promotes cell growth and metastasis by sponging mir4115p and mir4325p biochem biophys res commun “ li s gu h huang y circular rna 101368mir200a axis modulates the migration of hepatocellular carcinoma through hmgb1rage signaling cell cycle “ “ shang x li g liu h comprehensive circular rna profiling reveals that hsa_circ_0005075 a new circular rna biomarker is involved in hepatocellular carcinoma development medicine baltimore e3811 yao t chen q shao z circular rna as a new biomarker for hepatocellular carcinoma metastasis j clin lab anal e22572e9238327indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0c' Answer:

Colon_Cancer

collagen triple helix repeat containing1 cthrc1 anextracellular matrix ecm protein was identified in thescreening of diï¬erentially expressed sequences between balloon injury and normal arteries the evolution of cthrc1can be traced back to at least million years ago and theconserved genes were not found in invertebrates cthrc1has complicated interactions with various intracellular andextracellular matrices in diï¬erent ways of secretion [ ]cthrc1 increases the activity of collagen promoter throughbinding to ligands and could contribute to vascular remodeling by limiting collagen matrix deposition and promoting cellmigration cthrc1 promotes the recruitment of m2macrophages and regulates tgf and notch pathways toaccelerate wound healing in a mouse model of acute woundhealing as a coupling factor cthrc1 can be secretedby osteoclasts and ‚uence bone formation and remodelingby acting on osteoblasts and osteocytes [ ] cthrc1 maypromote il1induced apoptosis of chondrocytes by activating the jnk12 pathway the anti‚ammatoryeï¬ect of cthrc1 expressed on activated synovial cellswas also found in a collagen antibodyinduced arthritismodel besides cthrc1 can regulate physiologicalfunctions such as fat and glycogen synthesis and promoteautonomous activity [ ]therefore as a secreted protein cthrc1 is involved inmultiple pathophysiologies a remarkable eï¬ect is that thehigh expression of cthrc1 promotes tumorigenesis anddevelopment through positive regulation of tumor spreadinvasion migration adhesion and metastasis cthrc1exerts its eï¬ects through several signaling pathways such as 0cmediators of ‚ammationgpa gvp grd gsp gan gip gtp gip grd gfk gek gechydrophobic regiongxy repeatcysteinesnglycosylationnh2signal peptidecollagendomainc c c cc cc ccoohfigure the structure of the cthrc1 protein the construct of cthrc1 contains an nh2terminal peptide for extracellular secretion ashort collagen triple helix repeat of amino acids and a coohterminal globular domain the prolinerich hydrophobic domain liesbetween the 1st and 30th amino acids and serves as a signal peptide for transport to the endoplasmic reticulum cthrc1 comprises acollagen domain between amino acids and and the protein contains cysteine residues corresponding to about cysteine inthe final protein what is more its only amino acid posttranslational modification is the glycosylation of asparagine at position integrin faktgf wntsrcfak mekerkpi3kakterk hif1α and pkcδerk signaling pathways in this we focus on the advances in the signalingpathways mediated by cthrc1 in tumors the structural characteristics andexpression of cthrc1 the structural features of cthrc1 the cthrc1 geneis located at chromosome 8q223 and it contains five exonsin humans and four exons in mice it covers kb onthe direct strand and can be transcribed into kb mrnathe amino acid sequence identity between human and ratcthrc1 proteins was and no homologs were foundin lower species [ ]protein nglycosylationsecreted cthrc1 exists primarily as a dimer kdaand a trimer kda as well as multimers of the trimericcthrc1 kda and kda the construct of cthrc1contains an nh2terminal peptide for extracellular secretiona short collagen triple helix repeat of amino acids and acoohterminal globular domain [ ] similar molecularweight and structural characteristics to adiponectin alsoexplain why cthrc1 can form high molecular weightcomplexes the biological activity of cthrc1 isrestricted to the highly conserved amino acids at thecterminal region and the cterminal region of cthrc1contains a putative nglycosylation site that stabilizescthrc1promotescthrc1 to tether to the cell membrane which promotesactin polymerization and cell polarity a short collagen motif with glyxy repeats presents in c1qtumornecrosis factorαrelated proteins ctrps which appearsto be responsible for the trimerization of protein and renders molecule susceptible to cleavage by collagenase seefigure however dimeric cthrc1 would not be susceptible to cleavage by collagenase [ ] the molecularweight of secreted cthrc1 kda appears to be largerthan that of cellular cthrc1 kda cthrc1 has fourdiï¬erentabout kda to kda the fulllength of cthrc1 accountsfor both secreted and cellular cthrc1 glycosylatedprotein cthrc1 with a signal sequence is related to ecmisoforms with molecular weights ofalsoand contains a variable short collagenlike motif intriguingly cthrc1 plays a role in inhibiting structural proteins unlike other members of the collagen family moreover leclair found that cthrc1 cleaved atthe nterminus by plasmin shows better inhibition of collagen synthesis compared to fulllength cthrc1 in thepac1 cell line these studies suggested that cthrc1might obtain biologicalthrough proteolyticprocessingactivity the expression of cthrc1 cthrc1 is transientlyexpressed by fibroblasts in remodeling adventitia and bysmooth muscle cells in the neointima of injured tissuehowever cthrc1 is not detected in normal arteries ininjured arteries and skin the expression of cthrc1 isassociated with myofibroblasts and locates in the sites ofcollagen matrix deposition in micethe first exon ofcthrc1 was targeted to be replaced with a galactosidase expression construct which demonstrated the expression of cthrc1 in inner ear hair cells there iscthrc1 expression in many mesenchymalderived cellsduring body growth and tissue repair in mouseembryos cthrc1 is expressed in visceral endodermnotochord neuraltube developing kidney and heartabundant expression of cthrc1 is observed in developincluding cartilage primordia growth plateing skeletoncartilagein adultscthrc1 is expressed only in bone matrix and periosteum cthrc1 is also found in the matrix of calcifyingatherosclerotic plaques and mineralized bone of skeletaltissues in humans in other tissues the sites of cthrc1expression overlap considerably with interstitial collagensand transforming growth factor tgf family membersparticularly bone morphogenetic proteins bmps the sitesof cthrc1 expression are characterized by the presence ofactive tgf and abundant collagen synthesis cthrc1mrna expression levels are increased in response to bmp4bmp2 and tgf furthermore tgf signaling could leadto a significant increase in neointimal lesion formation the expression of cthrc1 is also positively correlatedwith tumor lymph node metastasis tumornodemetastasistnm stage and disease prognosis however its potentialand periosteumbone matrix 0cmediators of ‚ammationregulatory mechanisms in the tumor environment have notyet been elucidated the molecules that regulate theexpression of cthrc1cthrc1 is abnormally expressed in several solid tumorsespecially in gastric cancer pancreatic cancer hepatocellularcarcinoma keloid breast cancer colorectal cancer crcepithelial ovarian cancer esophageal squamous cell carcinoma escc cervical cancer nonsmallcell lung carcinoma nsclc melanoma and so on [ “] andmolecules that regulate the expression of cthrc1 includemirnas lncrnas waif1 and dpagt1 mirnas microribonucleic acids mirnas which canregulate gene expression are a class of noncoding singlestranded small rnas of about nucleotides that can inhibitthe mrna translation process by exclusively promoting thedegradation of several mrnas in many tumors mirnas such as mir30c mir9 mir520d5p mir1555pmir98 let7b mir155 mir101 and mir217 can regulate the expression of cthrc1mir30c could regulate cthrc1 at a posttranscriptionallevelin breast cancer it downregulates the cthrc1mediated gsk3catenin signal and inhibits tumor cellproliferation invasion and migration in addition mir30ccan also upregulate baxcaspase9caspase3 a downstreamsignal of cthrc1 inhibiting apoptosis in hepatocellular carcinoma cthrc1 downregulates mir1555p throughthe activation of gsk3involved wntcatenin signalingto promote tumor formation and mir98 dramaticallydownregulates cthrc1 by directly targeting the ²utr ofcthrc1 suppressing hepatocellular carcinoma formation mir9 could inhibit the migration of schwann cell bytargeting cthrc1 following sciatic nerve injury therebyinactivating downstream rac1 gtpase mir520d5pis significantly downexpressed and suppresses cell proliferation migration and invasion by targeting cthrc1 in crc as the second mirna following lin4 in caenorhabditiselegans let7b may directly target cthrc1 and function asa tumor suppressor gene in gastric cancer [ ] in esccmir101 and mir217 could inhibitthe expression ofcthrc1 mir30 could downregulate the expressionof cthrc1 and downstream signal molecules such as mmp and mmp2 to inhibit the invasion and migration ofnsclc cells a recent study found that mir155 downregulation and cthrc1 upregulation were observed incrc moreover overexpression of mir155 can silencedownstream cthrc1 thereby inhibiting cell proliferationand inducing apoptosis of cells to prevent tumor progressionand metastasis in conclusion the negative regulation ofcthrc1 by mirna has the potential to become a noveldirection for cancer treatment in the futurelncrnas metastasisassociated lung adenocarcinomatranscript i malat1 is a large infrequently spliced longnoncoding rna lncrna which could genetically increasecthrc1 activity to regulate lung cancer cell migration the silence of malat1 could also inhibit the expression ofcthrc1 which is a positive regulator of escc furtheranother lncrna named nonmmug014387 could also regulate cthrc1 and activate the wntpcp pathway to promote schwann cell proliferation at the site of injury waif1 wntactivated inhibitory factor waif1 issilenced by promoter hypermethylation in various cancers[ ] lcmsms analysis using liquid chromatographyand mass spectrometry analysis of samples of cthrc1binding membrane proteins indicates that the largest partof cthrc1 binds the waif1 receptor recent researchsuggests that waifi expression is activated by suppressingmethylation of its promoter activated waif1 downregulates the expression of wntcatenin target genes to inhibitthe development of endometrial adenocarcinoma thebinding of cthrc1 to waif1 could promote osteoblast differentiation therefore cthrc1waif1 interactioncan be a potential therapeutic target in the futurepromoter hypomethylation dpagt1 nglycosylation is essential for the migrationpattern of immune cells and its dysregulation is related tovarious diseases including cancer in human escc the overexpression of cthrc1 is associated with hyperglycosylationandincreased nglycosylation is associated with preferential localization ofcthrc1 in wound cells and nglycosylation facilitatesthe promigratory function of cthrc1 dolichylphosphatenacetylglucosaminephosphotransferase dpagt1 thegene that encodes the first enzyme and ratelimiting enzymein the assembly of lipidlinked oligosaccharide precursors inthe endoplasmic reticulum is related to the formation ofmature intercellular adhesion complexes as anupstream regulator of nglycosylation status of ecadherindpagt1 could upregulate cthrc1 by increasing proteinturnover indicating that nglycosylation can also stabilizecthrc1 besides tgf and fak could also regulate the expression of cthrc1 in diï¬erent signaling pathways it should behighlighted that cthrc1 not only is the result of tumor progression but also plays a predominant regulatory role in theprogression and metastasis of many solid tumors [ “]in summary many molecules can regulate the expressionand activity of cthrc1 and together with cthrc1 as novelantitumor molecular targets for the treatment of cancer inthe future signaling pathways mediated by cthrc1involved in the progression andmetastasis of tumorthe ‚uence of cthrc1 on various events in tumor progression is based on its regulation of various signaling pathways such as tgf wntintegrin fak srcfakmekerk pi3kakterk hif1α and pkcδerk signaling pathways see figure these properties™ pathwaysaï¬ected by cthrc1 play an essential role not only in tissueremodeling after injury regulation of ossification and other 0cmediators of ‚ammationcthrc1tgf𝛽t𝛽r2wnt3alrp56cthrc1wnt5aror12cthrc1cthrc1𝛽𝛼𝛼𝛽integrincxrc4t𝛽r1p ppsmad23smad4cthrc1dvlpdvlaxinapc𝛽cateninck1𝛼gsk3dpagt1stabilization𝛽cateninpkc𝛿daamrac1rhoajnkrockpsrcfakpaxgrb2rasrafmekerkpfakpi3kaktmtorfra1crebmmpmekerkhif𝛼vegfecminvasionpsmad23smad4𝛽catenintcflefcjunap1snailcyclin d1g1mg2smetastasisangiogenesisproliferationfigure signaling pathways mediated by cthrc1 involved in the progression and metastasis of tumor tgf signaling pathway isquite complex especially in terms of its eï¬ects which are often contradictory depending on location and time there exists a criticalnegative feedback regulatory loop between tgfsmad23 signaling pathway and cthrc1 wnt signaling includes wntcatenincanonical pathway and cateninindependent noncanonical pathway in the canonical wnt signaling fzd receptor and lrp5lrp6coreceptor are transduced to catenin signaling cascade for the maintenance of stem and progenitor cells in the noncanonical wntsignaling fzd receptor and ror2ptk7ryk coreceptor are transduced to rhoa jnk signaling cascades for the control of tissuepolarity cell adhesion or cell movement the downstream molecules of the wntpcp pathway mainly include the small gtpase familysuch as rac1 rhoa and jnk which play essential roles in cancer cell migration and invasion cthrc1 signal via waif1 canactivate pkcδ which is an essential component of the wntpcp pathway furthermore pkcδ is responsible for the activation of thecthrc1induced erk signaling pathway in cthrc1integrin signaling pathway the upregulation of cthrc1 is related to theprogression and metastasis of several cancers through the activation of several key signaling molecules including src fak paxillin mekerk and rac1 fak promotes cancer cell migration by regulating focal adhesion formation and turnover which involve activation of srcand paxillin fra1 is activated by cthrc1 through the mapkmekerk signaling which leads to the upregulation of cyclin d1 andthat promotes cell proliferation fra1 also induces snail1mediated mmp14 expression to facilitate escc cell invasion migration andmetastasis pi3kakt signaling pathway induces emt change and mmp2mmp9 expression hif1α and vegf are activated bycthrc1 through activating the pi3kaktmtor signaling pathway which promotes tumor angiogenesis cthrc1 also participates intumor cell migration and invasion through hif1αcxcr4 signalsphysiological processes but also in the development of cancerand metastasis negative feedback regulation of cthrc1 and cell typespecific tgf signaling pathway as the most potentgrowth factor involved in wound healing tgf is releasedby platelets at the site of injury ‚uencing ‚ammatoryresponse angiogenesis reepithelialization ecm and remodeling tgf superfamily members include tgf activin and bmps smad158 mediates bmp signaling whilesmad23 mediates tgf and activin signalingcthrc1 has been reported to have a relationship withthe tgf family since its discovery as their expression sitesoverlap significantly tgf1 and bmp4 can induce thetranscription and expression of cthrc1 in nih3t3 cells cthrc1 can activate tgf signaling via an elevationin smad2smad3 phosphorylation activated smad23 formsa complex with smad4 and accumulates in the nucleus causing an increase in collagen type i deposition during vascularremodeling [“] there exists a critical negative feedbackregulatory loop between tgf1 and cthrc1 the conserved region of amino acids in cthrc1 proteincan bind to phosphosmad3 cthrc1 is induced by tgf1 via phosphosmad3 binding to the promoter with subsequenttranscription activation and in turn cthrc1inhibits tgf1 signaling by accelerating proteasomal degradation of phosphosmad3 which inhibits collagen deposition tgf can enhance the migration and invasioncharacteristics of endothelial cells by regulating the secretionand expression of mmp2 and mmp9 therefore inhibiting cthrc1mediated tgf signaling pathways mayeï¬ectively suppress the invasion and angiogenesis of cancercells [ ]however the mechanism of tgf involved in tumorprogression is very complex even in the same tumor typetgf has many diï¬erent roles in tumor progression forexample the activation of nuclear factor of activated tcellsnfats can drive the switch of the tumorsuppressive function of tgf towards tumor progression [ ] tgfincreases the level of cthrc1 in crc cells highly 0cmediators of ‚ammationexpressed cthrc1 promotes epithelialmesenchymal transition emt and tumor metastasis through the smad2smad3 activation of tgf pathway cthrc1 can alsoinhibit the tgfsmad pathway and yap nuclear translocation thereby inhibiting type i collagen synthesis metabolites such as bile acid may induce cthrc1 to activatethe tgfsmad2smad3 pathway to mediate liver fibrosisand may progress towards hepatocellular carcinoma in the polyvinyl alcohol sponge model cthrc1 activates tgf and notch pathways to promote the recruitment of m2 macrophages however cthrc1 maydownregulate tgf expression during the late remodelingphase of wound healing tgf regulates the expressionof cthrc1 in a concentrationdependent manner inkeloids and excess cthrc1 reverses collagen synthesis therefore these results of the regulation betweencthrc1 and tgf are not contradictory other than thatcthrc1 has no inhibitory eï¬ect on tgf signaling inendothelial cells these results indicate that the regulation of tgf by cthrc1 may play a role in other interstitial cells of the tumor microenvironment and that thisregulation is cell typespecific the further exploration ofdetailed molecular mechanism by which cthrc1 activatesthe tgf pathway may resolve these disputes mutual regulation between cthrc1 and wnt pathwaysto promote tumor progression and metastasis wnt familyare secreted glycoproteins include wnt1 wnt1 wnt3awnt4 wnt5a wnt5b wnt6 wnt7a and wnt7b andparticipate in the process of numerous oncogenic and development progress [“] wnt5a is a member of the wntprotein family and plays an essential role in the pathologicalprocess of neuropathy and malignant tumors [“] wntproteins activate the wntcatenin canonical pathway andcateninindependent noncanonicalamongwhich the planar cell polarity pcp pathway and wntcalcium ca2 pathway are the most widely studied [“]current reports indicate that cthrc1 is mainly involvedin tumor progression through the canonical wntcateninand noncanonical wntpcp pathwayspathway wntcatenin canonical signaling pathway in thewntcatenin canonical pathway wnt proteins bind tofrizzled fzd receptor and lipoprotein receptorrelatedprotein lrp56 coreceptor in the absence of wntsignaling the cytoplasmic catenin form the œdestructioncomplex composed with the casein kinase 1α ck1αglycogen synthase kinase 3 gsk3 adenomatous polyposis coli apc and axin which activates the emt topromotethroughcthrc1wntcatenin [ ] the level of cateninis maintained as low by the series of eventsincludingpriming phosphorylation by ck1α at ser45 and subsequently at thr41 ser37 and ser33 by gsk3 [ ]when secreted wnt ligands are accumulated wnt combines with fzd receptor and lrp56 coreceptors lead toactivation of dishevelled dvl protein the activateddvl is phosphorylated and translocated to the fzd recepthe catenintorcausing the dissociation ofand metastases cancerinvasionœdestruction complex and the cytosolic accumulation ofcatenin as the cytosolic catenin accumulates rasproteins are accumulated due to the absence of gsk3mediated phosphorylation the stabilized ras proteins atthe plasma membrane activate rafmitogenactivatedprotein kinase mekextracellular signalregulated kinaseerk cascade besides cytosolic catenin subsequently translocates into the nucleus and forms a complexwith the tcell factor tcf or lymphoid enhancer factorlef the complex activates the expression oftargetgenes involving proliferation and transformation such ascmyc cjun ccnd1 gene encoding cyclin d1 epidermal growth factor receptor egfr cd44 cd133 andleucinerich repeatcontaining g proteincoupled receptor lgr5 [“]the wntcatenin signaling pathway plays an indispensable role in the occurrence and development of manytypes of cancer cthrc1induced nuclear translocation ofcatenin was observed in nclh23 cells and luciferaseassay showed that catenintcf transcriptional activitywas enhanced in contrast the knockdown of cthrc1reduced the catenintcf transcriptional activity whichshows that cthrc1 regulates the invasiveness of nsclccells through the wntcatenin pathway similarlycthrc1 activates snail1 through the wntcatenin signaling pathway to promote emt in epithelial ovarian cancer during the development and metastasis of crccthrc1 may promote the activation of the wnt signalingpathway through anos1 it can also participate in thewntcatenin pathway to regulate the malignant behaviorof hepatocellular carcinoma with gsk3 many cancers usually metastasize to bone in advanced stages cthrc1secreted by osteoclasts promotes basic fibroblast growth factor bfgf expression in osteoblasts by stimulating wntcatenin signaling which may induce the development of cancerous bone lesions but not mediate vascular production the constitutive activation of the wntcatenin pathway leads to carcinogenesis in tumors cthrc1 promotes catenin nucleartranslocation and inducestranscription of downstream target genes such as cmycand cyclin d1 in the nucleus reduces cell adhesion and promotes cell proliferation subsequently tumor cell invasion and metastasis occurredinterestingly another reported that catenincould act on the cthrc1 promoter region and promotetranscription nglycosylation stabilizes cthrc1 in oralsquamous cell carcinoma oscc specimens by reducingprotein turnover rate and cthrc1 is positively feedbackregulated by the dpagt1canonical wnt pathway therebyactivating noncanonical wnt pathways to drive tumor cellmigration and invasion in contrast the overexpressionof cthrc1 in hek293t cell and gastrointestinal stromaltumor gist cell significantly inhibited the canonical wntpathway but activated the noncanonical wntpcp pathway[ ] based on the evidence reviewed above it can be indicated that crosstalk between the canonical wntcateninpathway and noncanonical wntpcp pathway and themutual regulation of wntcatenin and cthrc1 acceleratethe process of tumor progression 0cmediators of ‚ammation wntpcp noncanonical signaling pathway earlyreports suggest that cthrc1 activates the pcp pathwayduring inner ear development cthrc1 can interactwith multiple extracellular components of wnt signalingfzd proteins and wntpcp coreceptor ror2 the components form a cthrc1wntfzdror2 complex to activatethe wntpcp pathway selectively and transmit signals fromthe cellsurface complex to the nucleus by dvlrhoarac1jnkatf2cjun cascade promoting cancer cell protrusionsproliferation migration and invasion [ “]cthrc1 is capable of coordinating three small rho gtpasesrac1 rhoa and cdc42 which are the leading performers ofwntpcp to promote cell migration in cervical cancercthrc1 cooperates with e6e7 human papillomavirushpv to activate the noncanonical wntpcp pathway whichaggravates tumor malignancy in pancreatic cancer andhuman urothelial carcinoma wnt5aror2 signaling is associated with emt and promotes tumor cellinvasion andmetastasis [ ] in gist cthrc1 appears to activatethe wntpcp pathway in a dosedependent manner andwnt5apcprho axis determinesinvasionpromoting activity of cthrc1 a recent study demonstrated that cthrc1 could promote erk and jnkphosphorylation by activating pcp signaling pathways inhuman umbilical vein endothelial cells huvecs and promote tumor angiogenesis whatit wasobserved that the paracrine cthrc1 controls the expression of ang2 via noncanonical wnt pathway activationof erkdependent ap1 in huvecs hence overand above that associated with the canonical wntcateninpathway noncanonical wnt signaling pathways interactwith other signaling pathwaysis moretumorthe cthrc1 binds integrin and triggers a series ofsignaling cascades to promote tumor progressionand metastasis integrin faksrc signaling pathway integrins aretransmembrane receptors that promote cellecm adhesion with two subtypes of α and it can participate ina variety of physiological activities such as tumor progression and migration cthrc1 promotes hepatocellular carcinoma cell invasion by activating the rhoarhoassociated kinase rock pathway and facilitates adhesionof hepatocellular carcinoma cells to ecm through induction of integrin 1 expression and activation of focal adhesion kinase fak another study of hepatocellularcarcinoma suggests that cthrc1 inhibits anoikis andincreases tumor cell survival by activating integrin expression cell adhesion to fibronectin is mediatedby integrin 1 previous researches have demonstrated that targeting the integrin 3fak signaling couldenhance the antitumor activity and attenuate cancermetastasiscancernsclc and escc [“] guo found that phosphorylated fak was significantly reduced in mice witheoc xenograft tumors and inhibition of fak did notinterfere with integrin 3 expression in vivo howeverthe overexpression of cthrc1 leads to the upregulationincluding melanomaendometrialof integrin 3 in model mice proving that cthrc1 interacts with integrin 3 and promotes fak phosphorylationat tyr397 thereby promoting ovarian cancer cell adhesionmigration and invasion the high level of cthrc1 is connected with the progression and metastasis of pancreatic cancers through the activation of several key signaling molecules including the steroidreceptor coactivator src fak paxillin mek erk andrasrelated c3 botulinum toxin substrate rac1 srcfak signaling cascade takes a regulative role in regulating the formation of protein complexes at focal adhesionsin the migration and metastasis of cancer cells srccan correspond to integrinecm interaction and is recruitedto form the srcfak complex which permits fak to beactive [ ] then fak activates src and paxillin by regulating focal adhesion formation and turnover paxillin a focaladhesion adaptor molecule serves as a scaï¬oldfor the organization and the activation of raf mek anderk [ ] furthermore paxillin can stimulate rac1which is a ras superfamily member of small guanosine triphosphatase gtpase and a critical factor in cytoskeleton reorganization regulation of gene expression and cell proliferationand cellular transformation [“]erk2mediated paxillin phosphorylation promotes fakadhesion to focal adhesions additionally the inhibitionof fakpaxillin interaction results in decreased phosphorylation of fak and its targets which in turn changes cell adhesion and migration thisinspired thedevelopment of anticancer drugs targeting fak faksrc complex plays essential functions in tgfinduced hepatocyte emt models such as upregulating mmp9 and fibronectin and downregulating ecadherin evidence has mekerk and pi3kakterk signaling pathwaycthrc1 interacts with integrin to trigger a series of signalcascades because src can phosphorylate other fak sites itcan recruit proteins containing src homology sh2domains such as grb2 subsequentlythe downstreamrasmapk pathway and the phosphatidylinositol kinase pi3k akt cascades are activated to participatein cellular response cthrc1 activates fosrelatedantigen1 fra1 through mapkmekerk signalingwhich leads to the upregulation of cyclin d1 and promotescell proliferation fra1 a fos family transcription factoralso induces snail1mediated mmp14 expression to facilitate escc cellinvasion migration and metastasis snail1 transcriptionaltriggeringemt and inducing tumor cell invasion knockingdown cthrc1 will change the phosphorylation level oferk12 and thus regulate the pathological process of endometriosisfrequent upregulation ofcthrc1 observed in human colon cancer cells may bedue to a cpg demethylation event in the exon regionof the gene kim tested the luciferase reporter geneusing the erkresponsive elk1 promoter proposing thatcthrc1 upregulates mmp9 through erk activation further treatment with mek12 inhibitors can reduce tumorcell invasion and erk activation and aggressiveness arereduced by knocking down cthrc1 theis essentialfactorforem 0cmediators of ‚ammationcthrc1 promotes invasiveness and metastasis of hepatocellular carcinoma through the activation of pi3kproteinkinase b pkbakterkcamp responseelementbinding protein creb signaling pathway which inducesemt change and mmp2mmp9 expression cthrc1is highly expressed in hepatitis b virus hbv associatedhepatocellular carcinoma hbv activates nuclear factorkappa b nfκb and creb through the erkcjun nterminal kinase cjnk pathway to stimulate cthrc1expression in addition hypoxiainducible factor 1α hif1α and vascular endothelial growth factor vegf are activated by cthrc1 through activating the pi3kaktmammalian target of rapamycin mtor signaling pathwaywhich promotesis morecthrc1 enhances colony formation migration and invasion of hepatocellular carcinoma cells by downregulatingtumor suppressor p53 and stimulating invasionassociatedfactor mmp9 tumor angiogenesis whatstudies on myocardial infarction mi have also foundthat activation of infarct repair cardiac fibroblasts ircfinvolves cthrc1 expression and pi3kakt signaling pathway in ovarian cancer cells gene silencing cthrc1 doesnot alter mmp9 expression or phosphorylate mek theinvasionpromoting eï¬ect of cthrc1 on eoc cells dependson downstream pi3kakt and erk12 signaling dominatedby egfr besides the invasion and metastasis of endometrial cancer are closely related to the upregulation ofcthrc1mediated cx3cr1 in macrophages this processregulates the integrin 3pi3kakt pathway which alsopromotes the recruitment of m2like macrophages cthrc1 activates hifα pathway and contributes totumor angiogenesis as mentioned above cthrc1 in hepatocellular carcinoma can induce hif1α to promote tumorangiogenesis and regulate downstream mmps and tumorsuppressor gene p53 by activating the pi3kakt signalingpathway in human squamous cell carcinoma hif1αoverexpression stimulates vegfc upregulation and induceslymphangiogenesis and tumor cell invasion ding pinpointed that cthrc1 and hif1α were upregulated inthe nucleus of cthrc1 overexpressed gc cells hif1αinhibitors reduced cthrc1induced cxcr4 expressionfurthermore it was found that inhibition of hif1α expression and inhibition of cxcl12cxcr4 signals all alleviatetumor cell migration and invasion therefore cthrc1 canparticipate in tumor cell migration and invasion throughhif1αcxcr4 signals in gc in short cthrc1 canaï¬ect the expression of hif1α which is not only related tolymphangiogenesis but also closely related to tumor progression and invasion a novel signaling pathway the potential role of pkcδerk in tumors protein kinase c δ pkcδ has beenimplicated in various epithelial tumors such as prostatebreast and crc [ ] activated pkcδ causes angiogenesis and tumor growth of prostate tumors by increasingnadph oxidase activity and hif1α expression levels pkcδ can also inhibit the wntcatenin pathwayin colon cancer cells however a recent study illustrated that mek and pkcδ inhibitors could blockcthrc1induced erk phosphorylation and that pkcδphosphorylation was not inhibited by mek inhibition surprisingly inhibition of plc a membraneassociated enzymethat activates pkcδ to promote bone formation in noncanonical wnt signals did not inhibit cthrc1induced alkaline phosphatase alp activity therefore waif1 bound bycthrc1 activates pkcδ and erk to stimulate osteoblastdiï¬erentiation which is a novel signaling pathway unrelatedto the noncanonical wnt pathway therefore pkcδsignal may explain the role of cthrc1 in tumor progressions such as angiogenesis and bone metastasisto put it briefly cthrc1 may be involved in manyother signaling pathways including mirna and lncrnawhich interact with or crosstalk with the tgf wnt andintegrin erk pathways and jointly participate in tumordevelopment and metastasis see table conclusiontumor development and metastasis a complex processinvolving cell adhesion and proteolytic degradation of theecm depends not only on the cancer cells but also on theinteraction between the cancer cells and their microenvironment complementary dna microarray analysis also demonstrated that the cthrc1 gene is expressed in mosthuman solid cancers as we all know cthrc1 is a secretedecm protein which can inhibit collagen deposition and participate in tumor invasion and metastasis even thoughcthrc1 was first discovered more than a decad

cancer7539

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: collagen triple helix repeat containing1 cthrc1 anextracellular matrix ecm protein was identified in thescreening of diï¬erentially expressed sequences between balloon injury and normal arteries the evolution of cthrc1can be traced back to at least million years ago and theconserved genes were not found in invertebrates cthrc1has complicated interactions with various intracellular andextracellular matrices in diï¬erent ways of secretion [ ]cthrc1 increases the activity of collagen promoter throughbinding to ligands and could contribute to vascular remodeling by limiting collagen matrix deposition and promoting cellmigration cthrc1 promotes the recruitment of m2macrophages and regulates tgf and notch pathways toaccelerate wound healing in a mouse model of acute woundhealing as a coupling factor cthrc1 can be secretedby osteoclasts and ‚uence bone formation and remodelingby acting on osteoblasts and osteocytes [ ] cthrc1 maypromote il1induced apoptosis of chondrocytes by activating the jnk12 pathway the anti‚ammatoryeï¬ect of cthrc1 expressed on activated synovial cellswas also found in a collagen antibodyinduced arthritismodel besides cthrc1 can regulate physiologicalfunctions such as fat and glycogen synthesis and promoteautonomous activity [ ]therefore as a secreted protein cthrc1 is involved inmultiple pathophysiologies a remarkable eï¬ect is that thehigh expression of cthrc1 promotes tumorigenesis anddevelopment through positive regulation of tumor spreadinvasion migration adhesion and metastasis cthrc1exerts its eï¬ects through several signaling pathways such as 0cmediators of ‚ammationgpa gvp grd gsp gan gip gtp gip grd gfk gek gechydrophobic regiongxy repeatcysteinesnglycosylationnh2signal peptidecollagendomainc c c cc cc ccoohfigure the structure of the cthrc1 protein the construct of cthrc1 contains an nh2terminal peptide for extracellular secretion ashort collagen triple helix repeat of amino acids and a coohterminal globular domain the prolinerich hydrophobic domain liesbetween the 1st and 30th amino acids and serves as a signal peptide for transport to the endoplasmic reticulum cthrc1 comprises acollagen domain between amino acids and and the protein contains cysteine residues corresponding to about cysteine inthe final protein what is more its only amino acid posttranslational modification is the glycosylation of asparagine at position integrin faktgf wntsrcfak mekerkpi3kakterk hif1α and pkcδerk signaling pathways in this we focus on the advances in the signalingpathways mediated by cthrc1 in tumors the structural characteristics andexpression of cthrc1 the structural features of cthrc1 the cthrc1 geneis located at chromosome 8q223 and it contains five exonsin humans and four exons in mice it covers kb onthe direct strand and can be transcribed into kb mrnathe amino acid sequence identity between human and ratcthrc1 proteins was and no homologs were foundin lower species [ ]protein nglycosylationsecreted cthrc1 exists primarily as a dimer kdaand a trimer kda as well as multimers of the trimericcthrc1 kda and kda the construct of cthrc1contains an nh2terminal peptide for extracellular secretiona short collagen triple helix repeat of amino acids and acoohterminal globular domain [ ] similar molecularweight and structural characteristics to adiponectin alsoexplain why cthrc1 can form high molecular weightcomplexes the biological activity of cthrc1 isrestricted to the highly conserved amino acids at thecterminal region and the cterminal region of cthrc1contains a putative nglycosylation site that stabilizescthrc1promotescthrc1 to tether to the cell membrane which promotesactin polymerization and cell polarity a short collagen motif with glyxy repeats presents in c1qtumornecrosis factorαrelated proteins ctrps which appearsto be responsible for the trimerization of protein and renders molecule susceptible to cleavage by collagenase seefigure however dimeric cthrc1 would not be susceptible to cleavage by collagenase [ ] the molecularweight of secreted cthrc1 kda appears to be largerthan that of cellular cthrc1 kda cthrc1 has fourdiï¬erentabout kda to kda the fulllength of cthrc1 accountsfor both secreted and cellular cthrc1 glycosylatedprotein cthrc1 with a signal sequence is related to ecmisoforms with molecular weights ofalsoand contains a variable short collagenlike motif intriguingly cthrc1 plays a role in inhibiting structural proteins unlike other members of the collagen family moreover leclair found that cthrc1 cleaved atthe nterminus by plasmin shows better inhibition of collagen synthesis compared to fulllength cthrc1 in thepac1 cell line these studies suggested that cthrc1might obtain biologicalthrough proteolyticprocessingactivity the expression of cthrc1 cthrc1 is transientlyexpressed by fibroblasts in remodeling adventitia and bysmooth muscle cells in the neointima of injured tissuehowever cthrc1 is not detected in normal arteries ininjured arteries and skin the expression of cthrc1 isassociated with myofibroblasts and locates in the sites ofcollagen matrix deposition in micethe first exon ofcthrc1 was targeted to be replaced with a galactosidase expression construct which demonstrated the expression of cthrc1 in inner ear hair cells there iscthrc1 expression in many mesenchymalderived cellsduring body growth and tissue repair in mouseembryos cthrc1 is expressed in visceral endodermnotochord neuraltube developing kidney and heartabundant expression of cthrc1 is observed in developincluding cartilage primordia growth plateing skeletoncartilagein adultscthrc1 is expressed only in bone matrix and periosteum cthrc1 is also found in the matrix of calcifyingatherosclerotic plaques and mineralized bone of skeletaltissues in humans in other tissues the sites of cthrc1expression overlap considerably with interstitial collagensand transforming growth factor tgf family membersparticularly bone morphogenetic proteins bmps the sitesof cthrc1 expression are characterized by the presence ofactive tgf and abundant collagen synthesis cthrc1mrna expression levels are increased in response to bmp4bmp2 and tgf furthermore tgf signaling could leadto a significant increase in neointimal lesion formation the expression of cthrc1 is also positively correlatedwith tumor lymph node metastasis tumornodemetastasistnm stage and disease prognosis however its potentialand periosteumbone matrix 0cmediators of ‚ammationregulatory mechanisms in the tumor environment have notyet been elucidated the molecules that regulate theexpression of cthrc1cthrc1 is abnormally expressed in several solid tumorsespecially in gastric cancer pancreatic cancer hepatocellularcarcinoma keloid breast cancer colorectal cancer crcepithelial ovarian cancer esophageal squamous cell carcinoma escc cervical cancer nonsmallcell lung carcinoma nsclc melanoma and so on [ “] andmolecules that regulate the expression of cthrc1 includemirnas lncrnas waif1 and dpagt1 mirnas microribonucleic acids mirnas which canregulate gene expression are a class of noncoding singlestranded small rnas of about nucleotides that can inhibitthe mrna translation process by exclusively promoting thedegradation of several mrnas in many tumors mirnas such as mir30c mir9 mir520d5p mir1555pmir98 let7b mir155 mir101 and mir217 can regulate the expression of cthrc1mir30c could regulate cthrc1 at a posttranscriptionallevelin breast cancer it downregulates the cthrc1mediated gsk3catenin signal and inhibits tumor cellproliferation invasion and migration in addition mir30ccan also upregulate baxcaspase9caspase3 a downstreamsignal of cthrc1 inhibiting apoptosis in hepatocellular carcinoma cthrc1 downregulates mir1555p throughthe activation of gsk3involved wntcatenin signalingto promote tumor formation and mir98 dramaticallydownregulates cthrc1 by directly targeting the ²utr ofcthrc1 suppressing hepatocellular carcinoma formation mir9 could inhibit the migration of schwann cell bytargeting cthrc1 following sciatic nerve injury therebyinactivating downstream rac1 gtpase mir520d5pis significantly downexpressed and suppresses cell proliferation migration and invasion by targeting cthrc1 in crc as the second mirna following lin4 in caenorhabditiselegans let7b may directly target cthrc1 and function asa tumor suppressor gene in gastric cancer [ ] in esccmir101 and mir217 could inhibitthe expression ofcthrc1 mir30 could downregulate the expressionof cthrc1 and downstream signal molecules such as mmp and mmp2 to inhibit the invasion and migration ofnsclc cells a recent study found that mir155 downregulation and cthrc1 upregulation were observed incrc moreover overexpression of mir155 can silencedownstream cthrc1 thereby inhibiting cell proliferationand inducing apoptosis of cells to prevent tumor progressionand metastasis in conclusion the negative regulation ofcthrc1 by mirna has the potential to become a noveldirection for cancer treatment in the futurelncrnas metastasisassociated lung adenocarcinomatranscript i malat1 is a large infrequently spliced longnoncoding rna lncrna which could genetically increasecthrc1 activity to regulate lung cancer cell migration the silence of malat1 could also inhibit the expression ofcthrc1 which is a positive regulator of escc furtheranother lncrna named nonmmug014387 could also regulate cthrc1 and activate the wntpcp pathway to promote schwann cell proliferation at the site of injury waif1 wntactivated inhibitory factor waif1 issilenced by promoter hypermethylation in various cancers[ ] lcmsms analysis using liquid chromatographyand mass spectrometry analysis of samples of cthrc1binding membrane proteins indicates that the largest partof cthrc1 binds the waif1 receptor recent researchsuggests that waifi expression is activated by suppressingmethylation of its promoter activated waif1 downregulates the expression of wntcatenin target genes to inhibitthe development of endometrial adenocarcinoma thebinding of cthrc1 to waif1 could promote osteoblast differentiation therefore cthrc1waif1 interactioncan be a potential therapeutic target in the futurepromoter hypomethylation dpagt1 nglycosylation is essential for the migrationpattern of immune cells and its dysregulation is related tovarious diseases including cancer in human escc the overexpression of cthrc1 is associated with hyperglycosylationandincreased nglycosylation is associated with preferential localization ofcthrc1 in wound cells and nglycosylation facilitatesthe promigratory function of cthrc1 dolichylphosphatenacetylglucosaminephosphotransferase dpagt1 thegene that encodes the first enzyme and ratelimiting enzymein the assembly of lipidlinked oligosaccharide precursors inthe endoplasmic reticulum is related to the formation ofmature intercellular adhesion complexes as anupstream regulator of nglycosylation status of ecadherindpagt1 could upregulate cthrc1 by increasing proteinturnover indicating that nglycosylation can also stabilizecthrc1 besides tgf and fak could also regulate the expression of cthrc1 in diï¬erent signaling pathways it should behighlighted that cthrc1 not only is the result of tumor progression but also plays a predominant regulatory role in theprogression and metastasis of many solid tumors [ “]in summary many molecules can regulate the expressionand activity of cthrc1 and together with cthrc1 as novelantitumor molecular targets for the treatment of cancer inthe future signaling pathways mediated by cthrc1involved in the progression andmetastasis of tumorthe ‚uence of cthrc1 on various events in tumor progression is based on its regulation of various signaling pathways such as tgf wntintegrin fak srcfakmekerk pi3kakterk hif1α and pkcδerk signaling pathways see figure these properties™ pathwaysaï¬ected by cthrc1 play an essential role not only in tissueremodeling after injury regulation of ossification and other 0cmediators of ‚ammationcthrc1tgf𝛽t𝛽r2wnt3alrp56cthrc1wnt5aror12cthrc1cthrc1𝛽𝛼𝛼𝛽integrincxrc4t𝛽r1p ppsmad23smad4cthrc1dvlpdvlaxinapc𝛽cateninck1𝛼gsk3dpagt1stabilization𝛽cateninpkc𝛿daamrac1rhoajnkrockpsrcfakpaxgrb2rasrafmekerkpfakpi3kaktmtorfra1crebmmpmekerkhif𝛼vegfecminvasionpsmad23smad4𝛽catenintcflefcjunap1snailcyclin d1g1mg2smetastasisangiogenesisproliferationfigure signaling pathways mediated by cthrc1 involved in the progression and metastasis of tumor tgf signaling pathway isquite complex especially in terms of its eï¬ects which are often contradictory depending on location and time there exists a criticalnegative feedback regulatory loop between tgfsmad23 signaling pathway and cthrc1 wnt signaling includes wntcatenincanonical pathway and cateninindependent noncanonical pathway in the canonical wnt signaling fzd receptor and lrp5lrp6coreceptor are transduced to catenin signaling cascade for the maintenance of stem and progenitor cells in the noncanonical wntsignaling fzd receptor and ror2ptk7ryk coreceptor are transduced to rhoa jnk signaling cascades for the control of tissuepolarity cell adhesion or cell movement the downstream molecules of the wntpcp pathway mainly include the small gtpase familysuch as rac1 rhoa and jnk which play essential roles in cancer cell migration and invasion cthrc1 signal via waif1 canactivate pkcδ which is an essential component of the wntpcp pathway furthermore pkcδ is responsible for the activation of thecthrc1induced erk signaling pathway in cthrc1integrin signaling pathway the upregulation of cthrc1 is related to theprogression and metastasis of several cancers through the activation of several key signaling molecules including src fak paxillin mekerk and rac1 fak promotes cancer cell migration by regulating focal adhesion formation and turnover which involve activation of srcand paxillin fra1 is activated by cthrc1 through the mapkmekerk signaling which leads to the upregulation of cyclin d1 andthat promotes cell proliferation fra1 also induces snail1mediated mmp14 expression to facilitate escc cell invasion migration andmetastasis pi3kakt signaling pathway induces emt change and mmp2mmp9 expression hif1α and vegf are activated bycthrc1 through activating the pi3kaktmtor signaling pathway which promotes tumor angiogenesis cthrc1 also participates intumor cell migration and invasion through hif1αcxcr4 signalsphysiological processes but also in the development of cancerand metastasis negative feedback regulation of cthrc1 and cell typespecific tgf signaling pathway as the most potentgrowth factor involved in wound healing tgf is releasedby platelets at the site of injury ‚uencing ‚ammatoryresponse angiogenesis reepithelialization ecm and remodeling tgf superfamily members include tgf activin and bmps smad158 mediates bmp signaling whilesmad23 mediates tgf and activin signalingcthrc1 has been reported to have a relationship withthe tgf family since its discovery as their expression sitesoverlap significantly tgf1 and bmp4 can induce thetranscription and expression of cthrc1 in nih3t3 cells cthrc1 can activate tgf signaling via an elevationin smad2smad3 phosphorylation activated smad23 formsa complex with smad4 and accumulates in the nucleus causing an increase in collagen type i deposition during vascularremodeling [“] there exists a critical negative feedbackregulatory loop between tgf1 and cthrc1 the conserved region of amino acids in cthrc1 proteincan bind to phosphosmad3 cthrc1 is induced by tgf1 via phosphosmad3 binding to the promoter with subsequenttranscription activation and in turn cthrc1inhibits tgf1 signaling by accelerating proteasomal degradation of phosphosmad3 which inhibits collagen deposition tgf can enhance the migration and invasioncharacteristics of endothelial cells by regulating the secretionand expression of mmp2 and mmp9 therefore inhibiting cthrc1mediated tgf signaling pathways mayeï¬ectively suppress the invasion and angiogenesis of cancercells [ ]however the mechanism of tgf involved in tumorprogression is very complex even in the same tumor typetgf has many diï¬erent roles in tumor progression forexample the activation of nuclear factor of activated tcellsnfats can drive the switch of the tumorsuppressive function of tgf towards tumor progression [ ] tgfincreases the level of cthrc1 in crc cells highly 0cmediators of ‚ammationexpressed cthrc1 promotes epithelialmesenchymal transition emt and tumor metastasis through the smad2smad3 activation of tgf pathway cthrc1 can alsoinhibit the tgfsmad pathway and yap nuclear translocation thereby inhibiting type i collagen synthesis metabolites such as bile acid may induce cthrc1 to activatethe tgfsmad2smad3 pathway to mediate liver fibrosisand may progress towards hepatocellular carcinoma in the polyvinyl alcohol sponge model cthrc1 activates tgf and notch pathways to promote the recruitment of m2 macrophages however cthrc1 maydownregulate tgf expression during the late remodelingphase of wound healing tgf regulates the expressionof cthrc1 in a concentrationdependent manner inkeloids and excess cthrc1 reverses collagen synthesis therefore these results of the regulation betweencthrc1 and tgf are not contradictory other than thatcthrc1 has no inhibitory eï¬ect on tgf signaling inendothelial cells these results indicate that the regulation of tgf by cthrc1 may play a role in other interstitial cells of the tumor microenvironment and that thisregulation is cell typespecific the further exploration ofdetailed molecular mechanism by which cthrc1 activatesthe tgf pathway may resolve these disputes mutual regulation between cthrc1 and wnt pathwaysto promote tumor progression and metastasis wnt familyare secreted glycoproteins include wnt1 wnt1 wnt3awnt4 wnt5a wnt5b wnt6 wnt7a and wnt7b andparticipate in the process of numerous oncogenic and development progress [“] wnt5a is a member of the wntprotein family and plays an essential role in the pathologicalprocess of neuropathy and malignant tumors [“] wntproteins activate the wntcatenin canonical pathway andcateninindependent noncanonicalamongwhich the planar cell polarity pcp pathway and wntcalcium ca2 pathway are the most widely studied [“]current reports indicate that cthrc1 is mainly involvedin tumor progression through the canonical wntcateninand noncanonical wntpcp pathwayspathway wntcatenin canonical signaling pathway in thewntcatenin canonical pathway wnt proteins bind tofrizzled fzd receptor and lipoprotein receptorrelatedprotein lrp56 coreceptor in the absence of wntsignaling the cytoplasmic catenin form the œdestructioncomplex composed with the casein kinase 1α ck1αglycogen synthase kinase 3 gsk3 adenomatous polyposis coli apc and axin which activates the emt topromotethroughcthrc1wntcatenin [ ] the level of cateninis maintained as low by the series of eventsincludingpriming phosphorylation by ck1α at ser45 and subsequently at thr41 ser37 and ser33 by gsk3 [ ]when secreted wnt ligands are accumulated wnt combines with fzd receptor and lrp56 coreceptors lead toactivation of dishevelled dvl protein the activateddvl is phosphorylated and translocated to the fzd recepthe catenintorcausing the dissociation ofand metastases cancerinvasionœdestruction complex and the cytosolic accumulation ofcatenin as the cytosolic catenin accumulates rasproteins are accumulated due to the absence of gsk3mediated phosphorylation the stabilized ras proteins atthe plasma membrane activate rafmitogenactivatedprotein kinase mekextracellular signalregulated kinaseerk cascade besides cytosolic catenin subsequently translocates into the nucleus and forms a complexwith the tcell factor tcf or lymphoid enhancer factorlef the complex activates the expression oftargetgenes involving proliferation and transformation such ascmyc cjun ccnd1 gene encoding cyclin d1 epidermal growth factor receptor egfr cd44 cd133 andleucinerich repeatcontaining g proteincoupled receptor lgr5 [“]the wntcatenin signaling pathway plays an indispensable role in the occurrence and development of manytypes of cancer cthrc1induced nuclear translocation ofcatenin was observed in nclh23 cells and luciferaseassay showed that catenintcf transcriptional activitywas enhanced in contrast the knockdown of cthrc1reduced the catenintcf transcriptional activity whichshows that cthrc1 regulates the invasiveness of nsclccells through the wntcatenin pathway similarlycthrc1 activates snail1 through the wntcatenin signaling pathway to promote emt in epithelial ovarian cancer during the development and metastasis of crccthrc1 may promote the activation of the wnt signalingpathway through anos1 it can also participate in thewntcatenin pathway to regulate the malignant behaviorof hepatocellular carcinoma with gsk3 many cancers usually metastasize to bone in advanced stages cthrc1secreted by osteoclasts promotes basic fibroblast growth factor bfgf expression in osteoblasts by stimulating wntcatenin signaling which may induce the development of cancerous bone lesions but not mediate vascular production the constitutive activation of the wntcatenin pathway leads to carcinogenesis in tumors cthrc1 promotes catenin nucleartranslocation and inducestranscription of downstream target genes such as cmycand cyclin d1 in the nucleus reduces cell adhesion and promotes cell proliferation subsequently tumor cell invasion and metastasis occurredinterestingly another reported that catenincould act on the cthrc1 promoter region and promotetranscription nglycosylation stabilizes cthrc1 in oralsquamous cell carcinoma oscc specimens by reducingprotein turnover rate and cthrc1 is positively feedbackregulated by the dpagt1canonical wnt pathway therebyactivating noncanonical wnt pathways to drive tumor cellmigration and invasion in contrast the overexpressionof cthrc1 in hek293t cell and gastrointestinal stromaltumor gist cell significantly inhibited the canonical wntpathway but activated the noncanonical wntpcp pathway[ ] based on the evidence reviewed above it can be indicated that crosstalk between the canonical wntcateninpathway and noncanonical wntpcp pathway and themutual regulation of wntcatenin and cthrc1 acceleratethe process of tumor progression 0cmediators of ‚ammation wntpcp noncanonical signaling pathway earlyreports suggest that cthrc1 activates the pcp pathwayduring inner ear development cthrc1 can interactwith multiple extracellular components of wnt signalingfzd proteins and wntpcp coreceptor ror2 the components form a cthrc1wntfzdror2 complex to activatethe wntpcp pathway selectively and transmit signals fromthe cellsurface complex to the nucleus by dvlrhoarac1jnkatf2cjun cascade promoting cancer cell protrusionsproliferation migration and invasion [ “]cthrc1 is capable of coordinating three small rho gtpasesrac1 rhoa and cdc42 which are the leading performers ofwntpcp to promote cell migration in cervical cancercthrc1 cooperates with e6e7 human papillomavirushpv to activate the noncanonical wntpcp pathway whichaggravates tumor malignancy in pancreatic cancer andhuman urothelial carcinoma wnt5aror2 signaling is associated with emt and promotes tumor cellinvasion andmetastasis [ ] in gist cthrc1 appears to activatethe wntpcp pathway in a dosedependent manner andwnt5apcprho axis determinesinvasionpromoting activity of cthrc1 a recent study demonstrated that cthrc1 could promote erk and jnkphosphorylation by activating pcp signaling pathways inhuman umbilical vein endothelial cells huvecs and promote tumor angiogenesis whatit wasobserved that the paracrine cthrc1 controls the expression of ang2 via noncanonical wnt pathway activationof erkdependent ap1 in huvecs hence overand above that associated with the canonical wntcateninpathway noncanonical wnt signaling pathways interactwith other signaling pathwaysis moretumorthe cthrc1 binds integrin and triggers a series ofsignaling cascades to promote tumor progressionand metastasis integrin faksrc signaling pathway integrins aretransmembrane receptors that promote cellecm adhesion with two subtypes of α and it can participate ina variety of physiological activities such as tumor progression and migration cthrc1 promotes hepatocellular carcinoma cell invasion by activating the rhoarhoassociated kinase rock pathway and facilitates adhesionof hepatocellular carcinoma cells to ecm through induction of integrin 1 expression and activation of focal adhesion kinase fak another study of hepatocellularcarcinoma suggests that cthrc1 inhibits anoikis andincreases tumor cell survival by activating integrin expression cell adhesion to fibronectin is mediatedby integrin 1 previous researches have demonstrated that targeting the integrin 3fak signaling couldenhance the antitumor activity and attenuate cancermetastasiscancernsclc and escc [“] guo found that phosphorylated fak was significantly reduced in mice witheoc xenograft tumors and inhibition of fak did notinterfere with integrin 3 expression in vivo howeverthe overexpression of cthrc1 leads to the upregulationincluding melanomaendometrialof integrin 3 in model mice proving that cthrc1 interacts with integrin 3 and promotes fak phosphorylationat tyr397 thereby promoting ovarian cancer cell adhesionmigration and invasion the high level of cthrc1 is connected with the progression and metastasis of pancreatic cancers through the activation of several key signaling molecules including the steroidreceptor coactivator src fak paxillin mek erk andrasrelated c3 botulinum toxin substrate rac1 srcfak signaling cascade takes a regulative role in regulating the formation of protein complexes at focal adhesionsin the migration and metastasis of cancer cells srccan correspond to integrinecm interaction and is recruitedto form the srcfak complex which permits fak to beactive [ ] then fak activates src and paxillin by regulating focal adhesion formation and turnover paxillin a focaladhesion adaptor molecule serves as a scaï¬oldfor the organization and the activation of raf mek anderk [ ] furthermore paxillin can stimulate rac1which is a ras superfamily member of small guanosine triphosphatase gtpase and a critical factor in cytoskeleton reorganization regulation of gene expression and cell proliferationand cellular transformation [“]erk2mediated paxillin phosphorylation promotes fakadhesion to focal adhesions additionally the inhibitionof fakpaxillin interaction results in decreased phosphorylation of fak and its targets which in turn changes cell adhesion and migration thisinspired thedevelopment of anticancer drugs targeting fak faksrc complex plays essential functions in tgfinduced hepatocyte emt models such as upregulating mmp9 and fibronectin and downregulating ecadherin evidence has mekerk and pi3kakterk signaling pathwaycthrc1 interacts with integrin to trigger a series of signalcascades because src can phosphorylate other fak sites itcan recruit proteins containing src homology sh2domains such as grb2 subsequentlythe downstreamrasmapk pathway and the phosphatidylinositol kinase pi3k akt cascades are activated to participatein cellular response cthrc1 activates fosrelatedantigen1 fra1 through mapkmekerk signalingwhich leads to the upregulation of cyclin d1 and promotescell proliferation fra1 a fos family transcription factoralso induces snail1mediated mmp14 expression to facilitate escc cellinvasion migration and metastasis snail1 transcriptionaltriggeringemt and inducing tumor cell invasion knockingdown cthrc1 will change the phosphorylation level oferk12 and thus regulate the pathological process of endometriosisfrequent upregulation ofcthrc1 observed in human colon cancer cells may bedue to a cpg demethylation event in the exon regionof the gene kim tested the luciferase reporter geneusing the erkresponsive elk1 promoter proposing thatcthrc1 upregulates mmp9 through erk activation further treatment with mek12 inhibitors can reduce tumorcell invasion and erk activation and aggressiveness arereduced by knocking down cthrc1 theis essentialfactorforem 0cmediators of ‚ammationcthrc1 promotes invasiveness and metastasis of hepatocellular carcinoma through the activation of pi3kproteinkinase b pkbakterkcamp responseelementbinding protein creb signaling pathway which inducesemt change and mmp2mmp9 expression cthrc1is highly expressed in hepatitis b virus hbv associatedhepatocellular carcinoma hbv activates nuclear factorkappa b nfκb and creb through the erkcjun nterminal kinase cjnk pathway to stimulate cthrc1expression in addition hypoxiainducible factor 1α hif1α and vascular endothelial growth factor vegf are activated by cthrc1 through activating the pi3kaktmammalian target of rapamycin mtor signaling pathwaywhich promotesis morecthrc1 enhances colony formation migration and invasion of hepatocellular carcinoma cells by downregulatingtumor suppressor p53 and stimulating invasionassociatedfactor mmp9 tumor angiogenesis whatstudies on myocardial infarction mi have also foundthat activation of infarct repair cardiac fibroblasts ircfinvolves cthrc1 expression and pi3kakt signaling pathway in ovarian cancer cells gene silencing cthrc1 doesnot alter mmp9 expression or phosphorylate mek theinvasionpromoting eï¬ect of cthrc1 on eoc cells dependson downstream pi3kakt and erk12 signaling dominatedby egfr besides the invasion and metastasis of endometrial cancer are closely related to the upregulation ofcthrc1mediated cx3cr1 in macrophages this processregulates the integrin 3pi3kakt pathway which alsopromotes the recruitment of m2like macrophages cthrc1 activates hifα pathway and contributes totumor angiogenesis as mentioned above cthrc1 in hepatocellular carcinoma can induce hif1α to promote tumorangiogenesis and regulate downstream mmps and tumorsuppressor gene p53 by activating the pi3kakt signalingpathway in human squamous cell carcinoma hif1αoverexpression stimulates vegfc upregulation and induceslymphangiogenesis and tumor cell invasion ding pinpointed that cthrc1 and hif1α were upregulated inthe nucleus of cthrc1 overexpressed gc cells hif1αinhibitors reduced cthrc1induced cxcr4 expressionfurthermore it was found that inhibition of hif1α expression and inhibition of cxcl12cxcr4 signals all alleviatetumor cell migration and invasion therefore cthrc1 canparticipate in tumor cell migration and invasion throughhif1αcxcr4 signals in gc in short cthrc1 canaï¬ect the expression of hif1α which is not only related tolymphangiogenesis but also closely related to tumor progression and invasion a novel signaling pathway the potential role of pkcδerk in tumors protein kinase c δ pkcδ has beenimplicated in various epithelial tumors such as prostatebreast and crc [ ] activated pkcδ causes angiogenesis and tumor growth of prostate tumors by increasingnadph oxidase activity and hif1α expression levels pkcδ can also inhibit the wntcatenin pathwayin colon cancer cells however a recent study illustrated that mek and pkcδ inhibitors could blockcthrc1induced erk phosphorylation and that pkcδphosphorylation was not inhibited by mek inhibition surprisingly inhibition of plc a membraneassociated enzymethat activates pkcδ to promote bone formation in noncanonical wnt signals did not inhibit cthrc1induced alkaline phosphatase alp activity therefore waif1 bound bycthrc1 activates pkcδ and erk to stimulate osteoblastdiï¬erentiation which is a novel signaling pathway unrelatedto the noncanonical wnt pathway therefore pkcδsignal may explain the role of cthrc1 in tumor progressions such as angiogenesis and bone metastasisto put it briefly cthrc1 may be involved in manyother signaling pathways including mirna and lncrnawhich interact with or crosstalk with the tgf wnt andintegrin erk pathways and jointly participate in tumordevelopment and metastasis see table conclusiontumor development and metastasis a complex processinvolving cell adhesion and proteolytic degradation of theecm depends not only on the cancer cells but also on theinteraction between the cancer cells and their microenvironment complementary dna microarray analysis also demonstrated that the cthrc1 gene is expressed in mosthuman solid cancers as we all know cthrc1 is a secretedecm protein which can inhibit collagen deposition and participate in tumor invasion and metastasis even thoughcthrc1 was first discovered more than a decad Answer:

Colon_Cancer

antibioticresistant pathogen strainlevel investigations are beginning to reveal themolecular mechanisms used by vrefm to colonize regions of the human bowelhowever the role of commensal bacteria during vrefm colonizationin particularfollowing antibiotic treatment remains largely unknown we employed amplicon16s rrna gene sequencing and metabolomics in a murine model system to try andinvestigate functional roles of the gut microbiome during vrefm colonization firstorder taxonomic shifts between bacteroidetes and tenericutes within the gut microbial community composition were detected both in response to pretreatment usingceftriaxone and to subsequent vrefm challenge using neural networking approaches to find cooccurrence profiles of bacteria and metabolites we detected keymetabolome features associated with butyric acid during and after vrefm colonization these metabolite features were associated with bacteroides indicative of a transition toward a preantibiotic naive microbiome this study shows the impacts of antibiotics on the gut ecosystem and the progression of the microbiome in responseto colonization with vrefm our results offer insights toward identifying potentialnonantibiotic alternatives to eliminate vrefm through metabolic reengineering topreferentially select for bacteroidesimportance this study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions betweencommensal gut bacteria and a specific pathogen knowledge from this researchwillinform gut microbiome engineering strategies with the aim of translatingobservations from animal models to humanrelevant therapeutic applicationscitation mu a carter gp li l isles ns vrbanacaf morton jt jarmusch ak de souza dpnarayana vk kanojia k nijagal b mcconvillemj knight r howden bp stinear tp microbemetabolite associations linked to therebounding murine gut microbiomepostcolonization with vancomycinresistantenterococcus faecium msystems 5e0045220101128msystems0045220editor manuel liebeke max planck institutefor marine microbiologycopyright mu this is an openaccess distributed under the terms ofthe creative commons attribution international licenseaddress correspondence to andre muandremuunimelbeduaureceived may accepted july published august julyaugust volume issue e0045220msystemsasm 0cmu keywords microbiome multiomics metagenomics metabolomics gutmicrobiome vancomycinresistant enterococci colonization antimicrobial resistanceceftriaxonevancomycinresistant enterococcus faecium vrefm is a significant health careassociated pathogen vrefm infections can be difficult to treat due to their intrinsicand acquired resistance to nearly all classes of antibiotics the world healthanization categorizes vrefm as a œhigh priority bacterial pathogen advocatingresearch to stop the global increase in antibiotic resistance recent studies highlightthe importance of the gut microbiota in modulating the growth and virulence of vrefmin the gastrointestinal ecosystem for instance the depletion of normal gut flora usingantibiotics exacerbates the severity of vrefm infection whereas transplant ofcommensal species including a consortium of clostridium bolteae blautia productablautia sartorii and parabacteroides distasonis can drive established vrefm colonization to below levels of culture detection specifically b producta”a colonizer ofthe colon”reduces vrefm growth in vivo by secreting a lantibiotic these observations raise the intriguing possibility that metabolic traits act in concert betweenpathogen and select gut commensals to confer mutual benefits during pathogenpersistence these findings also highlight the greater risk posed to immunocompromised patients when colonized with vrefm for instance allogeneic hematopoietic celltransplantation patients have gastrointestinal tracts that are dominated by vrefm as aresult of losing a large portion of the intestinal commensal microbiota upon receivingbroadspectrum antibiotics as pretreatment hildebrand discovered longtermecological impacts to the gut microbiome with strong bacterial species turnover afterceftriaxone treatment in humans further mice receiving broadspectrum antibioticscombination of metronidazole neomycin and vancomycin showed markedly increased vrefm colonization of the cecum and colon the compromised intestinal innateimmune defenses in these animals allowed proliferation of vrefm caused by theantibiotic exposure and subsequently reduced the expression of antimicrobial molecules produced by bacteria in the intestinal mucosa the problem with vrefm is further complicated by the fact that enterococci aremembers of the gastrointestinal tract microbiota a key reservoir of antimicrobialresistance amr genes and potentially facilitating gene transfer within the gut microbiome for example the vanb resistance gene was detected in human fecalspecimens that did not contain culturable vre and instead demonstrated that isolatescarrying the resistance transposon are anaerobic commensal bacteria eggerthella lentaand clostridium innocuum colonization of and persistence in the gastrointestinaltract therefore presents as a key mechanism for de novo vre and may lead to severeinvasive diseasethe current study aimed to understand the impact of antibiotics on the murine gutmicrobiota and the subsequent colonization pattern of vrefm to this extent wedesigned a murine model timeseries study that consisted of two main perturbativephases i antibiotic pretreatment with ceftriaxone and ii vrefm challenge our 16srrna gene profiling analyses highlighted a firstorder shift in bacterial biodiversitycomposition across time a secondorder clustering of samples associated with theexperimental phases and the transition of the postvrefm colonization gut microbiotaand its metabolome toward resembling an asymptomatic carriagelike microbiomephenotype this research provides support for engineering the metabolic potential ofthe gut microbiome using for example prebiotics as a nonantibiotic alternative fortreating multidrugresistant bacterial infectionsresultsexperimental design the following experimental design was developed to address the hypothesis that there are specific murine gut microbiome factors thatfacilitate vrefm colonization three groups of three c57bl6 mice cocaged wildtypejulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vretable summary of samples analyzed in this studyday of exptphase of exptannnnnnabxtxabxtxabxwnvreevreevreevreevrelvrelamplicon 16s rrnagene databœ“œ“œ“«º«ºœ“œ“œ“œ“œ“«º«ºœ“œ“œ“metabolomicsbœ“«º«º«º«ºœ“œ“œ“œ“œ“«º«º«º«ºœ“avg no ofobserved sotusc«º«º«º«ºathe key phases of the experiment where n represents naive abxtx represents antibiotic treatment abxwn represents antibiotic weaning vree represents earlyphase postvrefm colonization and vrelrepresents latephase postvrefm colonizationbsymbols œ“ sample processed «º data unavailablecthe average number of sotus observed across all mice for each day of the experimentmales were monitored and fecal samples were collected over a 14day period with twointervention time points including i ceftriaxone treatment administered at 05gliter indrinking water across a 2day period and ii colonization via oral gavage with «» vrefm st796 per mouse postantibiotic treatment at a single time point mice werehoused in groups of five and samples were collected from the same three mice torepresent technical replicates per cage herein each group of cohoused mice will bereferred to as group a group b and group c the remaining two mice per group werereserved for microbiological assays table highlights samples and data sets collectedamplicon 16s rrna gene sequencing revealed firstorder shifts in bacterialcommunity composition amplicon 16s rrna gene sequencing was performed tocapture the bacterial community composition in an effort to track changes in responseto antibiotic pretreatment and vrefm colonization bacterial community profiles wereassessed in fecal samples from nine mice before during and after the two interventionstable a total of of reads reads passed quality control with reads on average per sample and a total of exact variant sequence typesie features with an average of features per sample and an upper bound of features when rarefied to reads alpha rarefaction analysis demonstratedsufficient sequencing depth to capture microbial diversity to saturation see fig s1 inthe supplemental materialthe biodiversity profiles of each sample were compared and showed that keysuboperational taxonomic units sotus were differentially abundant throughout thecourse of the experiment fig there was a shift in the dominance of bacteroidiabacteroidetes light green colored bars during the naive phase of the experiment tomollicutes tenericutes fuschia colored bars in response to ceftriaxone treatment witha return to the predominance of bacteroidia during the late phase of the experimentafter vrefm colonization ie days to of note is the predominance of lactobacillales in mouse to from group a fig the murine gut microbiota responds to antibiotics and microbial communityrichness begins to rebound days after vre colonization principalcoordinateanalysis pcoa of the unweighted unifrac distances was used to assess clusteringof fecal samples based on bacterial composition this assessment showed that the fecalmicrobiota from samples collected from each phase clustered together but were clearlyseparated between phases after exposure to ceftriaxone and challenge with vrefmpostantibiotic treatment fig 2a permutationbased statistical testing demonstratesthe groups are significantly different from one another fig s2 temporal tracking ofjulyaugust volume issue e0045220msystemsasm 0cmu fig biodiversity plot of sotus as relative frequencies at the taxonomic level of class firstorder shifts in microbial communitycomposition as revealed by 16s rrna gene community profiling from a predominance of bacteroidetes to tenericutes and return tobacteroidetes was observed each column displays the relative bacterial community composition in a mouse fecal sample collecteddaily and sorted by the chronology of the experiment ie day of experiment table the columns are further sorted by group iegroup a group b and group c and individual mice within each group mouse mouse and mouse stacked bars are presentedas relative frequencies at the taxonomical level of class however annotations of key taxa are at the phylum level bacteroidetes [green]firmicutes [gray] and tenericutes [fuschia] or order level lactobacillales [yellow]the changing microbiomes against each mouse on the pcoa sample space demonstrated a clear unidirectional trajectory that followed the chronology of the experiment106084m9figshare12775859 procrustes analyses of weighted andunweighted unifrac distances showed that the same general patterns on the samplespace were preserved meaning that there is congruency in global spatial patternsbetween qualitative and quantitative measures of community dissimilarity fig s3analysis of community diversity faith™s phylogenetic diversity index revealed astable and rich microbial community during the naive phase preceding a sharpdecrease following antibiotic treatment and a further decrease immediately followingvrefm colonization fig 2b of note is the responsiveness of the microbiota within h to the removal of antibiotics at the end of day community richness began torebound at approximately days after vrefm colonization ie day with group ademonstrating a higher rate of rebound compared to groups b and c calculating thedistances of dissimilarity unweighted unifrac distances of each mouse microbiotatime point relative to day a proxy for the naive bacterial community phenotyperevealed a small dissimilarity distance for samples collected during the naive phase andan increasing dissimilarity distance following antibiotic treatment day and vrefmcolonization day fig 2c there was a downward trajectory in distance scores daysafter vrefm colonization ie day group a followed a sharper return to a microbiotaresembling day these observations suggest that mice were transitioning toward apersistent carrierlike state and that the rebounding community richness toward levelsrepresentative of the naive phase was by a microbial community structure that resembled the naive phase additional studies where the time frame of postvrefm challengeextends beyond week of monitoring are needed to understand whether the perturbed microbiome will return to resemble an absolute naive state or arrive at a newaltered statemultinomial regression identifies sotus most positively associated with vrefmcolonization multinomial regression using songbird was employed to identify sotusjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig diversity analyses a principalcoordinate analysis plot of unweighted unifrac distances data points areprojected onto the sample space and colored by prevrefm colonization red and postvrefm colonization bluenote that circles and ellipses function to highlight the separation of experimental phases and do not indicatestatistical confidence intervals principal coordinate axis explains of the variation observed between thenaive microbiota and those from the postvrefm colonization phase b community richness of the murine gutmicrobiome as measured by faith™s phylogenetic diversity in response to ceftriaxone treatment and challengewith vrefm c community dissimilarity distances as calculated by unweighted unifrac of each time point relativeto day naive phasethat were most positively and negatively associated with the postvrefm colonizationphase fig the five most positively associated sotus were enterococcus bacteroideserysipelotrichaceae catabacter and lachnospiraceae while the five most negativelyassociated were clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridiales temporal tracking of exact sequence variants esvs demonstrated that theesv feature classified as enterococcus”and identified as the most positively associatedwith the postvrefm colonization phase”was most abundant on days of challengeconfirming that this esv likely was the st796 vrefm colonization challenge anismsfig there were a further eight esv features classified as enterococcus however theywere absent during the days representing vrefm colonization and lacked positiveassociations with the postvrefm colonization phase suggesting that these featuresrepresent murine gut commensal enterococcijulyaugust volume issue e0045220msystemsasm 0cmu fig multinomial regression multinomial regression identified an enterococcus exact sequence variant as the most positivelyassociated with the colonization phase log fold change score of read counts for the enterococcus esv tracked daily acrossthe experiment showing high abundance during the days of vrefm challengemolecular networking identifies differential metabolome profiles duplicatefecal samples from key time points throughout the experiment ie days and were analyzed by datadependent tandem mass spectrometry msms performed on a liquid chromatography quadrupole time of flight lcqtof system tomonitor changes in the murine gut metabolome table polar metabolite analysiswas given preference in an effort to broadly capture primary metabolites that play a keyrole in œmetabolic handoffs that define interspecies interactions analysis of the globalmetabolome profile of each sample was measured based on their overlapping molecules and a pcoa plot using a binary jaccard distance metric through the global naturalproducts social molecular networking gnps platform a separation of metaboliteprofiles along pcoa1 was observed fig 4a metabolomes from the naive andlate vrefm colonization phase tended to cluster together while samples from thepostantibiotic phases including the early vrefm colonization phase clustered togethersupporting pairwise permutational multivariate analysis of variance permanovatesting fig s4 highlights that naive and early vre samples are significantly differentwhile late vre has a lower distance to naive samples compared to antibiotictreatedantibiotic wean and early vre samplesrandom forest analysis of spectrum profiles from lcmsms was used to predictexperimental phase and rank the importance of metabolite association with eachexperimental phase the top metabolite features for each experimental phase arehighlighted in fig 4b unique profiles of metabolite features were observed for eachphase of the experiment importantly the late vrefm colonization phase capturesan unknown metabolite feature with a masstocharge ratio mz of and retention time rt of this metabolite is exclusively present during whatrepresents the transition toward resembling the naive microbiome manual curationoffeature in positiveion mode predicts a molecular formula c5h8n4o3with ±–10 ppm in mass error the major peaks in the msms spectrum for feature are precursor ion [m«¹h]«¹ assumed precursor ion [h2o] product ion [likely c2h2o] [c2h2o«¹h] plus product ionfurther supporting neutral loss of c2h2o given the summation of results the chemicalstructure of feature is likely to contain a nacetylated hydroxyl group peakquantification values indicate its presence during the late phase of vre colonizationfig 4c further manual curation of msms data identified ceftriaxone as feature julyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig metabolomic analyses a emperor plot displaying principalcoordinate analysis of binary jaccard distances of metabolomic profiles samples are colorcoded and the colors represent the naive orange antibiotic treatment red antibiotic weaning blue early vre colonization green and late vrefmcolonization purple phases b random forest classifier identifying metabolite features spectra for each phase of the experiment the heatmap is color codedfrom low ranking score white ie lowest importance to high ranking score dark blue highest importance metabolite features are labeled by theirmasscharge ratios and retention times for the reason that current databases do not capture their chemical structure andor identifications abx tx antibiotictreatment c peak quantification values for feature mz «½ and rt «½ present in abundance during vre colonization late phase dpeak quantification values for ceftriaxone mz «½ and rt «½ tracked across the experiment ceftriaxone values are highest during antibiotictreatment phase and begins to wane during antibiotic weaning phase julyaugust volume issue e0045220msystemsasm 0cmu with an mz of and rt of and mostly abundant during days of antibioticexposure fig 4dbacteroidalesassociated metabolites implicated in latephase postvrefm colonization a distinct profile shift in microbe and metabolite abundances as calculatedby multinomial regression was observed particularly during latephase vrefm colonization fig s6 shallow neural networking analysis with mmvec was used to predictmicrobemetabolite interactions through their cooccurrence probabilities fig sequential biplots captured the shift in experimental phases and highlighted the cooccurrences of microbiota and metabolomic data sets fig 5a to c there was a strongenterococcus effect as indicated by the magnitude of the corresponding arrow and therebounding species during the latephase vrefm colonization are predominantly bacteroidales sotus fig 5c with cooccurring metabolite features mz rt and mz rt metabolite feature mz rt was ranked asbeing highly associated with the postvre colonization phase these results integratemicrobial and metabolite data sets to reveal which microbes may be responsible fordetected metabolites in this instance the metabolite present during the phase representing a transition toward a microbiome approximating the naive state feature mz and rt fig 4b is linked with bacteroidales fig 5adiscussionin this study of the murine gut ecosystem we employed a mouse model ofgastrointestinal tract colonization that replicates the shift in bacterial compositionwhen patients enter the health care system develop an imbalance in their microbiomeas a result of pretreatment eg antibiotic treatment and are subsequently colonizedwith a hospital superbug the resolution of current studies describes a consortiumof commensal microbes that can for example reduce the magnitude of vrefm colonization however understanding the key metabolic shifts relative to the gutmicrobiota remains challenging here we employed amplicon 16s rrna genesequencing and highresolution mass spectrometry metabolomics in an effort towarddetermining microbiotametabolome interactions during vrefm colonization we demonstrated clear changes in the gut microbiome in response to ceftriaxone and vrefmchallengeconceptual and statistical advances in analysis of amplicon 16s rrna gene data whereby otus are clustered at a nucleotide similarity threshold allows for theidentification of exact sequence variants esvs query against an errorcorrecteddatabase can detect multiple esvs that may be classified to the same taxonomicrank for example our analyses identified multiple esvs classified as enterococcihowever when the relative abundances were tracked across the chronology of theexperiment only one enterococcus esv was dominant in relative abundances and mostpositively associated with the days of postvrefm challenge fig this highlights theresolving power to differentiate between commensal and pathogenic strains of enterococci when the composition of the microbial community is considered the factthat this was achievable at the level of amplicon 16s rrna gene sequencing alludes tothe possibility of implementing microbiota screenings as routine diagnostics for patients entering health care systems further firstorder level shifts in microbial community composition was observed in response to ceftriaxone and subsequent vrefmchallenge fig three days after vrefm colonization ie day the microbiomerichness begins to rebound suggesting that mice are transitioning toward a persistentcarrierlike state interestingly the group a cohort exhibited a higher rate of reboundthat may be facilitated by their initially higher microbial community richness andpredominance of lactobacillales on the day of vrefm challenge fig 2b this observation supports the need to prescreen œbaseline microbiota profiles of patients uponadmission into hospital for the reason that it is not necessarily which microbialpopulations are removed postperturbation eg antibiotic pretreatment but insteadwhich populations persist that drives the responding phenotype we can begin toassess patients from across different wards eg intensive care unit oncology neuroljulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig microbemetabolite vector biplots sequential biplots highlighting the changing metabolitedifferentials across each key phase of the experiment abx tx is the antibiotic treatment phase and abxwean is the period when antibiotics were removed for a 24h period prior to colonization with vrefmeach point on the sample space represents metabolites and arrows represent microbes microbe andcontinued on next pagejulyaugust volume issue e0045220msystemsasm 0cmu ogy and healthy cohorts and build a database of microbiome profiles that can be usedas biomarkers to predict i the susceptibility of patients to develop persistent bacterialcolonization and ii propensity to clear the pathogen once colonized the clinicalimplication is that new patients are screened and identified via betadiversity metaanalyses by these biomarkers and placed in bedding cohorts accordingly therebyimproving infectious disease management and isolation precautions within healthcareassociated ecosystemsthe shortlist of microbes ranked as most negatively associated with the colonizationphase clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridialesfig are hypothesized to play a role in maintaining the health of the animals indeedamong the microbes identified are known shortchain fatty acid eg butyric acidproducers which supports and expands upon those previously identified bycaballero further the use of deblur to identify esvs facilitates the temporaltracking of their relative abundances to inform selection of primary fecal samples thatwill provide the best probability ie highest relative abundance of culturing targettaxa for downstream screening of probiotic potential however translating animalderived observations from experimental animal models to human clinical situationsremains challenging particularly where the key microbes are rodentspecific microbesone solution may be to integrate metabolomics to reveal shared metabolic capacityamong taxonomically divergent microbes our supervised classifying approaches suggests an altered metabolome composition during the late phase of vrefm challengethat may facilitate the apparent œsuppression of vrefm to levels below the limit ofdetection by culture despite the caveat of poor resolution in current databases to linkmetabolite features to associated chemical structures microbemetabolite vector analysis linked metabolite feature mz «½ and rt «½ to bacteroidesfig our efforts toward manually identifying feature suggests a chemicalformula of c5h8n4o3 and a structure likely to contain a nacetylated hydroxyl group aputative annotation through pubchem search is 3hydroxy4nitrosocyanamido butyramide butyramide is the amide group of butyric acid a shortchain fatty acid thathas been shown to play a key role in colonization resistance against intestinal pathogens “ further research to comprehensively characterize interactions betweenmicrobe and metabolites will be critical to address the gaps in our understanding of thebiochemical parameters that define interspecies microbiome interactions during antibiotic pretreatment and persistent infectionsthe resolution of our results provides the basis in which to begin to identifynonantibiotic alternatives to engineer the gut microbiome through prebiotic interventions eg butyric acid and translating animal studies to humanrelevant therapeuticapplications by delineating taxonomically diverse microbes with shared metaboliccapacity here achieving integrative omics to link microbemetabolite associations ourfindings add support to the incorporation of microbiome profiling approaches intoroutine clinical microbiology particularly in the context of monitoring the impacts ofantibiotic usematerials and methodsmouse gastrointestinal colonization model sixweekold wildtype c57bl6 male mice were usedto establish an animal model of gastrointestinal colonization with vrefm mice were cohoused and hadfree access to food ordinary chow and water and had environmental enrichment eg fun tunnels chewblocks and tissue paper the lightdark cycle was 12h light12h dark and cages were changed weeklyfig legend continuedmetabolite features are fixed upon the sample space with gradient coloring of metabolites indicating thetransition across key phases of the experiment the distance between each point is indicative ofmetabolite cooccurrence frequency and the angle between arrows indicates microbial cooccurrencethe directionality of the arrows describes the variation in the metabolites explained by the microbesrepresented by the arrows for example metabolite feature mz and rt isdemonstrated to cooccur with bacteroides information about the abundances of these cooccurringfeatures are provided as heatmaps in fig s6 in the supplemental materialjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vremice were pretreated with gliter ceftriaxone in drinking water for days followed by an antibioticwean period of h mice were then challenged with «» cfu vrefm st796genomic dna extraction and sequencing wholecommunity genomic dna gdna was extractedfrom mouse fecal samples using the qiagen powersoil dna extraction kit formerly mobio following themanufacturer™s protocol a preprocessing step of mechanicallysis was incorporated using a bertintechnologies precellysis machine for one round of a 40s cycle at rpm the v4 region of thebacterial 16s rrna gene was amplified using small subunit forward golaybarcoded and ssu806reverse primers following the earth microbiome project protocol and sequenced using the illuminamiseq platform v2 cycles illumina inc san diego ca usa further primary derived data egbiom tables used to produce results can be found within qiita study id amplicon 16s rrna gene profiling analyses sequence data were processed within the qiitav010 framework for quality control split libraries v q2191 demultiplexing trimming sequencereads to a length of nucleotides nt and picking suboperational taxonomic units sotus usingdeblur v110 to resolve singlenucleotide community sequencing patterns ie feature identification ofsotus [] the output biom files were further processed using qiime2 v20197 for downstreamstatistical analyses alpha rarefaction curves were generated to determine whether each sample hadbeen sequenced to saturation the feature table was subsequently rarefied to reads per sampletaxonomy was assigned using the sklearn classifier and greengenes otus from 515f806rregion of sequences classifier available from docsqiime220184dataresources furthermorerelative abundances of each taxa were visualized as bar plots using the qiime2 taxa plugin a phylogenetic tree was constructed using fragment insertion qiime fragmentinsertion sepp [] to guidephylogeneticaware statistical analyses generated using the qiime2 plugin q2diversity coremetricsphylogenetic key metrics computed by default include both alphadiversity eg shannon™s diversityindex faith™s phylogenetic diversity and evenness and betadiversity eg braycurtis distance andunweighted unifrac distance metrics the unweighted unifrac distance matrix was used tocompute first distances and calculate distances relative to day as the baseline between sequentialstates qiime longitudinal firstdistances ggplot2 r v360 ggplot2tidyverse was used tovisualize the distance scores as line plots emperor was used to visualize principalcoordinate analysisplots of unweighted unifrac distances permutationbased statistical testing permanova on unweighted unifrac distances was used to determine whether samples grouped by phase of experimentwere significantly different from one another q2betagroupsignificance songbird githubcommortonjtsongbird was employed to determine the importance ie fold change of each sotu inrelation to a given metadata variable eg vrefm colonization microbial features from all samples weresplit into training and test sets for supervised learning classifier analyses of input samples wereallocated to train the random forest classifier within qiime2 the estimator method used for sampleprediction the different experimental phases were the response variables while the 16s rrna gene datawere the featuresmetabolite extraction and liquid chromatographytandem mass spectrometry analysis duplicate fecal samples as outlined in table were processed for polar metabolite extraction and analysisdays and feces were metabolically arrested by immediate collection into dry ice andstored at “ °c until further processing metabolite extraction from the fecal samples was undertaken bythe addition of \u242el per sample of methanolwater solution [volvol] containing \u242em [13c]sorbitoland \u242em [13c15n]valine and \u242em [13c]leucine as internal standards fecal samples were homogenizedat rpm for min in a thermomixer maintained at °c mechanically disrupted and incubated fora further min in the thermomixer samples were randomized for metabolite extractionmetabolite analysis of the extracted samples pooled biological quality control pbqc samples and mixtures of authentic standard mixes was performed by liquid chromatographymass spectrometrylcms using hydrophilic interaction column zicphilic and highresolution agilent seriesquadrupole time of flight mass spectrometry qtof ms as described previously pcoa of binaryjaccard distances of test standard mixes and pbqc samples are presented in fig s5 in the supplementalmaterial ions were analyzed in positive mode with full scan range of to mz and in datadependent tandem ms mode to facilitate downstream metabolite identificationmetabolomic analyses

cancer7540

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: antibioticresistant pathogen strainlevel investigations are beginning to reveal themolecular mechanisms used by vrefm to colonize regions of the human bowelhowever the role of commensal bacteria during vrefm colonizationin particularfollowing antibiotic treatment remains largely unknown we employed amplicon16s rrna gene sequencing and metabolomics in a murine model system to try andinvestigate functional roles of the gut microbiome during vrefm colonization firstorder taxonomic shifts between bacteroidetes and tenericutes within the gut microbial community composition were detected both in response to pretreatment usingceftriaxone and to subsequent vrefm challenge using neural networking approaches to find cooccurrence profiles of bacteria and metabolites we detected keymetabolome features associated with butyric acid during and after vrefm colonization these metabolite features were associated with bacteroides indicative of a transition toward a preantibiotic naive microbiome this study shows the impacts of antibiotics on the gut ecosystem and the progression of the microbiome in responseto colonization with vrefm our results offer insights toward identifying potentialnonantibiotic alternatives to eliminate vrefm through metabolic reengineering topreferentially select for bacteroidesimportance this study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions betweencommensal gut bacteria and a specific pathogen knowledge from this researchwillinform gut microbiome engineering strategies with the aim of translatingobservations from animal models to humanrelevant therapeutic applicationscitation mu a carter gp li l isles ns vrbanacaf morton jt jarmusch ak de souza dpnarayana vk kanojia k nijagal b mcconvillemj knight r howden bp stinear tp microbemetabolite associations linked to therebounding murine gut microbiomepostcolonization with vancomycinresistantenterococcus faecium msystems 5e0045220101128msystems0045220editor manuel liebeke max planck institutefor marine microbiologycopyright mu this is an openaccess distributed under the terms ofthe creative commons attribution international licenseaddress correspondence to andre muandremuunimelbeduaureceived may accepted july published august julyaugust volume issue e0045220msystemsasm 0cmu keywords microbiome multiomics metagenomics metabolomics gutmicrobiome vancomycinresistant enterococci colonization antimicrobial resistanceceftriaxonevancomycinresistant enterococcus faecium vrefm is a significant health careassociated pathogen vrefm infections can be difficult to treat due to their intrinsicand acquired resistance to nearly all classes of antibiotics the world healthanization categorizes vrefm as a œhigh priority bacterial pathogen advocatingresearch to stop the global increase in antibiotic resistance recent studies highlightthe importance of the gut microbiota in modulating the growth and virulence of vrefmin the gastrointestinal ecosystem for instance the depletion of normal gut flora usingantibiotics exacerbates the severity of vrefm infection whereas transplant ofcommensal species including a consortium of clostridium bolteae blautia productablautia sartorii and parabacteroides distasonis can drive established vrefm colonization to below levels of culture detection specifically b producta”a colonizer ofthe colon”reduces vrefm growth in vivo by secreting a lantibiotic these observations raise the intriguing possibility that metabolic traits act in concert betweenpathogen and select gut commensals to confer mutual benefits during pathogenpersistence these findings also highlight the greater risk posed to immunocompromised patients when colonized with vrefm for instance allogeneic hematopoietic celltransplantation patients have gastrointestinal tracts that are dominated by vrefm as aresult of losing a large portion of the intestinal commensal microbiota upon receivingbroadspectrum antibiotics as pretreatment hildebrand discovered longtermecological impacts to the gut microbiome with strong bacterial species turnover afterceftriaxone treatment in humans further mice receiving broadspectrum antibioticscombination of metronidazole neomycin and vancomycin showed markedly increased vrefm colonization of the cecum and colon the compromised intestinal innateimmune defenses in these animals allowed proliferation of vrefm caused by theantibiotic exposure and subsequently reduced the expression of antimicrobial molecules produced by bacteria in the intestinal mucosa the problem with vrefm is further complicated by the fact that enterococci aremembers of the gastrointestinal tract microbiota a key reservoir of antimicrobialresistance amr genes and potentially facilitating gene transfer within the gut microbiome for example the vanb resistance gene was detected in human fecalspecimens that did not contain culturable vre and instead demonstrated that isolatescarrying the resistance transposon are anaerobic commensal bacteria eggerthella lentaand clostridium innocuum colonization of and persistence in the gastrointestinaltract therefore presents as a key mechanism for de novo vre and may lead to severeinvasive diseasethe current study aimed to understand the impact of antibiotics on the murine gutmicrobiota and the subsequent colonization pattern of vrefm to this extent wedesigned a murine model timeseries study that consisted of two main perturbativephases i antibiotic pretreatment with ceftriaxone and ii vrefm challenge our 16srrna gene profiling analyses highlighted a firstorder shift in bacterial biodiversitycomposition across time a secondorder clustering of samples associated with theexperimental phases and the transition of the postvrefm colonization gut microbiotaand its metabolome toward resembling an asymptomatic carriagelike microbiomephenotype this research provides support for engineering the metabolic potential ofthe gut microbiome using for example prebiotics as a nonantibiotic alternative fortreating multidrugresistant bacterial infectionsresultsexperimental design the following experimental design was developed to address the hypothesis that there are specific murine gut microbiome factors thatfacilitate vrefm colonization three groups of three c57bl6 mice cocaged wildtypejulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vretable summary of samples analyzed in this studyday of exptphase of exptannnnnnabxtxabxtxabxwnvreevreevreevreevrelvrelamplicon 16s rrnagene databœ“œ“œ“«º«ºœ“œ“œ“œ“œ“«º«ºœ“œ“œ“metabolomicsbœ“«º«º«º«ºœ“œ“œ“œ“œ“«º«º«º«ºœ“avg no ofobserved sotusc«º«º«º«ºathe key phases of the experiment where n represents naive abxtx represents antibiotic treatment abxwn represents antibiotic weaning vree represents earlyphase postvrefm colonization and vrelrepresents latephase postvrefm colonizationbsymbols œ“ sample processed «º data unavailablecthe average number of sotus observed across all mice for each day of the experimentmales were monitored and fecal samples were collected over a 14day period with twointervention time points including i ceftriaxone treatment administered at 05gliter indrinking water across a 2day period and ii colonization via oral gavage with «» vrefm st796 per mouse postantibiotic treatment at a single time point mice werehoused in groups of five and samples were collected from the same three mice torepresent technical replicates per cage herein each group of cohoused mice will bereferred to as group a group b and group c the remaining two mice per group werereserved for microbiological assays table highlights samples and data sets collectedamplicon 16s rrna gene sequencing revealed firstorder shifts in bacterialcommunity composition amplicon 16s rrna gene sequencing was performed tocapture the bacterial community composition in an effort to track changes in responseto antibiotic pretreatment and vrefm colonization bacterial community profiles wereassessed in fecal samples from nine mice before during and after the two interventionstable a total of of reads reads passed quality control with reads on average per sample and a total of exact variant sequence typesie features with an average of features per sample and an upper bound of features when rarefied to reads alpha rarefaction analysis demonstratedsufficient sequencing depth to capture microbial diversity to saturation see fig s1 inthe supplemental materialthe biodiversity profiles of each sample were compared and showed that keysuboperational taxonomic units sotus were differentially abundant throughout thecourse of the experiment fig there was a shift in the dominance of bacteroidiabacteroidetes light green colored bars during the naive phase of the experiment tomollicutes tenericutes fuschia colored bars in response to ceftriaxone treatment witha return to the predominance of bacteroidia during the late phase of the experimentafter vrefm colonization ie days to of note is the predominance of lactobacillales in mouse to from group a fig the murine gut microbiota responds to antibiotics and microbial communityrichness begins to rebound days after vre colonization principalcoordinateanalysis pcoa of the unweighted unifrac distances was used to assess clusteringof fecal samples based on bacterial composition this assessment showed that the fecalmicrobiota from samples collected from each phase clustered together but were clearlyseparated between phases after exposure to ceftriaxone and challenge with vrefmpostantibiotic treatment fig 2a permutationbased statistical testing demonstratesthe groups are significantly different from one another fig s2 temporal tracking ofjulyaugust volume issue e0045220msystemsasm 0cmu fig biodiversity plot of sotus as relative frequencies at the taxonomic level of class firstorder shifts in microbial communitycomposition as revealed by 16s rrna gene community profiling from a predominance of bacteroidetes to tenericutes and return tobacteroidetes was observed each column displays the relative bacterial community composition in a mouse fecal sample collecteddaily and sorted by the chronology of the experiment ie day of experiment table the columns are further sorted by group iegroup a group b and group c and individual mice within each group mouse mouse and mouse stacked bars are presentedas relative frequencies at the taxonomical level of class however annotations of key taxa are at the phylum level bacteroidetes [green]firmicutes [gray] and tenericutes [fuschia] or order level lactobacillales [yellow]the changing microbiomes against each mouse on the pcoa sample space demonstrated a clear unidirectional trajectory that followed the chronology of the experiment106084m9figshare12775859 procrustes analyses of weighted andunweighted unifrac distances showed that the same general patterns on the samplespace were preserved meaning that there is congruency in global spatial patternsbetween qualitative and quantitative measures of community dissimilarity fig s3analysis of community diversity faith™s phylogenetic diversity index revealed astable and rich microbial community during the naive phase preceding a sharpdecrease following antibiotic treatment and a further decrease immediately followingvrefm colonization fig 2b of note is the responsiveness of the microbiota within h to the removal of antibiotics at the end of day community richness began torebound at approximately days after vrefm colonization ie day with group ademonstrating a higher rate of rebound compared to groups b and c calculating thedistances of dissimilarity unweighted unifrac distances of each mouse microbiotatime point relative to day a proxy for the naive bacterial community phenotyperevealed a small dissimilarity distance for samples collected during the naive phase andan increasing dissimilarity distance following antibiotic treatment day and vrefmcolonization day fig 2c there was a downward trajectory in distance scores daysafter vrefm colonization ie day group a followed a sharper return to a microbiotaresembling day these observations suggest that mice were transitioning toward apersistent carrierlike state and that the rebounding community richness toward levelsrepresentative of the naive phase was by a microbial community structure that resembled the naive phase additional studies where the time frame of postvrefm challengeextends beyond week of monitoring are needed to understand whether the perturbed microbiome will return to resemble an absolute naive state or arrive at a newaltered statemultinomial regression identifies sotus most positively associated with vrefmcolonization multinomial regression using songbird was employed to identify sotusjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig diversity analyses a principalcoordinate analysis plot of unweighted unifrac distances data points areprojected onto the sample space and colored by prevrefm colonization red and postvrefm colonization bluenote that circles and ellipses function to highlight the separation of experimental phases and do not indicatestatistical confidence intervals principal coordinate axis explains of the variation observed between thenaive microbiota and those from the postvrefm colonization phase b community richness of the murine gutmicrobiome as measured by faith™s phylogenetic diversity in response to ceftriaxone treatment and challengewith vrefm c community dissimilarity distances as calculated by unweighted unifrac of each time point relativeto day naive phasethat were most positively and negatively associated with the postvrefm colonizationphase fig the five most positively associated sotus were enterococcus bacteroideserysipelotrichaceae catabacter and lachnospiraceae while the five most negativelyassociated were clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridiales temporal tracking of exact sequence variants esvs demonstrated that theesv feature classified as enterococcus”and identified as the most positively associatedwith the postvrefm colonization phase”was most abundant on days of challengeconfirming that this esv likely was the st796 vrefm colonization challenge anismsfig there were a further eight esv features classified as enterococcus however theywere absent during the days representing vrefm colonization and lacked positiveassociations with the postvrefm colonization phase suggesting that these featuresrepresent murine gut commensal enterococcijulyaugust volume issue e0045220msystemsasm 0cmu fig multinomial regression multinomial regression identified an enterococcus exact sequence variant as the most positivelyassociated with the colonization phase log fold change score of read counts for the enterococcus esv tracked daily acrossthe experiment showing high abundance during the days of vrefm challengemolecular networking identifies differential metabolome profiles duplicatefecal samples from key time points throughout the experiment ie days and were analyzed by datadependent tandem mass spectrometry msms performed on a liquid chromatography quadrupole time of flight lcqtof system tomonitor changes in the murine gut metabolome table polar metabolite analysiswas given preference in an effort to broadly capture primary metabolites that play a keyrole in œmetabolic handoffs that define interspecies interactions analysis of the globalmetabolome profile of each sample was measured based on their overlapping molecules and a pcoa plot using a binary jaccard distance metric through the global naturalproducts social molecular networking gnps platform a separation of metaboliteprofiles along pcoa1 was observed fig 4a metabolomes from the naive andlate vrefm colonization phase tended to cluster together while samples from thepostantibiotic phases including the early vrefm colonization phase clustered togethersupporting pairwise permutational multivariate analysis of variance permanovatesting fig s4 highlights that naive and early vre samples are significantly differentwhile late vre has a lower distance to naive samples compared to antibiotictreatedantibiotic wean and early vre samplesrandom forest analysis of spectrum profiles from lcmsms was used to predictexperimental phase and rank the importance of metabolite association with eachexperimental phase the top metabolite features for each experimental phase arehighlighted in fig 4b unique profiles of metabolite features were observed for eachphase of the experiment importantly the late vrefm colonization phase capturesan unknown metabolite feature with a masstocharge ratio mz of and retention time rt of this metabolite is exclusively present during whatrepresents the transition toward resembling the naive microbiome manual curationoffeature in positiveion mode predicts a molecular formula c5h8n4o3with ±–10 ppm in mass error the major peaks in the msms spectrum for feature are precursor ion [m«¹h]«¹ assumed precursor ion [h2o] product ion [likely c2h2o] [c2h2o«¹h] plus product ionfurther supporting neutral loss of c2h2o given the summation of results the chemicalstructure of feature is likely to contain a nacetylated hydroxyl group peakquantification values indicate its presence during the late phase of vre colonizationfig 4c further manual curation of msms data identified ceftriaxone as feature julyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig metabolomic analyses a emperor plot displaying principalcoordinate analysis of binary jaccard distances of metabolomic profiles samples are colorcoded and the colors represent the naive orange antibiotic treatment red antibiotic weaning blue early vre colonization green and late vrefmcolonization purple phases b random forest classifier identifying metabolite features spectra for each phase of the experiment the heatmap is color codedfrom low ranking score white ie lowest importance to high ranking score dark blue highest importance metabolite features are labeled by theirmasscharge ratios and retention times for the reason that current databases do not capture their chemical structure andor identifications abx tx antibiotictreatment c peak quantification values for feature mz «½ and rt «½ present in abundance during vre colonization late phase dpeak quantification values for ceftriaxone mz «½ and rt «½ tracked across the experiment ceftriaxone values are highest during antibiotictreatment phase and begins to wane during antibiotic weaning phase julyaugust volume issue e0045220msystemsasm 0cmu with an mz of and rt of and mostly abundant during days of antibioticexposure fig 4dbacteroidalesassociated metabolites implicated in latephase postvrefm colonization a distinct profile shift in microbe and metabolite abundances as calculatedby multinomial regression was observed particularly during latephase vrefm colonization fig s6 shallow neural networking analysis with mmvec was used to predictmicrobemetabolite interactions through their cooccurrence probabilities fig sequential biplots captured the shift in experimental phases and highlighted the cooccurrences of microbiota and metabolomic data sets fig 5a to c there was a strongenterococcus effect as indicated by the magnitude of the corresponding arrow and therebounding species during the latephase vrefm colonization are predominantly bacteroidales sotus fig 5c with cooccurring metabolite features mz rt and mz rt metabolite feature mz rt was ranked asbeing highly associated with the postvre colonization phase these results integratemicrobial and metabolite data sets to reveal which microbes may be responsible fordetected metabolites in this instance the metabolite present during the phase representing a transition toward a microbiome approximating the naive state feature mz and rt fig 4b is linked with bacteroidales fig 5adiscussionin this study of the murine gut ecosystem we employed a mouse model ofgastrointestinal tract colonization that replicates the shift in bacterial compositionwhen patients enter the health care system develop an imbalance in their microbiomeas a result of pretreatment eg antibiotic treatment and are subsequently colonizedwith a hospital superbug the resolution of current studies describes a consortiumof commensal microbes that can for example reduce the magnitude of vrefm colonization however understanding the key metabolic shifts relative to the gutmicrobiota remains challenging here we employed amplicon 16s rrna genesequencing and highresolution mass spectrometry metabolomics in an effort towarddetermining microbiotametabolome interactions during vrefm colonization we demonstrated clear changes in the gut microbiome in response to ceftriaxone and vrefmchallengeconceptual and statistical advances in analysis of amplicon 16s rrna gene data whereby otus are clustered at a nucleotide similarity threshold allows for theidentification of exact sequence variants esvs query against an errorcorrecteddatabase can detect multiple esvs that may be classified to the same taxonomicrank for example our analyses identified multiple esvs classified as enterococcihowever when the relative abundances were tracked across the chronology of theexperiment only one enterococcus esv was dominant in relative abundances and mostpositively associated with the days of postvrefm challenge fig this highlights theresolving power to differentiate between commensal and pathogenic strains of enterococci when the composition of the microbial community is considered the factthat this was achievable at the level of amplicon 16s rrna gene sequencing alludes tothe possibility of implementing microbiota screenings as routine diagnostics for patients entering health care systems further firstorder level shifts in microbial community composition was observed in response to ceftriaxone and subsequent vrefmchallenge fig three days after vrefm colonization ie day the microbiomerichness begins to rebound suggesting that mice are transitioning toward a persistentcarrierlike state interestingly the group a cohort exhibited a higher rate of reboundthat may be facilitated by their initially higher microbial community richness andpredominance of lactobacillales on the day of vrefm challenge fig 2b this observation supports the need to prescreen œbaseline microbiota profiles of patients uponadmission into hospital for the reason that it is not necessarily which microbialpopulations are removed postperturbation eg antibiotic pretreatment but insteadwhich populations persist that drives the responding phenotype we can begin toassess patients from across different wards eg intensive care unit oncology neuroljulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig microbemetabolite vector biplots sequential biplots highlighting the changing metabolitedifferentials across each key phase of the experiment abx tx is the antibiotic treatment phase and abxwean is the period when antibiotics were removed for a 24h period prior to colonization with vrefmeach point on the sample space represents metabolites and arrows represent microbes microbe andcontinued on next pagejulyaugust volume issue e0045220msystemsasm 0cmu ogy and healthy cohorts and build a database of microbiome profiles that can be usedas biomarkers to predict i the susceptibility of patients to develop persistent bacterialcolonization and ii propensity to clear the pathogen once colonized the clinicalimplication is that new patients are screened and identified via betadiversity metaanalyses by these biomarkers and placed in bedding cohorts accordingly therebyimproving infectious disease management and isolation precautions within healthcareassociated ecosystemsthe shortlist of microbes ranked as most negatively associated with the colonizationphase clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridialesfig are hypothesized to play a role in maintaining the health of the animals indeedamong the microbes identified are known shortchain fatty acid eg butyric acidproducers which supports and expands upon those previously identified bycaballero further the use of deblur to identify esvs facilitates the temporaltracking of their relative abundances to inform selection of primary fecal samples thatwill provide the best probability ie highest relative abundance of culturing targettaxa for downstream screening of probiotic potential however translating animalderived observations from experimental animal models to human clinical situationsremains challenging particularly where the key microbes are rodentspecific microbesone solution may be to integrate metabolomics to reveal shared metabolic capacityamong taxonomically divergent microbes our supervised classifying approaches suggests an altered metabolome composition during the late phase of vrefm challengethat may facilitate the apparent œsuppression of vrefm to levels below the limit ofdetection by culture despite the caveat of poor resolution in current databases to linkmetabolite features to associated chemical structures microbemetabolite vector analysis linked metabolite feature mz «½ and rt «½ to bacteroidesfig our efforts toward manually identifying feature suggests a chemicalformula of c5h8n4o3 and a structure likely to contain a nacetylated hydroxyl group aputative annotation through pubchem search is 3hydroxy4nitrosocyanamido butyramide butyramide is the amide group of butyric acid a shortchain fatty acid thathas been shown to play a key role in colonization resistance against intestinal pathogens “ further research to comprehensively characterize interactions betweenmicrobe and metabolites will be critical to address the gaps in our understanding of thebiochemical parameters that define interspecies microbiome interactions during antibiotic pretreatment and persistent infectionsthe resolution of our results provides the basis in which to begin to identifynonantibiotic alternatives to engineer the gut microbiome through prebiotic interventions eg butyric acid and translating animal studies to humanrelevant therapeuticapplications by delineating taxonomically diverse microbes with shared metaboliccapacity here achieving integrative omics to link microbemetabolite associations ourfindings add support to the incorporation of microbiome profiling approaches intoroutine clinical microbiology particularly in the context of monitoring the impacts ofantibiotic usematerials and methodsmouse gastrointestinal colonization model sixweekold wildtype c57bl6 male mice were usedto establish an animal model of gastrointestinal colonization with vrefm mice were cohoused and hadfree access to food ordinary chow and water and had environmental enrichment eg fun tunnels chewblocks and tissue paper the lightdark cycle was 12h light12h dark and cages were changed weeklyfig legend continuedmetabolite features are fixed upon the sample space with gradient coloring of metabolites indicating thetransition across key phases of the experiment the distance between each point is indicative ofmetabolite cooccurrence frequency and the angle between arrows indicates microbial cooccurrencethe directionality of the arrows describes the variation in the metabolites explained by the microbesrepresented by the arrows for example metabolite feature mz and rt isdemonstrated to cooccur with bacteroides information about the abundances of these cooccurringfeatures are provided as heatmaps in fig s6 in the supplemental materialjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vremice were pretreated with gliter ceftriaxone in drinking water for days followed by an antibioticwean period of h mice were then challenged with «» cfu vrefm st796genomic dna extraction and sequencing wholecommunity genomic dna gdna was extractedfrom mouse fecal samples using the qiagen powersoil dna extraction kit formerly mobio following themanufacturer™s protocol a preprocessing step of mechanicallysis was incorporated using a bertintechnologies precellysis machine for one round of a 40s cycle at rpm the v4 region of thebacterial 16s rrna gene was amplified using small subunit forward golaybarcoded and ssu806reverse primers following the earth microbiome project protocol and sequenced using the illuminamiseq platform v2 cycles illumina inc san diego ca usa further primary derived data egbiom tables used to produce results can be found within qiita study id amplicon 16s rrna gene profiling analyses sequence data were processed within the qiitav010 framework for quality control split libraries v q2191 demultiplexing trimming sequencereads to a length of nucleotides nt and picking suboperational taxonomic units sotus usingdeblur v110 to resolve singlenucleotide community sequencing patterns ie feature identification ofsotus [] the output biom files were further processed using qiime2 v20197 for downstreamstatistical analyses alpha rarefaction curves were generated to determine whether each sample hadbeen sequenced to saturation the feature table was subsequently rarefied to reads per sampletaxonomy was assigned using the sklearn classifier and greengenes otus from 515f806rregion of sequences classifier available from docsqiime220184dataresources furthermorerelative abundances of each taxa were visualized as bar plots using the qiime2 taxa plugin a phylogenetic tree was constructed using fragment insertion qiime fragmentinsertion sepp [] to guidephylogeneticaware statistical analyses generated using the qiime2 plugin q2diversity coremetricsphylogenetic key metrics computed by default include both alphadiversity eg shannon™s diversityindex faith™s phylogenetic diversity and evenness and betadiversity eg braycurtis distance andunweighted unifrac distance metrics the unweighted unifrac distance matrix was used tocompute first distances and calculate distances relative to day as the baseline between sequentialstates qiime longitudinal firstdistances ggplot2 r v360 ggplot2tidyverse was used tovisualize the distance scores as line plots emperor was used to visualize principalcoordinate analysisplots of unweighted unifrac distances permutationbased statistical testing permanova on unweighted unifrac distances was used to determine whether samples grouped by phase of experimentwere significantly different from one another q2betagroupsignificance songbird githubcommortonjtsongbird was employed to determine the importance ie fold change of each sotu inrelation to a given metadata variable eg vrefm colonization microbial features from all samples weresplit into training and test sets for supervised learning classifier analyses of input samples wereallocated to train the random forest classifier within qiime2 the estimator method used for sampleprediction the different experimental phases were the response variables while the 16s rrna gene datawere the featuresmetabolite extraction and liquid chromatographytandem mass spectrometry analysis duplicate fecal samples as outlined in table were processed for polar metabolite extraction and analysisdays and feces were metabolically arrested by immediate collection into dry ice andstored at “ °c until further processing metabolite extraction from the fecal samples was undertaken bythe addition of \u242el per sample of methanolwater solution [volvol] containing \u242em [13c]sorbitoland \u242em [13c15n]valine and \u242em [13c]leucine as internal standards fecal samples were homogenizedat rpm for min in a thermomixer maintained at °c mechanically disrupted and incubated fora further min in the thermomixer samples were randomized for metabolite extractionmetabolite analysis of the extracted samples pooled biological quality control pbqc samples and mixtures of authentic standard mixes was performed by liquid chromatographymass spectrometrylcms using hydrophilic interaction column zicphilic and highresolution agilent seriesquadrupole time of flight mass spectrometry qtof ms as described previously pcoa of binaryjaccard distances of test standard mixes and pbqc samples are presented in fig s5 in the supplementalmaterial ions were analyzed in positive mode with full scan range of to mz and in datadependent tandem ms mode to facilitate downstream metabolite identificationmetabolomic analyses Answer:

Colon_Cancer

" chronic colorectal inflammation has been asso‘ciated with colorectal cancer crc however its exact molecular mechanisms remain unclear the present study aimed to investigate the effect of toll‘like receptor tlr9 on the development of colitis‘associated crc cac through its regulation of the nf‘κb signaling pathway by using a cac mouse model and immunohistochemistry the present study discovered that the protein expression levels of tlr9 were gradually upregulated during the development of crc in addition the expression levels of tlr9 were revealed to be positively correlated with nf‘κb and ki67 expression levels in vitro inhibiting tlr9 expression levels using chloroquine decreased the cell viability proliferation and migration of the crc cell line ht29 and further experiments indicated that this may occur through downregulating the expression levels of nf‘κb proliferating cell nuclear antigen and bcl‘xl in the findings of the present study suggested that tlr9 may serve an important role in the development of cac by regulating nf‘κb signalingcorrespondence to professor youxiang chen or dr chunyan zeng department of gastroenterology the first affiliated hospital of nanchang university yongwaizheng street nanchang jiangxi pr chinae‘mail chenyx102126com e‘mail zcy896163com abbreviations aif acute inflammation aom azoxymethane cac chronic inflammation crc colorectal cancer dai disease activity index dss dextran sodium sulfate ibd inflammatory bowel disease iecs immunohistochemistry pcna proliferating cell nuclear antigen tlr9 toll‘like receptor uc ulcerative colitiskey words colitis‘associated crc aom dss tlr9 nf‘κbintestinal epithelial cells ihc colitis‘associated colorectal cancer cif introductionthe number of patients with colorectal cancer crc world‘wide is increasing annually with an incidence rate of in chronic inflammation is the leading cause of immune cell infiltration and proliferation and it has been suggested to be a high‘risk factor for colitis‘associated crc cac inflammatory bowel disease ibd which encompasses both ulcerative colitis uc and crohn's disease was established as an important precursor to crc for example the incidence of ibd‘associated crc in patients with uc was reported to have a cumulative risk rate of at years at years and at years of disease duration therefore further in vivo studies are required for researchers to gain an improved understanding of the molecular mechanisms of cac which may provide more exact molecular targets for the diagnosis and treatment of cac during the early stages toll‘like receptor tlr9 a member of the tlr family is located in the cytoplasm and intracellular endosomes and can be activated by unmethylated bacterial cpg dna the activation of the tlr9 signaling pathway induces a type t helper cell immune response and stimulates the proliferation of b cells thus protecting the host from external microbial invasion ‘ multiple studies have revealed that abnormal tlr9 expression levels were involved in the pathogenesis and progression of uc in addition abnormal expression levels of tlr9 were also identified during the tumorigenesis and development of crc ‘nf‘κb is an important transcription factor involved in various biological processes including inflammatory reac‘tions immune responses apoptosis and proliferation in fact nf‘κb is regarded as a molecular hub that links inflam‘mation and cancer it was previously suggested that nf‘κb may serve an important role in colorectal carcinogenesis by regulating matrix metalloproteinase‘ expression ‘ previous studies have revealed that tlr9 was related to the biological characteristics of various types of cancer including bladder lung and prostate cancer such as cell proliferation invasion tumor growth and progression ‘ in fact one previous study reported that tlr9 regulated the expression levels of interleukin il‘ through the myeloid differentiation primary response protein myd88 myd88nf‘κb signaling 0cluo tlr9 promotes colorectal carcinogenesispathway in myeloid cells to promote tumor recurrence after irradiation including in melanoma bladder carcinoma and colorectal carcinoma the current study aimed to investigate the effect of tlr9 on the development of cac through its regulation of the nf‘κb signaling pathway owing to the synergistic effects of azoxy‘methane aom a tumor‘inducing agent and dextran sodium sulfate dss a tumor‘promoting agent the present study established cac model mice by co‘administering aom and dss to analyze the expression levels of tlr9 nf‘κb and ki67 in cac tissuesmaterials and methodsreagents and antibodies aom cat no a5486 and chloro‘quine tlr9 inhibitor cat no c6628‘25g were obtained from sigma‘aldrich merck kgaa dss cat no ‘100g was purchased from mp biomedicals llc anti‘tlr9 cat no ab134368 and anti‘myd88 cat no ab135693 primary antibodies were obtained from abcam anti‘nf‘κb nf‘κb p65 cat no 8242s anti‘bcl‘xl cat no and anti‘ki67 cat no 9449s primary antibodies were obtained from cell signaling technology inc the anti‘proliferating cell nuclear antigen pcna cat no sc‘ primary anti‘body was obtained from santa cruz biotechnology inc and the anti‘gapdh cat no ta309157 primary antibody was obtained from origene technologies inc horseradish peroxidase secondary goat anti‘rabbit cat no zb‘ and goat anti‘mouse cat no zb‘ antibodies used for western blotting and goat anti‘mouserabbit antibodies cat no ta130001ta130015 used for immunohistochem‘istry ihc were obtained from origene technologies inccell lines and culture the human crc cell line ht29 was obtained from the american type culture collection and cultured in mccoy's 5a modified medium gibco thermo fisher scientific inc supplemented with fbs gibco thermo fisher scientific inc mgml penicillin and mgml streptomycin maintained in a humidified atmo‘sphere of co2 at ˚cwestern blotting cells were lysed in ripa lysis buffer beijing solarbio science technology co ltd at ˚c for min the protein concentration was measured following centrifugation at x g for min at ˚c and quantified using a bicinchoninic acid protein assay kit beijing solarbio science technology co ltd an equal amount of protein µglane was separated via sds‘page and electrophoretically transferred onto polyvinylidene difluo‘ride membranes emd millipore the membranes were blocked with skimmed milk for h at room temperature and were subsequently incubated overnight at ˚c with the following primary antibodies anti‘tlr9 anti‘nf‘κb anti‘bcl‘xl anti‘pcna anti‘myd88 and anti‘gapdh following the primary antibody incubation the membranes were washed times with pbs for min each and incubated with the corresponding goat anti‘rabbit or goat anti‘mouse secondary antibodies at ˚c for h protein bands were visualized using an ecl reagent thermo fisher scientific inc on a gel doc xr system bio‘rad laboratories inc and analyzed using image lab version software bio‘rad laboratories incwound healing assay a wound healing assay was performed to analyze the cell migratory ability briefly ht29 cells 3x105well were seeded into six‘well plates and cultured to ‘ confluence subsequently a ‘µl pipette tip was used to scratch a wound in the cell monolayer fresh serum‘free mccoy's 5a modified medium containing different concen‘trations of chloroquine or µgml was added to each well and cultured for h in a humidified atmosphere of co2 at ˚c images of each well were captured at and h using a light phase contrast microscope olympus zkx‘ olympus corporation with a magnification of x100 the wound‘healing areas were assessed using imagej 152a software national institutes of health the migratory rate of cells wound area at h‘wound area at harea at hcolony formation assay ht29 cells 12x105well were cultured in ‘well plates for h in medium containing different concentrations of chloroquine or µgml at ˚c and then seeded into ‘cm cell culture dishes cellsdish and incubated in complete medium at ˚c after days the cells were fixed with paraformaldehyde for min and stained with crystal violet for min both at room temperature the number of colonies defined as cellscolony was counted manually using a light microscope with a magnifica‘tion of x100 relative colony number number of colonies in observed groupnumber of colonies in control group all of the experiments were repeated ‰¥ timescell viability assay the viability of ht29 cells was analyzed using an mtt assay briefly ht29 cells were seeded at a density of cellswell in a ‘well plate and treated for or h with chloroquine or µgml at ˚c and then analyzed using an mtt assay as previously described animal studies all animal experiments were approved by the ethics committee of the first affiliated hospital of nanchang university nanchang china a total of female balbc mice weight ‘ g age ‘ weeks were obtained from beijing vital river laboratory animal technology co ltd laboratory animal license no scxk ‘ the animals were maintained with a normal diet and tap water ad libitum at a temperature of ±Ëšc and a relative humidity of ‘ and artificially illuminated on an approximate h lightdark cycle at the animal care facility in the medical college of nanchang university all of the mice experiments were approved by the animal care and use committee of nanchang university the total experiment lasted for weeks the mice were divided into four groups micegroup i group a aom dss which was intraperitoneally injected without anesthesia with mgkg aom once on the first day followed by dss given in the drinking water for week and then weeks of distilled water one cycle which was repeated for two additional cycles ii group b aom which was intraperitoneally injected with mgkg aom once on the first day and provided with distilled drinking water during weeks ‘ iii group c dss which was treated with dss 0concology letters as described for group a but without the aom treatment and iv group d blank control which received neither dss nor aom treatment and was provided with distilled drinking water for the first weeks all the mice were provided with a normal diet and tap water during weeks ‘ fig 1athe disease activity index dai was evaluated at the end of the experiment using the numerical system described by tian the dai parameters included total body weight loss none ‘ ‘ ‘ stool consistency well‘formed pellets loose stool diarrhoea and the presence of fecal occult blood negative positive gross bleedingseveral mice were randomly sacrificed at certain times points following aom injection the 1st 2nd 3rd 6th 9th 12th 18th and 23rd weeks six mice were sacrificed at weeks and one mouse was sacrificed at week and four mice were sacrificed at weeks and in each group additionally six mice in group d were randomly sacrificed at week as control all remaining mice were sacrificed at week after the large bowels were resected and washed with pbs they were carefully examined photographed and fixed in formalin at room temperature for h further histological examinations were subsequently performedhistopathological analysis and immunohistochemistry ihc paraffin‘embedded colorectal sections ‘µm‘thick were stained with hematoxylin for min and eosin for min at room temperature to analyze the degree of inflammation using a light microscope with magnifications of x40 and x100 briefly the severity of inflammation the thickness of inflammation the severity of epithelial damage and the extent of the lesions were each graded from to by two investigators who were blinded to the treatment groups as previously described the severity of inflammation was adapted from the grading system developed by truelove and richards as follows i grade no neutrophil infiltration in the lamina propria ii grade i infiltration of a small number of neutrophils [ cellshigh power field hpf] in the lamina propria involving a few crypts iii grade ii obvious neutrophil infiltration in the lamina propria ‘ cellshpf involving of the crypts iv grade iii infiltration of neutrophils cellshpf in the lamina propria with crypt abscess and v grade iv obvious acute inflammation in the lamina propria with ulcer formation grade i was classi‘fied as mild grade ii was classified as moderate and grades iii and iv were classified as severe the severity of inflammation ranged from to no inflammation mild moderate severe the thickness of inflammation ranged from to no inflammation mucosa mucosa plus submucosa transmural the severity of epithelial damage ranged from to intact epithelium disruption of architectural structure erosion ulceration and the extent of lesions ranged from to no lesions punctuate multifocal diffuseihc was performed as described in our previous study colorectal tissues were fixed in formalin for h at room temperature formalin‘fixed and paraffin‘embedded tissue blocks were cut into ‘µm‘thick sections and mounted on glass slides slides were heated in an oven at ˚c for min and deparaffined in xylene twice for min each at room tempera‘ture rehydrated in a descending ethanol series and ethanol for min each at room temperature and incubated in h2o2 for min at room temperature to block endogenous peroxidase antigen retrieval was performed by heating in a microwave at ˚c in sodium citrate buffer mm ph for min slides were blocked with bovine serum albumin beijing solarbio science technology co ltd for h at room temperature to block non‘specific antibody binding and incubated overnight at ˚c with the following primary anti‘bodies anti‘tlr9 anti‘nf‘κb and anti‘ki67 following the primary antibody incubation the sections were washed three times with pbs and incubated with horse‘radish peroxidase secondary goat anti‘mouserabbit antibodies ready to use at ˚c for min the sections were stained with '‘diaminobenzidine at room temperature the duration of staining was based on the staining observed under a light microscope with a magnification of x100 and the reaction was terminated when the staining was yellowish‘brown the sections were then counterstained with hematoxylin for min at room temperature the slides were observed under a light microscope with a magnification of x100 the widely accepted german semi‘quantitative scoring system was used to determine the staining intensity and area of staining according to the recommendations of remmele and stegner each specimen was assigned a score according to the intensity of the nucleic cytoplasmic andor membrane staining no staining not detected0 weak staining light yellow1 moderate staining yellowish brown2 strong staining brown3 and the extent of stained cells no staining ‘ ‘ ‘ ‘ the final immunoreactive score was determined by multiplying the intensity score with the extent of stained cells score ranging from the minimum to the maximumco‘immunoprecipitation assay ht29 cells were lysed in ripa lysis buffer beijin solarbio science technology co ltd at ˚c for min whole‘cell lysates were pelleted via centrifugation at x g for min at ˚c the super‘natant was incubated with an anti‘tlr9 antibody or goat anti‘mouse igg cat no zb‘ origene technologies inc together with protein ag plus‘agarose beads santa cruz biotechnology at ˚c overnight the beads were washed three times with the non‘lubrol lysis buffer at x g centrifugation for min at ˚c then subjected to sds‘page and subsequent western blotting analysis as aforementioned whole cell lysate was used as a controlstatistical analysis statistical analysis was performed using spss software ibm corp the data are presented as the mean ± sd all experiments were performed at least in triplicate a one‘way anova followed by a tukey's post hoc test was used to determine the statistical differences between groups whereas an unpaired student's t‘test were used to determine the statistical differences between groups a spearman's rank correlation test was used to determine the correlation between the expression levels of tlr9 nf‘κb and ki67 in crc tissues p005 was considered to indicate a statistically significant differenceresultsconstruction of the cac model mice in groups a aom dss and c dss the body weights of the mice decreased with 0cluo tlr9 promotes colorectal carcinogenesisfigure construction of the colitis‘associated‘colorectal cancer model in mice a schematic diagram of the experimental protocol for colitis‘associated colorectal cancer model mice in groups a‘d arrow indicates mgkg azoxymethane administration via intraperitoneal injection black square indicates administration of dextran sodium sulfate in drinking water for week continuous line indicates duration fed a normal diet and distilled water dotted line indicates duration fed a normal diet and tap water and triangle indicates timepoint of sacrifice b body weights of the mice in each group p0001 c disease activity index of groups a and c data are presented as the mean ± sd group a mgkg azoxymethane and dextran sodium sulfate water group b mgkg azoxymethane group c dextran sodium sulfate water group d blank controltime compared with group d blank control group fig 1b the average weight of the mice in group c declined from ± g at the beginning of the experiment to ± g by week while the average weight of the mice in group a decreased from ± g at the beginning of the experiment to ± g by week the differences between groups a and d and groups c and d were statistically significant at week p005 the dai which reflects the severity of colitis was also markedly increased in groups a and c after dss administration on the 1st 3rd and the 6th week fig 1c no weight loss or any signs of inflammation such as loose stool or diarrhea positive fecal occult blood or gross bleeding were observed in groups b aom and d blank control the dai of groups b and d was zero throughout the modeling process and is therefore not shown in fig 1c the lengths of the large bowels in groups a and c were decreased compared with groups b and d fig 2a notably the lengths of the large bowels were significantly decreased in groups a and c from week compared with the control group mice sacrificed in group d at week or with group dp005 fig 2b however the severity of inflamma‘tion was significantly increased in group a compared with in group c at week and the extent of inflammation was signifi‘cantly increased in group a compared with in group c after week p005 while there was no significant differences in the thickness of inflammation and the severity of epithelial damage between groups a and c fig 2cby the 12th week colorectal tumors were only observed in the mice of group a fig 2d pathological results further revealed that the mice in group a presented with acute inflam‘mation aif chronic inflammation cif adenoma and adenocarcinoma of the colorectum at weeks and respectively fig 2d and eexpression levels of tlr9 and nf‘κb are simultaneously upregulated during the development of chronic colitis in crc ihc staining revealed that tlr9 was located in the cytoplasm of the intestinal epithelial cells iecs and inflammatory cells in the lamina propria fig 3a tlr9 expression levels were significantly upregulated in aif cif adenoma and adenocarcinoma tissues compared with the corresponding tissues from control mice group d p00334 p00379 p00437 and p00008 respectively fig 3b furthermore the protein expression levels of tlr9 was significantly upregulated in the adenocarcinoma tissue compared with the aif p00077 cif p00278 and adenoma p00273 tissue fig 3bthe positive nf‘κb region was mainly confined to the cytoplasm of the iecs and inflammatory cells fig 3a the ihc results revealed that the expression levels of nf‘κb were significantly upregulated in the aif cif adenoma and adenocarcinoma tissues compared with the corresponding tissues from control mice p00061 p00043 p00019 and 0concology letters figure colorectal pathological results in mice a length of the large bowels of mice in each group at week representative bowels from ‘ independent animals are shown b mean length of large bowel of mice in each group p005 p001 p0001 vs control mice sacrificed in group d at week or group d bottom graph c histological score of severity thickness damage and extent of lesions of the colorectums from mice in groups a and c p005 d large bowels were retrieved at week and from mice in group a to determine the numbers of tumors formed p005 p001 e histological analysis of various pathological changes in the colons of mice in group a were determined using hematoxylin and eosin staining the control groups represents tissues taken at week from group d scale bars ‘µm upper and ‘µm lower inflammatory cells green arrows and intestinal epithelial cells red arrows are indicated group a mgkg azoxymethane and dextran sodium sulfate water group c dextran sodium sulfate water aif acute inflammation cif chronic inflammationp00001 respectively fig 3d moreover nf‘κb expression levels were significantly upregulated in the adenocarcinoma tissue compared with the aif p00001 and adenoma p00161 tissues fig 3dki67 expression levels were observed in the nuclei of iecs and inflammatory cells fig 3a the ihc results revealed that the ki67 expression levels were gradually upregulated across the intestinal lesions including in the aif cif adenoma and 0cluo tlr9 promotes colorectal carcinogenesisfigure tlr9 and nf‘κb are simultaneously upregulated as cac develops a ihc was used to analyze the expression levels and localization of tlr9 nf‘κb and ki67 in colorectal sections obtained from mice in group a inflammatory cells green arrows and intestinal epithelial cells red arrows are indicated ihc staining scores of b tlr9 c ki67 and d nf‘κb in sections of normal control group d aif cif adenoma and adenocarcinoma tissues which were obtained as described in the methods section scale bar ‘µm p005 p001 and p0001 vs control p005 p001 and p0001 vs adenocarcinoma correlation analysis of e tlr9 and nf‘κb expression levels f ki67 and tlr9 expression levels and g nf‘κb and ki67 expression levels was conducted using spearman's rank correlation group a mgkg azoxymethane and dextran sodium sulfate water aif acute inflammation cif chronic inflammation tlr9 toll‘like receptor ihc immunohistochemistryadenocarcinoma tissues p00331 p00092 p00241 and p00006 respectively compared with the expression levels in the corresponding tissues from the control mice fig 3c interestingly the expression levels of tlr9 and nf‘κb were discovered to be significantly positively correlated with other rho08236 p00001 fig 3e in addition a significant positive correlation was also identified between tlr9 and ki67 expres‘sion levels rho05515 p0001 fig 3f and between nf‘κb and ki67 expression levels rho05103 p001 fig 3gdownregulated tlr9 expression levels reduces the migration viability and colony formation of ht29 cells to further investigate the role of tlr9 in crc the human crc cell line ht29 was treated with chloroquine an inhibitor of tlr9 in vitro the results revealed that suppressing tlr9 with chloroquine at both doses inhibited the migration viability and colony formation ability of ht29 cells in a dose‘dependent manner fig 4a‘e additionally lysates were collected from ht29 cells treated with either different concentrations of chloro‘quine for h or µgml chloroquine for different time periods fig 4f‘i and western blotting was performed the analysis revealed that the expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl were gradually downregulated in ht29 cells treated with increasing doses of chloroquine compared with the 0concology letters figure chloroquine inhibits the migration viability and colony formation of ht29 cells by inhibiting tlr9 chloroquine and µgml inhibited the migration of ht29 cells in a dose‘dependent manner at h compared with the control group which was determined using a wound healing assay a representative images were photographed magnification x100 and b migration rates were calculated proliferation rate of ht29 cells was reduced by chloroquine treatment and µgml which was determined using a colony formation assay c representative images were photographed and d the relative colony number was analyzed p005 p001 p0001 e viability of ht29 cells was decreased by chloroquine and µgml at and h compared with the control cells as determined using an mtt assay f expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl in lysates obtained from ht29 cells treated with numerous concentrations of chloroquine or µgml for h were analyzed using western blotting g semi‘quantification of the expression levels of the proteins presented in part f was performed using imagej software gapdh was used as the loading control and for normalization h expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl in lysates obtained from ht29 cells treated with µgml chloroquine for numerous durations or h were analyzed using western blotting i semi‘quantification of the expression levels of the proteins presented in part h was performed using imagej software gapdh was used as the loading control and for normalization j tlr9 interacted with the nf‘κb protein in ht29 cells which was determined using a co‘immunoprecipitation assay goat anti‘mouse igg antibody was used as the negative control all data are expressed as the mean ± sd of three independent experiments p005 p001 p0001 vs control0 h tlr9 toll‘like receptor pcna proliferating cell nuclear antigen myd88 myeloid differentiation primary response protein myd88 ip immunoprecipitated ib immunoblotting 0cluo tlr9 promotes colorectal carcinogenesiscontrol group fig 4f and g a similar trend was observed in the expression levels of these proteins as the duration of chlo‘roquine treatment increased fig 4h and i thus these results indicated that the expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl in ht29 cells treated with chloroquine may be downregulated in both a dose‘ and time‘dependent manner fig 4f‘i to verify whether tlr9 affected the colorectal carcinogenesis by interacting with nf‘κb co‘immunoprecipi‘tation assay was used to detect the interaction between tlr9 and nf‘κb in ht‘ cells the results revealed that there was an interaction between tlr9 and nf‘κb fig 4jdiscussioncolorectal carcinogenesis is a multi‘step process starting from normal crypts to aberrant crypt foci then to polyps adenoma and eventually adenocarcinoma it has been reported that individuals with ibd may have an increased risk of developing crc which is directly proportional to the extent and duration of their disease however the exact mechanism and duration required for chronic colitis to develop into adenoma and then adenocarcinoma remains unclear it has been suggested that patients with ibd have an increased risk of crc following the inflammation‘dysplasia‘carcinoma model including dysplasia and crc as primary conse‘quences of chronic inflammation however there are currently still no defined molecular biomarkers or existing monitoring protocols for detecting the occurrence of a malignant tumor except for frequent colonoscopy examinationsin the present study an acute colitis‘chronic colitis‘adenoma‘adenocarcinoma model was successfully constructed via aomdss induction using this model tlr9 expression levels were discovered to be upregulated as the severity of the colorectal lesions increased which indicated that tlr9 protein expression levels may be continuously activated during colitis‘crc development tlr9 is a critical protein associated with innate and acquired immunity and it has been demonstrated to serve a significant role in the development of colitis and sporadic crc however the mechanism by which tlr9 regulates the devel‘opment of crc remains to be elucidatedinterestingly ihc analysis revealed that the expression levels of nf‘κb and ki67 were simultaneously upregulated alongside tlr9 expression levels notably inhibiting tlr9 decreased the migration proliferation and viability of ht29 cells in vitro and tlr9 expression levels in vivo were identi‘fied to be significantly positively correlated with the expression levels of nf‘κb and ki67 a cell proliferation marker during the transition from colitis to crc the present study further revealed that the inhibition of tlr9 in vitro significantly downregulated the expression levels of nf‘κb myd88 pcna and the anti‘apoptotic protein bcl‘xl in a dose‘ and time‘dependent manner notably a previous study reported that tlr9 promoted the tumor‘propagating potential of prostate cancer cells via nf‘κb signaling thus the findings of the present study indicated that tlr9 may promote cac through nf‘κb signaling however these findings may be controver‘sial because other previous studies have revealed that tlr9 agonists exerted an antitumor effect in crc the majority of these studies primarily focused on the role of tlr9 in colitis or sporadic crc whereas the current study focused on the role of tlr9 in the pathogenesis of cac to provide novel targets for the treatment of cac for example altera‘tions in microtubule end‘binding protein were identified as a characteristic of sporadic but not uc‘associated crc and in another previous study the immune profiling patterns of patients with cac were significantly different compared with the patients with sporadic crc chloroquine a non‘specific inhibitor of tlr9 is an old antimalarial drug which has recently attracted significant interest for its potential antitumor properties for example numerous studies have reported that chloroquine directly regu‘lated cancer cells by inducing apoptosis inhibiting autophagy interacting with nucleotides eliminating cancer stem cells and enhancing the growth of cancer cells ‘ chloroquine also inhibited the expression levels of tlr9 by preventing the acidi‘fication and maturation of the endosomes and the trafficking of tlrs due to the multiple effects of chloroquine on tumor cells different concentrations of chloroquine were selected for use in the present study based on the lowest dose according to previous studies in order to obtain the best possible results with low toxicity to the cells in the future investiga‘tions using small interfering rna targeting tlr9 should be performed to determine the effect on the biological processes of crc cell lines to further verify the findings of the present study thus our future studies will focus on investigating the precise molecular mechanism by which tlr9 participates in the early occurrence of colorectal carcinogenesisin the present study developed a cac animal model the findings indicated that tlr9 may be closely asso‘ciated with the process of inflammation‘dysplasia‘carcinoma and may impact carcinogenesis by regulating the nf‘κb signaling pathway these results may provide promising potential to be developed into an early detection protocol or therapeutic molecular target for crcacknowledgementsnot applicablefundingthe present study was supported by grants from the national natural science foundation of china grant nos and the foundation of jiangxi educational committee grant no gjj170016 and the graduate student innovation funding program of nanchang university grant no cx2019119availability of data and materialsall data generated or analyzed during this study are included in this published article the original data are available from the corresponding author on reasonable requestauthors' contributionscz and ql designed the study and drafted the manuscript ql and lz performed the experiments ql ct and zz analyzed the data ql cz and yc revised the manuscript for important 0concology letters intellectual content cz and yc made substantial contribu‘tions to conception design and coordination of the study and gave final approval of the version to be published all authors have read and approved the final manuscriptethics approval and consent to participateall animal experiments were approved by the ethics committee of the first affiliated hospital of nanchang university nanchang china approval no ‘ pa

cancer7541

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " chronic colorectal inflammation has been asso‘ciated with colorectal cancer crc however its exact molecular mechanisms remain unclear the present study aimed to investigate the effect of toll‘like receptor tlr9 on the development of colitis‘associated crc cac through its regulation of the nf‘κb signaling pathway by using a cac mouse model and immunohistochemistry the present study discovered that the protein expression levels of tlr9 were gradually upregulated during the development of crc in addition the expression levels of tlr9 were revealed to be positively correlated with nf‘κb and ki67 expression levels in vitro inhibiting tlr9 expression levels using chloroquine decreased the cell viability proliferation and migration of the crc cell line ht29 and further experiments indicated that this may occur through downregulating the expression levels of nf‘κb proliferating cell nuclear antigen and bcl‘xl in the findings of the present study suggested that tlr9 may serve an important role in the development of cac by regulating nf‘κb signalingcorrespondence to professor youxiang chen or dr chunyan zeng department of gastroenterology the first affiliated hospital of nanchang university yongwaizheng street nanchang jiangxi pr chinae‘mail chenyx102126com e‘mail zcy896163com abbreviations aif acute inflammation aom azoxymethane cac chronic inflammation crc colorectal cancer dai disease activity index dss dextran sodium sulfate ibd inflammatory bowel disease iecs immunohistochemistry pcna proliferating cell nuclear antigen tlr9 toll‘like receptor uc ulcerative colitiskey words colitis‘associated crc aom dss tlr9 nf‘κbintestinal epithelial cells ihc colitis‘associated colorectal cancer cif introductionthe number of patients with colorectal cancer crc world‘wide is increasing annually with an incidence rate of in chronic inflammation is the leading cause of immune cell infiltration and proliferation and it has been suggested to be a high‘risk factor for colitis‘associated crc cac inflammatory bowel disease ibd which encompasses both ulcerative colitis uc and crohn's disease was established as an important precursor to crc for example the incidence of ibd‘associated crc in patients with uc was reported to have a cumulative risk rate of at years at years and at years of disease duration therefore further in vivo studies are required for researchers to gain an improved understanding of the molecular mechanisms of cac which may provide more exact molecular targets for the diagnosis and treatment of cac during the early stages toll‘like receptor tlr9 a member of the tlr family is located in the cytoplasm and intracellular endosomes and can be activated by unmethylated bacterial cpg dna the activation of the tlr9 signaling pathway induces a type t helper cell immune response and stimulates the proliferation of b cells thus protecting the host from external microbial invasion ‘ multiple studies have revealed that abnormal tlr9 expression levels were involved in the pathogenesis and progression of uc in addition abnormal expression levels of tlr9 were also identified during the tumorigenesis and development of crc ‘nf‘κb is an important transcription factor involved in various biological processes including inflammatory reac‘tions immune responses apoptosis and proliferation in fact nf‘κb is regarded as a molecular hub that links inflam‘mation and cancer it was previously suggested that nf‘κb may serve an important role in colorectal carcinogenesis by regulating matrix metalloproteinase‘ expression ‘ previous studies have revealed that tlr9 was related to the biological characteristics of various types of cancer including bladder lung and prostate cancer such as cell proliferation invasion tumor growth and progression ‘ in fact one previous study reported that tlr9 regulated the expression levels of interleukin il‘ through the myeloid differentiation primary response protein myd88 myd88nf‘κb signaling 0cluo tlr9 promotes colorectal carcinogenesispathway in myeloid cells to promote tumor recurrence after irradiation including in melanoma bladder carcinoma and colorectal carcinoma the current study aimed to investigate the effect of tlr9 on the development of cac through its regulation of the nf‘κb signaling pathway owing to the synergistic effects of azoxy‘methane aom a tumor‘inducing agent and dextran sodium sulfate dss a tumor‘promoting agent the present study established cac model mice by co‘administering aom and dss to analyze the expression levels of tlr9 nf‘κb and ki67 in cac tissuesmaterials and methodsreagents and antibodies aom cat no a5486 and chloro‘quine tlr9 inhibitor cat no c6628‘25g were obtained from sigma‘aldrich merck kgaa dss cat no ‘100g was purchased from mp biomedicals llc anti‘tlr9 cat no ab134368 and anti‘myd88 cat no ab135693 primary antibodies were obtained from abcam anti‘nf‘κb nf‘κb p65 cat no 8242s anti‘bcl‘xl cat no and anti‘ki67 cat no 9449s primary antibodies were obtained from cell signaling technology inc the anti‘proliferating cell nuclear antigen pcna cat no sc‘ primary anti‘body was obtained from santa cruz biotechnology inc and the anti‘gapdh cat no ta309157 primary antibody was obtained from origene technologies inc horseradish peroxidase secondary goat anti‘rabbit cat no zb‘ and goat anti‘mouse cat no zb‘ antibodies used for western blotting and goat anti‘mouserabbit antibodies cat no ta130001ta130015 used for immunohistochem‘istry ihc were obtained from origene technologies inccell lines and culture the human crc cell line ht29 was obtained from the american type culture collection and cultured in mccoy's 5a modified medium gibco thermo fisher scientific inc supplemented with fbs gibco thermo fisher scientific inc mgml penicillin and mgml streptomycin maintained in a humidified atmo‘sphere of co2 at ˚cwestern blotting cells were lysed in ripa lysis buffer beijing solarbio science technology co ltd at ˚c for min the protein concentration was measured following centrifugation at x g for min at ˚c and quantified using a bicinchoninic acid protein assay kit beijing solarbio science technology co ltd an equal amount of protein µglane was separated via sds‘page and electrophoretically transferred onto polyvinylidene difluo‘ride membranes emd millipore the membranes were blocked with skimmed milk for h at room temperature and were subsequently incubated overnight at ˚c with the following primary antibodies anti‘tlr9 anti‘nf‘κb anti‘bcl‘xl anti‘pcna anti‘myd88 and anti‘gapdh following the primary antibody incubation the membranes were washed times with pbs for min each and incubated with the corresponding goat anti‘rabbit or goat anti‘mouse secondary antibodies at ˚c for h protein bands were visualized using an ecl reagent thermo fisher scientific inc on a gel doc xr system bio‘rad laboratories inc and analyzed using image lab version software bio‘rad laboratories incwound healing assay a wound healing assay was performed to analyze the cell migratory ability briefly ht29 cells 3x105well were seeded into six‘well plates and cultured to ‘ confluence subsequently a ‘µl pipette tip was used to scratch a wound in the cell monolayer fresh serum‘free mccoy's 5a modified medium containing different concen‘trations of chloroquine or µgml was added to each well and cultured for h in a humidified atmosphere of co2 at ˚c images of each well were captured at and h using a light phase contrast microscope olympus zkx‘ olympus corporation with a magnification of x100 the wound‘healing areas were assessed using imagej 152a software national institutes of health the migratory rate of cells wound area at h‘wound area at harea at hcolony formation assay ht29 cells 12x105well were cultured in ‘well plates for h in medium containing different concentrations of chloroquine or µgml at ˚c and then seeded into ‘cm cell culture dishes cellsdish and incubated in complete medium at ˚c after days the cells were fixed with paraformaldehyde for min and stained with crystal violet for min both at room temperature the number of colonies defined as cellscolony was counted manually using a light microscope with a magnifica‘tion of x100 relative colony number number of colonies in observed groupnumber of colonies in control group all of the experiments were repeated ‰¥ timescell viability assay the viability of ht29 cells was analyzed using an mtt assay briefly ht29 cells were seeded at a density of cellswell in a ‘well plate and treated for or h with chloroquine or µgml at ˚c and then analyzed using an mtt assay as previously described animal studies all animal experiments were approved by the ethics committee of the first affiliated hospital of nanchang university nanchang china a total of female balbc mice weight ‘ g age ‘ weeks were obtained from beijing vital river laboratory animal technology co ltd laboratory animal license no scxk ‘ the animals were maintained with a normal diet and tap water ad libitum at a temperature of ±Ëšc and a relative humidity of ‘ and artificially illuminated on an approximate h lightdark cycle at the animal care facility in the medical college of nanchang university all of the mice experiments were approved by the animal care and use committee of nanchang university the total experiment lasted for weeks the mice were divided into four groups micegroup i group a aom dss which was intraperitoneally injected without anesthesia with mgkg aom once on the first day followed by dss given in the drinking water for week and then weeks of distilled water one cycle which was repeated for two additional cycles ii group b aom which was intraperitoneally injected with mgkg aom once on the first day and provided with distilled drinking water during weeks ‘ iii group c dss which was treated with dss 0concology letters as described for group a but without the aom treatment and iv group d blank control which received neither dss nor aom treatment and was provided with distilled drinking water for the first weeks all the mice were provided with a normal diet and tap water during weeks ‘ fig 1athe disease activity index dai was evaluated at the end of the experiment using the numerical system described by tian the dai parameters included total body weight loss none ‘ ‘ ‘ stool consistency well‘formed pellets loose stool diarrhoea and the presence of fecal occult blood negative positive gross bleedingseveral mice were randomly sacrificed at certain times points following aom injection the 1st 2nd 3rd 6th 9th 12th 18th and 23rd weeks six mice were sacrificed at weeks and one mouse was sacrificed at week and four mice were sacrificed at weeks and in each group additionally six mice in group d were randomly sacrificed at week as control all remaining mice were sacrificed at week after the large bowels were resected and washed with pbs they were carefully examined photographed and fixed in formalin at room temperature for h further histological examinations were subsequently performedhistopathological analysis and immunohistochemistry ihc paraffin‘embedded colorectal sections ‘µm‘thick were stained with hematoxylin for min and eosin for min at room temperature to analyze the degree of inflammation using a light microscope with magnifications of x40 and x100 briefly the severity of inflammation the thickness of inflammation the severity of epithelial damage and the extent of the lesions were each graded from to by two investigators who were blinded to the treatment groups as previously described the severity of inflammation was adapted from the grading system developed by truelove and richards as follows i grade no neutrophil infiltration in the lamina propria ii grade i infiltration of a small number of neutrophils [ cellshigh power field hpf] in the lamina propria involving a few crypts iii grade ii obvious neutrophil infiltration in the lamina propria ‘ cellshpf involving of the crypts iv grade iii infiltration of neutrophils cellshpf in the lamina propria with crypt abscess and v grade iv obvious acute inflammation in the lamina propria with ulcer formation grade i was classi‘fied as mild grade ii was classified as moderate and grades iii and iv were classified as severe the severity of inflammation ranged from to no inflammation mild moderate severe the thickness of inflammation ranged from to no inflammation mucosa mucosa plus submucosa transmural the severity of epithelial damage ranged from to intact epithelium disruption of architectural structure erosion ulceration and the extent of lesions ranged from to no lesions punctuate multifocal diffuseihc was performed as described in our previous study colorectal tissues were fixed in formalin for h at room temperature formalin‘fixed and paraffin‘embedded tissue blocks were cut into ‘µm‘thick sections and mounted on glass slides slides were heated in an oven at ˚c for min and deparaffined in xylene twice for min each at room tempera‘ture rehydrated in a descending ethanol series and ethanol for min each at room temperature and incubated in h2o2 for min at room temperature to block endogenous peroxidase antigen retrieval was performed by heating in a microwave at ˚c in sodium citrate buffer mm ph for min slides were blocked with bovine serum albumin beijing solarbio science technology co ltd for h at room temperature to block non‘specific antibody binding and incubated overnight at ˚c with the following primary anti‘bodies anti‘tlr9 anti‘nf‘κb and anti‘ki67 following the primary antibody incubation the sections were washed three times with pbs and incubated with horse‘radish peroxidase secondary goat anti‘mouserabbit antibodies ready to use at ˚c for min the sections were stained with '‘diaminobenzidine at room temperature the duration of staining was based on the staining observed under a light microscope with a magnification of x100 and the reaction was terminated when the staining was yellowish‘brown the sections were then counterstained with hematoxylin for min at room temperature the slides were observed under a light microscope with a magnification of x100 the widely accepted german semi‘quantitative scoring system was used to determine the staining intensity and area of staining according to the recommendations of remmele and stegner each specimen was assigned a score according to the intensity of the nucleic cytoplasmic andor membrane staining no staining not detected0 weak staining light yellow1 moderate staining yellowish brown2 strong staining brown3 and the extent of stained cells no staining ‘ ‘ ‘ ‘ the final immunoreactive score was determined by multiplying the intensity score with the extent of stained cells score ranging from the minimum to the maximumco‘immunoprecipitation assay ht29 cells were lysed in ripa lysis buffer beijin solarbio science technology co ltd at ˚c for min whole‘cell lysates were pelleted via centrifugation at x g for min at ˚c the super‘natant was incubated with an anti‘tlr9 antibody or goat anti‘mouse igg cat no zb‘ origene technologies inc together with protein ag plus‘agarose beads santa cruz biotechnology at ˚c overnight the beads were washed three times with the non‘lubrol lysis buffer at x g centrifugation for min at ˚c then subjected to sds‘page and subsequent western blotting analysis as aforementioned whole cell lysate was used as a controlstatistical analysis statistical analysis was performed using spss software ibm corp the data are presented as the mean ± sd all experiments were performed at least in triplicate a one‘way anova followed by a tukey's post hoc test was used to determine the statistical differences between groups whereas an unpaired student's t‘test were used to determine the statistical differences between groups a spearman's rank correlation test was used to determine the correlation between the expression levels of tlr9 nf‘κb and ki67 in crc tissues p005 was considered to indicate a statistically significant differenceresultsconstruction of the cac model mice in groups a aom dss and c dss the body weights of the mice decreased with 0cluo tlr9 promotes colorectal carcinogenesisfigure construction of the colitis‘associated‘colorectal cancer model in mice a schematic diagram of the experimental protocol for colitis‘associated colorectal cancer model mice in groups a‘d arrow indicates mgkg azoxymethane administration via intraperitoneal injection black square indicates administration of dextran sodium sulfate in drinking water for week continuous line indicates duration fed a normal diet and distilled water dotted line indicates duration fed a normal diet and tap water and triangle indicates timepoint of sacrifice b body weights of the mice in each group p0001 c disease activity index of groups a and c data are presented as the mean ± sd group a mgkg azoxymethane and dextran sodium sulfate water group b mgkg azoxymethane group c dextran sodium sulfate water group d blank controltime compared with group d blank control group fig 1b the average weight of the mice in group c declined from ± g at the beginning of the experiment to ± g by week while the average weight of the mice in group a decreased from ± g at the beginning of the experiment to ± g by week the differences between groups a and d and groups c and d were statistically significant at week p005 the dai which reflects the severity of colitis was also markedly increased in groups a and c after dss administration on the 1st 3rd and the 6th week fig 1c no weight loss or any signs of inflammation such as loose stool or diarrhea positive fecal occult blood or gross bleeding were observed in groups b aom and d blank control the dai of groups b and d was zero throughout the modeling process and is therefore not shown in fig 1c the lengths of the large bowels in groups a and c were decreased compared with groups b and d fig 2a notably the lengths of the large bowels were significantly decreased in groups a and c from week compared with the control group mice sacrificed in group d at week or with group dp005 fig 2b however the severity of inflamma‘tion was significantly increased in group a compared with in group c at week and the extent of inflammation was signifi‘cantly increased in group a compared with in group c after week p005 while there was no significant differences in the thickness of inflammation and the severity of epithelial damage between groups a and c fig 2cby the 12th week colorectal tumors were only observed in the mice of group a fig 2d pathological results further revealed that the mice in group a presented with acute inflam‘mation aif chronic inflammation cif adenoma and adenocarcinoma of the colorectum at weeks and respectively fig 2d and eexpression levels of tlr9 and nf‘κb are simultaneously upregulated during the development of chronic colitis in crc ihc staining revealed that tlr9 was located in the cytoplasm of the intestinal epithelial cells iecs and inflammatory cells in the lamina propria fig 3a tlr9 expression levels were significantly upregulated in aif cif adenoma and adenocarcinoma tissues compared with the corresponding tissues from control mice group d p00334 p00379 p00437 and p00008 respectively fig 3b furthermore the protein expression levels of tlr9 was significantly upregulated in the adenocarcinoma tissue compared with the aif p00077 cif p00278 and adenoma p00273 tissue fig 3bthe positive nf‘κb region was mainly confined to the cytoplasm of the iecs and inflammatory cells fig 3a the ihc results revealed that the expression levels of nf‘κb were significantly upregulated in the aif cif adenoma and adenocarcinoma tissues compared with the corresponding tissues from control mice p00061 p00043 p00019 and 0concology letters figure colorectal pathological results in mice a length of the large bowels of mice in each group at week representative bowels from ‘ independent animals are shown b mean length of large bowel of mice in each group p005 p001 p0001 vs control mice sacrificed in group d at week or group d bottom graph c histological score of severity thickness damage and extent of lesions of the colorectums from mice in groups a and c p005 d large bowels were retrieved at week and from mice in group a to determine the numbers of tumors formed p005 p001 e histological analysis of various pathological changes in the colons of mice in group a were determined using hematoxylin and eosin staining the control groups represents tissues taken at week from group d scale bars ‘µm upper and ‘µm lower inflammatory cells green arrows and intestinal epithelial cells red arrows are indicated group a mgkg azoxymethane and dextran sodium sulfate water group c dextran sodium sulfate water aif acute inflammation cif chronic inflammationp00001 respectively fig 3d moreover nf‘κb expression levels were significantly upregulated in the adenocarcinoma tissue compared with the aif p00001 and adenoma p00161 tissues fig 3dki67 expression levels were observed in the nuclei of iecs and inflammatory cells fig 3a the ihc results revealed that the ki67 expression levels were gradually upregulated across the intestinal lesions including in the aif cif adenoma and 0cluo tlr9 promotes colorectal carcinogenesisfigure tlr9 and nf‘κb are simultaneously upregulated as cac develops a ihc was used to analyze the expression levels and localization of tlr9 nf‘κb and ki67 in colorectal sections obtained from mice in group a inflammatory cells green arrows and intestinal epithelial cells red arrows are indicated ihc staining scores of b tlr9 c ki67 and d nf‘κb in sections of normal control group d aif cif adenoma and adenocarcinoma tissues which were obtained as described in the methods section scale bar ‘µm p005 p001 and p0001 vs control p005 p001 and p0001 vs adenocarcinoma correlation analysis of e tlr9 and nf‘κb expression levels f ki67 and tlr9 expression levels and g nf‘κb and ki67 expression levels was conducted using spearman's rank correlation group a mgkg azoxymethane and dextran sodium sulfate water aif acute inflammation cif chronic inflammation tlr9 toll‘like receptor ihc immunohistochemistryadenocarcinoma tissues p00331 p00092 p00241 and p00006 respectively compared with the expression levels in the corresponding tissues from the control mice fig 3c interestingly the expression levels of tlr9 and nf‘κb were discovered to be significantly positively correlated with other rho08236 p00001 fig 3e in addition a significant positive correlation was also identified between tlr9 and ki67 expres‘sion levels rho05515 p0001 fig 3f and between nf‘κb and ki67 expression levels rho05103 p001 fig 3gdownregulated tlr9 expression levels reduces the migration viability and colony formation of ht29 cells to further investigate the role of tlr9 in crc the human crc cell line ht29 was treated with chloroquine an inhibitor of tlr9 in vitro the results revealed that suppressing tlr9 with chloroquine at both doses inhibited the migration viability and colony formation ability of ht29 cells in a dose‘dependent manner fig 4a‘e additionally lysates were collected from ht29 cells treated with either different concentrations of chloro‘quine for h or µgml chloroquine for different time periods fig 4f‘i and western blotting was performed the analysis revealed that the expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl were gradually downregulated in ht29 cells treated with increasing doses of chloroquine compared with the 0concology letters figure chloroquine inhibits the migration viability and colony formation of ht29 cells by inhibiting tlr9 chloroquine and µgml inhibited the migration of ht29 cells in a dose‘dependent manner at h compared with the control group which was determined using a wound healing assay a representative images were photographed magnification x100 and b migration rates were calculated proliferation rate of ht29 cells was reduced by chloroquine treatment and µgml which was determined using a colony formation assay c representative images were photographed and d the relative colony number was analyzed p005 p001 p0001 e viability of ht29 cells was decreased by chloroquine and µgml at and h compared with the control cells as determined using an mtt assay f expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl in lysates obtained from ht29 cells treated with numerous concentrations of chloroquine or µgml for h were analyzed using western blotting g semi‘quantification of the expression levels of the proteins presented in part f was performed using imagej software gapdh was used as the loading control and for normalization h expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl in lysates obtained from ht29 cells treated with µgml chloroquine for numerous durations or h were analyzed using western blotting i semi‘quantification of the expression levels of the proteins presented in part h was performed using imagej software gapdh was used as the loading control and for normalization j tlr9 interacted with the nf‘κb protein in ht29 cells which was determined using a co‘immunoprecipitation assay goat anti‘mouse igg antibody was used as the negative control all data are expressed as the mean ± sd of three independent experiments p005 p001 p0001 vs control0 h tlr9 toll‘like receptor pcna proliferating cell nuclear antigen myd88 myeloid differentiation primary response protein myd88 ip immunoprecipitated ib immunoblotting 0cluo tlr9 promotes colorectal carcinogenesiscontrol group fig 4f and g a similar trend was observed in the expression levels of these proteins as the duration of chlo‘roquine treatment increased fig 4h and i thus these results indicated that the expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl in ht29 cells treated with chloroquine may be downregulated in both a dose‘ and time‘dependent manner fig 4f‘i to verify whether tlr9 affected the colorectal carcinogenesis by interacting with nf‘κb co‘immunoprecipi‘tation assay was used to detect the interaction between tlr9 and nf‘κb in ht‘ cells the results revealed that there was an interaction between tlr9 and nf‘κb fig 4jdiscussioncolorectal carcinogenesis is a multi‘step process starting from normal crypts to aberrant crypt foci then to polyps adenoma and eventually adenocarcinoma it has been reported that individuals with ibd may have an increased risk of developing crc which is directly proportional to the extent and duration of their disease however the exact mechanism and duration required for chronic colitis to develop into adenoma and then adenocarcinoma remains unclear it has been suggested that patients with ibd have an increased risk of crc following the inflammation‘dysplasia‘carcinoma model including dysplasia and crc as primary conse‘quences of chronic inflammation however there are currently still no defined molecular biomarkers or existing monitoring protocols for detecting the occurrence of a malignant tumor except for frequent colonoscopy examinationsin the present study an acute colitis‘chronic colitis‘adenoma‘adenocarcinoma model was successfully constructed via aomdss induction using this model tlr9 expression levels were discovered to be upregulated as the severity of the colorectal lesions increased which indicated that tlr9 protein expression levels may be continuously activated during colitis‘crc development tlr9 is a critical protein associated with innate and acquired immunity and it has been demonstrated to serve a significant role in the development of colitis and sporadic crc however the mechanism by which tlr9 regulates the devel‘opment of crc remains to be elucidatedinterestingly ihc analysis revealed that the expression levels of nf‘κb and ki67 were simultaneously upregulated alongside tlr9 expression levels notably inhibiting tlr9 decreased the migration proliferation and viability of ht29 cells in vitro and tlr9 expression levels in vivo were identi‘fied to be significantly positively correlated with the expression levels of nf‘κb and ki67 a cell proliferation marker during the transition from colitis to crc the present study further revealed that the inhibition of tlr9 in vitro significantly downregulated the expression levels of nf‘κb myd88 pcna and the anti‘apoptotic protein bcl‘xl in a dose‘ and time‘dependent manner notably a previous study reported that tlr9 promoted the tumor‘propagating potential of prostate cancer cells via nf‘κb signaling thus the findings of the present study indicated that tlr9 may promote cac through nf‘κb signaling however these findings may be controver‘sial because other previous studies have revealed that tlr9 agonists exerted an antitumor effect in crc the majority of these studies primarily focused on the role of tlr9 in colitis or sporadic crc whereas the current study focused on the role of tlr9 in the pathogenesis of cac to provide novel targets for the treatment of cac for example altera‘tions in microtubule end‘binding protein were identified as a characteristic of sporadic but not uc‘associated crc and in another previous study the immune profiling patterns of patients with cac were significantly different compared with the patients with sporadic crc chloroquine a non‘specific inhibitor of tlr9 is an old antimalarial drug which has recently attracted significant interest for its potential antitumor properties for example numerous studies have reported that chloroquine directly regu‘lated cancer cells by inducing apoptosis inhibiting autophagy interacting with nucleotides eliminating cancer stem cells and enhancing the growth of cancer cells ‘ chloroquine also inhibited the expression levels of tlr9 by preventing the acidi‘fication and maturation of the endosomes and the trafficking of tlrs due to the multiple effects of chloroquine on tumor cells different concentrations of chloroquine were selected for use in the present study based on the lowest dose according to previous studies in order to obtain the best possible results with low toxicity to the cells in the future investiga‘tions using small interfering rna targeting tlr9 should be performed to determine the effect on the biological processes of crc cell lines to further verify the findings of the present study thus our future studies will focus on investigating the precise molecular mechanism by which tlr9 participates in the early occurrence of colorectal carcinogenesisin the present study developed a cac animal model the findings indicated that tlr9 may be closely asso‘ciated with the process of inflammation‘dysplasia‘carcinoma and may impact carcinogenesis by regulating the nf‘κb signaling pathway these results may provide promising potential to be developed into an early detection protocol or therapeutic molecular target for crcacknowledgementsnot applicablefundingthe present study was supported by grants from the national natural science foundation of china grant nos and the foundation of jiangxi educational committee grant no gjj170016 and the graduate student innovation funding program of nanchang university grant no cx2019119availability of data and materialsall data generated or analyzed during this study are included in this published article the original data are available from the corresponding author on reasonable requestauthors' contributionscz and ql designed the study and drafted the manuscript ql and lz performed the experiments ql ct and zz analyzed the data ql cz and yc revised the manuscript for important 0concology letters intellectual content cz and yc made substantial contribu‘tions to conception design and coordination of the study and gave final approval of the version to be published all authors have read and approved the final manuscriptethics approval and consent to participateall animal experiments were approved by the ethics committee of the first affiliated hospital of nanchang university nanchang china approval no ‘ pa Answer:

Colon_Cancer

" drug resistance leads to tumor relapse and further progression during chemotherapy in lung cancer close homolog of l1 chl1 has been identified as a tumor suppressor in most malignancies however to the best of our knowledge whether chl1 mediates chemoresistance remains unknown the present study observed that chl1 was significantly downregulated in cisplatin ddp‘resistant cells a549ddp and paclitaxel ptx‘resistant cells a549ptx compared with a549 cells when treated with or without ddp and ptx silencing of chl1 in a549 cells promoted the cell survival rate and clone formation and decreased apoptosis whereas overexpression of chl1 in a549ddp and a549ptx cells impeded the cell survival and clone formation and promoted apoptosis additionally chl1 overexpression enhanced the chemosensitivity of a549ddp cells to ddp in vivo notably the chemoresistance induced by chl1 depletion was reversed by the akt inhibitor sc66 in a549 cells the results of the present study demonstrated that chl1 enhanced sensitivity of lung cancer cells by suppressing the akt pathway which suggested that chl1 may be a potential target for overcoming chemoresistance in lung cancerintroductionlung cancer is the most common human malignancy accounting for of all cancer‘associated deaths worldwide during in addition its morbidity and mortality rank the highest among all malignant tumor types worldwide according to the differentiation degree and morphological correspondence to dr rimao huang department of cardiothoracic surgery xiangya changde hospital moon avenue west of langzhou north road changde hunan pr chinae‘mail xyhuangrm163comkey words lung cancer close homolog of cisplatin paclitaxel chemosensitivitycharacteristics of cancer cells lung cancer can be roughly clas‘sified into non‘small‘cell lung cancer nsclc and small‘cell lung cancer among patients with lung cancer nearly are diagnosed as nsclc which manifests with earlier diffu‘sion and metastasis currently resection chemotherapy radiotherapy and targeted therapy are the primary treatments for lung cancer for patients with advanced nsclc or those who are clinically incapacitated for surgery chemo‘therapy is a remarkably important treatment cisplatin ddp is widely applied in the treatment of several malignan‘cies and it exhibits a broad spectrum of antitumor effects by inducing dna damage and hindering dna damage repair paclitaxel ptx another commonly used chemotherapeutic agent in the clinic targets the microtubule cytoskeleton and impedes cell division the majority of patients have a good initial response to chemotherapy agents however subse‘quent relapse is common and largely due to the emergence of drug resistance thus chemoresistance is considered one of the main factors of poor prognosis in patients with advanced nsclc therefore there is an urgent need to investigate the target and mechanism of chemoresistance in nsclcclose homolog of l1 chl1 is a member of the l1 family of nerve cell adhesion molecules and is located on the 3q26 locus as a nerve cell adhesion molecule chl1 serves an important role in the development regeneration and plasticity of the nervous system the absence or mutation of chl1 can trigger 3p syndrome and schizophrenia the abnormal expression of chl1 may lead to reduced working memory and social behavior mental damage and abnormal behavior chl1 has been reported to be involved in carcinogenesis and progression in a variety of human cancers in esophageal squamous cell carcinoma escc chl1 downregulation is associated with invasion lymph node metastasis and poor overall survival functional studies revealed that chl1 has anti‘proliferation and anti‘metastasis abilities the expression of chl1 is downregulated by hypermethylation in human breast cancer and its negative expression contributes to breast tumorigenesis and progression in thyroid cancer and colonic adenocarcinoma chl1 impedes cell proliferation and invasion and acts as a tumor suppressor in lung cancer hÓ§tzel evaluated chl1 expression 0ccai chl1 enhances the chemosensitivity of lung cancer cellsin nsclc cases based on a tissue microarray and it was reported that chl1 expression is associated with t stage in adenocarcinomas as well as with metastatic lymph node status and improved survival additionally by analyzing the gene expression omnibus dataset gse21656 submitted by sun microarray results demonstrated that chl1 expression in ddp‘resistant h460 cells is significantly lower compared with that in parental cells suggesting that chl1 may be involved in nsclc chemoresistance however to the best of our knowledge the underlying mechanism remains unknownin the present study the expression of chl1 in ddp‘ and ptx‘resistant a549 cells and the parental cells was assessed functional studies of chl1 were performed to investigate its potential role in chemoresistancematerials and methodsdata processing the human gse21656 microarray dataset was downloaded from the ncbi gene expression omnibus geo database wwwncbinlmnihgovgeo the available dataset gse21656 was based on the gpl6244 platform affymetrix human gene st array affymetrix thermo fisher scientific inc this data includes h460 cells and ddp‘resistant h460 cells sample and each cell has three repeats samples the online tool geo2r httpwwwncbinlmnihgovgeogeo2r was used to determine the differen‘tially expressed genes in h460 and ddp‘resistant h460 cells p005 and log2fold‘change‰¥ were set as cut‘off standardscell culture the human nsclc cell line a549 the ptx‘resistant cell line a549ptx and the ddp‘resistant cells a549ddp were purchased from procell life science technology co ltd the cells were cultured in ham's f‘12k medium supplemented with fetal bovine serum both purchased from thermo fisher scientific inc uml penicillin and uml strep‘tomycin cat no thermo fisher scientific inc in a ˚c humidified incubator with co2cell transfection the resistant cells a549ptx and a549ddp cells were transfected with µg chl1 recombinant expres‘sion plasmid cat no hg10143‘ny sino biological inc empty vector pcmv3‘sp‘n‘ha was used as the control a549 cells were transfected with pmol small interfering sirnas the sirna sequence for chl1 guangzhou ribobio co ltd were sirna‘ '‘gga gcu aau uug acc aua utt‘' sirna‘ '‘cag caa uau uag cga gua utt‘' and scrambled control '‘uuc ucc gaa cgu guc acg utt‘' plasmids and sirnas were transfected into cells using lipofectamine® thermo fisher scientific inc following the manufacturer's instructions the time interval between transfection and subsequent experimentation was h for the rescue experiments the chl1 silenced a549 cells were treated with the akt inhibitor sc66 cat no s5313 selleck chemicals along with ddp µgml or ptx ngml both purchased from selleck chemicals for h at ˚crna extraction and reverse transcription‘quantitative pcr rt‘qpcr assay total rnas were isolated using trizol reagent thermo fisher scientific inc according to the manufacturer's instructions and the mixed dnas were elim‘inated by dnase i new england biolabs inc first‘strand cdna synthesis was conducted using the goscripttm kit promega corporation according to the manufacturer's instructions the reaction conditions for reverse transcription were ˚c for min ˚c for min and ˚c for min the sybr green real‘time pcr master mix thermo fisher scientific inc was used to perform rt‘qpcr using a lightcycler480 system roche diagnostics gmbh the chl1 primer sequences were as follows forward '‘ggc ttg gtc tct tgc ttt cc‘' and reverse '‘atc ttc cct ccc ttt gca cg‘' and ‘actin forward '‘ttc ctt cct ggg cat gga gtc ‘' and reverse '‘tct tca ttg tgc tgg gtg cc‘' the following thermocycling conditions were used for qpcr min at ˚c followed by cycles at ˚c for sec sec at ˚c and a final extension at ˚c for sec each reaction was conducted in triplicate relative expression levels were calculated using the ‘δδcq method cell viability cell viability was detected by mtt assay a cell suspension µl was seeded into ‘well plates at a density of 1x104 cellswell and incubated overnight at ˚c the concentrations of ddp used to treat a549 cells were and µgml while the concentrations of ptx used to treat a549 cells were and ngml the concentrations of ddp used to treat a549ddp cells were and µgml while the concentrations of ptx used to treat a549ptx cells were and ngml after treating with different concentrations of ddp or ptx for h at ˚c µl mtt mgml solution was added to each well and incubated for h at ˚c subsequently µl dmso was added to each well to dissolve the blue formazan crystals and the absorbance was measured using a microplate reader biotek instruments inc at nmclone formation assay a total of 1x103 cells were seeded into a ‘mm dish in triplicate and maintained in f‘12k medium with or without ddp or ptx at ˚c for h a total of weeks later cells were fixed in paraformalde‘hyde for min at room temperature and stained with crystal violet dye at room temperature for min the rate of colony formation was calculated using the following equation number of coloniesnumber of seeded cells x100flow cytometry apoptosis was detected using a fitc annexin v apoptosis kit bd pharmingen bd biosciences according to the manufacturer's protocol cells 1x105 were collected and washed twice with pbs prior to being suspended in µl binding buffer subsequently cells were incubated with µl annexin v‘fitc and µl propidium iodide in the dark for min at room temperature and apoptosis was analyzed using a cytoflex flow cytometer beckman coulter inc data were analyzed using cytexpert software beckman coulter inc the ratio between early and late apoptosis was calculatedwestern blotting cells were collected washed twice with pbs and lysed with ripa lysis buffer thermo fisher scientific inc proteins were isolated from the cell lysis buffer and 0concology letters quantified using the piercetm„¢ bca protein assay kit cat no thermo fisher scientific inc with bovine serum album as a standard equal amount of protein µg proteins were separated by sds‘page gel next the proteins were transferred onto a polyvinylidene membrane thermo fisher scientific inc blocked with bsa thermo fisher scientific inc for h at ˚c and incubated overnight at ˚c with primary antibodies against chl1 cat no ‘‘ap proteintech inc multi‘drug resistance gene mdr1 cat no ‘‘ap proteintech inc multidrug resistance‘associated protein mrp cat no ‘‘ig proteintech inc low‘density lipoprotein receptor‘related protein lrp cat no ‘‘ap proteintech inc phosphorylated p‘akt cat no ab38449 abcam and akt cat no ab227385 abcam after washing three times with pbs the membrane was incubated with horseradish peroxide‘conjugated goat anti‘rabbit cat no ab6271 abcam_or rabbit anti‘mouse cat no ab6728 abcam secondary antibodies for h at room temperature and the blots were detected with enhanced chemiluminescence reagent thermo fisher scientific inc protein expression was quantified using image‘pro plus software media cybernetics incanimal experiments the animal experiments were approved by the medical ethics committee of xiangya changde hospital approval no and were performed in compliance with all regulatory institutional guidelines for animal welfare the national institutes of health publications no ‘ a total of male balbc‘nu mice ‘week‘old ± g hunan sja laboratory animals center of the chinese academy of sciences were used in this study all animals were kept at the spf level laboratory at ‘Ëšc a relative humidity of ‘ a h lightdark cycle and timesh of fresh air exchange all mice were given free access to food and water the bedding materials drinking water feeding cages and other items in contact with the animals were all autoclaved prior to use a549ddp cells 1x107 transfected with empty vector and chl1 overexpression vector using lipofectamine® reagent thermo fisher scientific inc were subcu‘taneously injected into the nude mice to establish xenograft models following anaesthesia with chloral hydrate mgkg xenografts were allowed to grow to mm3 over weeks and the mice were randomly divided into four groups n3group as follows i vector group a549ddp cells transfected with empty vector and treated with µl saline solution ii vector‘ddp group a549ddp cells trans‘fected with empty vector and treated with mgkg ddp iii chl1 group a549ddp cells transfected with chl1 overexpression vector and treated with µl saline solu‘tion and iv chl1‘ddp group a549ddp cells transfected with chl1 overexpression vector and treated with mgkg ddp ddp was administered by intraperitoneal injection every days for weeks the mice were observed daily and the tumors were measured by a vernier caliper every days the tumor volumes were calculated as length x width22 a total of weeks post‘injection mice were euthanized with co2 at volume displacement rate vdr per min using a programmable logic controller barry‘wehmiller design group inc mice were monitored continuously and once the mice were immobile except for breathing for min the vdr was provided at for min the animals remained in the euthanasia chamber for min and were then observed for an additional min breathing and heart rate were monitored to determine deathstatistical analysis all experiments were performed in tripli‘cate and data are presented as the mean ± standard deviation all experiments were performed at least three times paired student's t‘test was performed for comparisons between two groups and one‘way analysis of variance followed by tukey's multiple comparison post‘hoc analysis was performed for comparisons between multiple groups spss ibm corp was used to perform the analysis p005 was considered to indicate a statistically significant differenceresultschl1 is downregulated in a549ddp and a549ptx‘resistant cells in order to investigate the mechanism of chemore‘sistance in lung cancer the lung adenocarcinoma cell line a549 the ddp‘resistant cells a549ddp and ptx‘resistant cells a549ptx were used in the present study cells were exposed to different concentrations of ddp ‘ µgml and ptx ‘ ngml and mtt assay was used to detect the cell survival rate a549ddp and a549ptx cells demonstrated higher resistance to ddp and ptx compared with a549 cells fig 1a the half maximal inhibitory concentration ic50 of ddp was significantly higher in a549ddp cells ± µgml compared with a549 cells ± µgml and the ic50 of ptx was significantly higher in a549ptx cells ± ngml compared with a549 cells ± ngml fig 1b in addition the expression levels of the drug‘resistant markers mdr1 mrp and lrp were significantly higher in a549ddp and a549ptx cells compared with a549 cells fig 1c additionally the mrna and protein expression levels of chl1 were significantly lower in a549ddp and a549ptx cells compared with those in a549 cells fig 1d and e and this was also observed in h460 ddp‘resistant cells obtained from the geo dataset gse21656 fig 1f these results suggested that chl1 may be involved in regulating ddp and ptx resistance in nsclcknockdown of chl1 enhances resistance to ddp and ptx in a549 cells as chl1 was upregulated in a549 cells chl1 was silenced in a549 cells using sirnas chl1 expres‘sion was significantly reduced in the chl1 sirna groups compared with that of the scrambled control group fig 2a as sirna‘ demonstrated the greatest interference efficiency it was selected for use in the following experiments notably chl1‘knockdown enhanced the resistance to ddp and ptx in a549 cells fig 2b and c colony formation assay revealed that compared with the control group chl1‘knockdown significantly increased the rate of colony formation in the absence of chemotherapeutics and enhanced the resistance to ddp and ptx fig 2d flow cytometry results demon‘strated significantly reduced apoptosis in chl1‘knockdown cells after ddp and ptx treatment compared with that of the control group fig 2e 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure chl1 is downregulated in ddp and ptx‘resistant a549 cells a cell survival of a549 and a549‘resistant cells a549ddp and a549ptx treated with increasing concentrations of ddp and ptx as assessed by mtt assay b the ic50 values of ddp in a549ddp and a549 cells and the ic50 values of ptx in a549ptx and a549 cells p005 vs a549 cells c western blotting demonstrated the expression of drug resistance‘related proteins mdr1 mrp and lrp in a549 cells and a549‘resistant cells a549ddp and a549ptx p005 vs a549 cells the protein and mrna expression levels of chl1 in a549 cells and a549‘resistant cells a549ddp and a549ptx were analysed by d western blotting and e reverse transcription‘quantitative pcr respectively p005 vs a549 cells f the mrna expression of chl1 in h460 and h460ddp cells in the gse21656 dataset p005 vs h460 cells chl1 close homolog of l1 ddp cisplatin ptx paclitaxel mdr1 multi‘drug resistance gene mrp multidrug resistance‘associated protein lrp low‘density lipoprotein receptor‘related protein ic50 half maximal inhibitory concentration chl1 overexpression enhances the sensitivity of a549 resistant cells to ddp and ptx as chl1 is downregu‘lated in a549ddp and a549ptx cells the present study successfully overexpressed chl1 in these cells using chl1 recombinant expression plasmids fig 3a the results demonstrated that chl1 overexpression alleviated the resis‘tance to ddp and ptx compared with that of the control group fig 3b and c in addition chl1 overexpression inhibited colony formation in the absence or presence of ddp and ptx fig 3d additionally flow cytometry results demonstrated that restoration of chl1 expression promoted apoptosis in resistant cells following ddp and ptx treat‘ment fig 3eto further validate the effects of chl1 overexpression on ddp or ptx sensitivity xenograft mice model experi‘ments were performed the results demonstrated that chl1 overexpression or ddp treatment significantly impeded the tumor growth fig 3f and decreased the tumor weight fig 3g in addition chl1 overexpression further aggra‘vated ddp‘mediated repression on tumor growth fig 3f and g these data suggested that chl1 overexpression suppressed tumor growth and enhanced the chemosensitivity in nsclcchl1 mediates chemosensitivity by inhibiting akt activity recently studies have confirmed that chl1 inhibits akt activity in escc and neuroblastoma cell lines thus the present study investigated whether chl1 mediates chemoresistance via the akt pathway in nsclc in a549 cells compared with the scrambled group chl1‘knockdown elevated the expression of p‘aktser473 fig 4a by contrast restoring chl1 expression in a549ddp and a549ptx cells inhibited the akt phosphorylation compared with the control group fig 4a suggesting chl1 mediates chemosensitivity via the akt pathway subsequently chl1‘silenced a549 cells were treated with the akt inhibitor sc66 and it was demon‘strated that inhibiting akt activity significantly reduced the promotive effects on cell survival fig 4b and clone forma‘tion fig 4c and the inhibitory effects on apoptosis fig 4d induced by chl1‘depletion these results confirmed that chl1 mediates chemosensitivity in nsclc by inhibiting the akt pathway 0concology letters figure chl1‘knockdown increases a549 cell resistance to ddp and ptx a western blotting was performed to validate the efficiency of transfection with chl1 sirnas p005 vs scramble mtt assays were performed to determine the survival rate of chl1‘knockdown a549 cells treated with b ‘ µgml ddp or c ‘ ngml ddp d colony formation assay of a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was used to detect apoptosis in a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx p005 p0001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering discussionthe results of the present study demonstrated that chl1 was significantly downregulated in a549ddp and a549ptx cells compared with a549 cells the knockdown of chl1 in a549 cells facilitated the cell survival and clone formation and decreased apoptosis when treated with or without ddp and ptx whereas chl1 overexpression in a549ddp and a549ptx cells inhibited cell survival and clone formation and increased apoptosis the results of the present study also demonstrated that chl1 enhances nsclc chemosensitivity through inhibition of the akt pathway these data suggested that chl1 may be a promising target to improve the efficacy of chemosensitivity in nsclcchl1 belongs to the l1 family of nerve cell adhesion molecules it was initially cloned in mice and its expression in mouse development was analyzed by senchenko through cell‘cell interactions and mediating cell‘cell and cell‘matrix interactions chl1 has an important effect on the development regeneration and plasticity of the nervous system previous reports have demonstrated that chl1 also participates in carcinogenesis ‘ chl1 was observed to be significantly downregulated in up to types of tumor tissues compared with their adjacent normal tissues in most tumors chl1 is a potential tumor suppressor gene whose silencing is associated with tumor growth invasion and metastasis ‘ for example knockdown of chl1 expression results in enhanced cervical cancer cell invasion and migration a low expres‘sion of chl1 in patients with neuroblastoma predicts a poor prognosis and enhancing chl1 expression suppresses tumor progression in contrast chl1 has been reported to promote cell proliferation metastasis and migration in human gliomas however to the best of our knowledge research on chl1 and tumor chemoresistance has rarely been reported 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure overexpression of chl1 increases the sensitivity of resistant a549 cells to ddp and ptx a western blotting was performed to detect chl1 expression in a549ddp and a549ptx cells transfected with chl1 expression plasmids p005 vs vector effect of chl1 overexpression on resistant a549 cell survival rate when treated with b ‘ µgml ddp or c ‘ ngml ptx as determined by mtt assay d colony formation assays demon‘strated the number of colonies of resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was performed to assess apoptosis in resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx chl1 overexpression enhanced chemosensitivity of a549ddp cells to ddp in vivo which was demonstrated by the effect of ddp treatment or chl1 overexpression on the f growth and g weight of xenografts derived from a549ddp cells p005 p001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel the present study examined the differentially expressed genes in nsclc ddp‘resistant cells in a geo dataset chl1 was demonstrated to be upregulated in ddp‘resistant cells compared with parental cells suggesting that chl1 may be involved in nsclc chemotherapy resistance similarly a study that compared and analyzed the differentially expressed genes in chemosensitive tumors and chemoresistant ovarian adenocarcinomas tissues reported that the expression of chl1 in chemotherapy‘sensitive tumor tissues is higher compared with that in drug‘resistant tissues suggesting that chl1 may help to predict the efficacy of chemotherapy for ovarian cancer in addition aberrant methylation of chl1 may be associated with the recurrence of colorectal cancer crc following chemotherapy ‘azadc treatment restores ‘fluro‘uracil sensitivity in vitro which also suggests that chl1 may be involved in crc chemotherapy resistance the results of the present study demonstrated that chl1 was down‘regulated in a549ddp cells additionally as multiple drug resistance is a common characteristic another type of resistant cells a549tax cells were also used in the current study the results also demonstrated that chl1 was downregulated in a549ptx cells compared with control cells overexpression of chl1 significantly increased the sensitivity of cells resistant to ddp and ptx whereas knockdown of chl1 expression in 0concology letters figure chl1 mediates ddp and ptx sensitivity by inhibiting akt activity a western blotting was performed to detect the expression of p‘akt and total akt in chl‘silenced and ‘restored cell models p005 vs scramble or vector b mtt assays were performed to detect cell survival rates of a549 cells treated with chl1 sirna and akt inhibitor sc66 p005 c colony formation assays were performed in a549 cells treated with chl1 sirna and the akt inhibitor sc66 in the presence of ddp µgml or ptx ngml p005 vs si‘chl1 d apoptosis were measured in a549 cells treated with chl1 sirna and akt inhibitor sc66 in the presence of ddp µgml and ptx ngml p005 vs si‘chl1 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering p‘ phosphorylated parent a549 cells displayed the opposite results to the best of our knowledge this study is the first study to suggest that chl1 may be involved in chemosensitivity in lung cancer the concentration of ddp used in vivo is mgkg however this may not be in line with the concentrations that would be used in a clinical setting in a clinical trial the human initial dose was calculated from the no observed adverse effect levels noaels verified in animal experiments noael is the maximum dose level without significant adverse reactions the noael verified in animal experiments can be converted to a human equivalent dose according to the body surface area conversion which is based on the area standardization mgm2 proportional among different species in the present study the concentration of ddp used in vivo was not the noael thus it was not consistent with the concentrations used in clinical settingsakt is a serinethreonine protein kinase that is activated by phosphorylation as a key molecule of the pi3kakt signaling pathway p‘akt regulates cell survival cell growth cell motility and angiogenesis and prevents apoptosis additionally akt activation is associated with tumor chemore‘sistance the results of the present study demonstrated that compared with the control groups the expression of p‘akt was increased in chl1‘knockdown a549 cells and its expres‘sion was reduced in chl1 overexpressed a549ddp and a549ptx cells when akt activity was inhibited by the akt inhibitor the sensitivity to ddp and ptx in chl1‘knockdown a549 cells was restored this finding suggested that chl1 enhanced the chemosensitivity of nsclc by inhibiting the akt pathway considering numerous studies have confirmed that the akt pathway mediates chemoresistance via regulation of atp binding cassette abc members ‘ the present study didn't further investigate the specific abc members and mechanisms which was a of the limitation to the present study thus this research should be further investigated in vivoin summary the present study demonstrated that chl1 was downregulated in resistant cells a549ddp and a549ptx and upregulation of chl1 enhanced the chemosensitivity of nsclc via inhibiting the akt pathway to the best of our knowledge this was the first study to confirm the function and 0ccai chl1 enhances the chemosensitivity of lung cancer cellsmechanism of chl1 in mediating chemosensitivity in cancer thus the development of chl1‘based therapeutic strategies may improve the efficacy of chemosensitivity in nsclcacknowledgementsthe authors of the present study would like to thank mr dingliang li xiangya hospital changsha china for his guidance and assistance in flow cytometric analysisfundingno funding was receivedavailability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author upon reasonable requestauthors' contributionsrh conceived and designed the present study xc bh yh and pl performed experiments and collected the data sl zz and zh analyzed and interpreted the data ml and lz analyzed the data and prepared the figure xc ml and lz drafted the initial manuscript and revised it for intellectual content all authors read and approved the final manuscriptethics approval and consent to participatethe animal experiments were approved by the medical ethics committee of xiangya changde hospital changde china approval no patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences parascandola m and xiao l tobacco and the lung cancer epidemic in china transl lung cancer res suppl s21‘s30 chen w cancer statistics updated cancer burden in china chin j cancer res oser mg niederst mj sequist lv and engelman ja transformation from non‘small‘cell lung cancer to small‘cell lung cancer molecular drivers and cells of origin lancet oncol e165‘e172 thatcher n faivre‘finn c blackhall f anderson h and lorigan p sequential platinum‘based chemotherapy‘thoracic radiotherapy in early stage non‘small cell lung cancer clin cancer res suppl s5051‘s5056 yano t okamoto t fukuyama s and maehara y therapeutic strategy for postoperative recurrence in patients with non‘small cell lung cancer world j clin oncol ‘ fang z chen w yuan z liu x and jiang h lncrna‘malat1 contributes to the cisplatin‘resistance of lung cancer by upregulating mrp1 and mdr1 via stat3 activation biomed pharmacother ‘ cai y dong zy and wang jy lncrna nnt‘as1 is a major mediator of cisplatin chemoresistance in non‘small cell lung cancer through mapkslug pathway eur rev med pharmacol sci ‘ han ml zhao yf tan ch xiong yj wang wj wu f fei y wang l and liang zq cathepsin l upregulation‘induced emt phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in a549 cells acta pharmacol sin ‘ liu j meisner d kwong e wu xy and johnston mr translymphatic chemotherapy by intrapleural placement of gelatin sponge containing biodegradable paclitaxel colloids controls lymphatic metastasis in lung cancer cancer res ‘ hassan wa yoshida r kudoh s kameyama h hasegawa k niimori‘kita k and ito t notch1 controls cell chemoresistance in small cell lung carcinoma cells thorac cancer ‘ tang h jiang l zhu c liu r wu y yan q liu m jia y chen j qin y loss of cell adhesion molecule l1 like promotes tumor growth and metastasis in esophageal squamous cell carcinoma oncogene ‘ liu h focia pj and he x homophilic adhesion mechanism of neurofascin a member of the l1 family of neural cell adhesion molecules j biol chem ‘ tassano e biancheri r denegri l porta s novara f zuffardi o gimelli g and cuoco c heterozygous deletion of chl1 gene detailed array‘cgh and clinical characterization of a new case and review of the literature eur j med genet ‘ morellini f lepsveridze e kahler b dityatev a and schachner m reduced reactivity to novelty impaired social behavior and enhanced basal synaptic excitatory activity in perforant path projections to the dentate gyrus in young adult mice deficient in the neural cell adhesion molecule chl1 mol cell neurosci ‘ he lh ma q shi yh ge j zhao hm li sf and tong zs chl1 is involved in human breast tumorigenesis and progres‘sion biochem biophys res commun ‘ martín‘sánchez e mendaza s ulazia‘garmendia a monreal‘santesteban i blanco‘luquin i córdoba a vicente‘garcía f pérez‘janices n escors d megías d chl1 hypermethylation as a potential biomarker of poor prog‘nosis in breast cancer oncotarget ‘ zhu h fang j zhang j zhao z liu l wang j xi q and gu m mir‘ targets chl1 and controls tumor growth and invasion in papillary thyroid carcinoma biochem biophys res commun ‘ yu w zhu k wang y yu h and guo j overexpression of mir‘‘5p promotes proliferation and invasion of colon adeno‘carcinoma cells through targeting chl1 mol med hötzel j melling n müller j polonski a wolters‘eisfeld g izbicki jr karstens kf and tachezy m protein expression of close homologue of l1 chl1 is a marker for overall survival in non‘small cell lung cancer nsclc j cancer res clin oncol ‘ sun y zheng s torossian a speirs ck sc

cancer7542

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " drug resistance leads to tumor relapse and further progression during chemotherapy in lung cancer close homolog of l1 chl1 has been identified as a tumor suppressor in most malignancies however to the best of our knowledge whether chl1 mediates chemoresistance remains unknown the present study observed that chl1 was significantly downregulated in cisplatin ddp‘resistant cells a549ddp and paclitaxel ptx‘resistant cells a549ptx compared with a549 cells when treated with or without ddp and ptx silencing of chl1 in a549 cells promoted the cell survival rate and clone formation and decreased apoptosis whereas overexpression of chl1 in a549ddp and a549ptx cells impeded the cell survival and clone formation and promoted apoptosis additionally chl1 overexpression enhanced the chemosensitivity of a549ddp cells to ddp in vivo notably the chemoresistance induced by chl1 depletion was reversed by the akt inhibitor sc66 in a549 cells the results of the present study demonstrated that chl1 enhanced sensitivity of lung cancer cells by suppressing the akt pathway which suggested that chl1 may be a potential target for overcoming chemoresistance in lung cancerintroductionlung cancer is the most common human malignancy accounting for of all cancer‘associated deaths worldwide during in addition its morbidity and mortality rank the highest among all malignant tumor types worldwide according to the differentiation degree and morphological correspondence to dr rimao huang department of cardiothoracic surgery xiangya changde hospital moon avenue west of langzhou north road changde hunan pr chinae‘mail xyhuangrm163comkey words lung cancer close homolog of cisplatin paclitaxel chemosensitivitycharacteristics of cancer cells lung cancer can be roughly clas‘sified into non‘small‘cell lung cancer nsclc and small‘cell lung cancer among patients with lung cancer nearly are diagnosed as nsclc which manifests with earlier diffu‘sion and metastasis currently resection chemotherapy radiotherapy and targeted therapy are the primary treatments for lung cancer for patients with advanced nsclc or those who are clinically incapacitated for surgery chemo‘therapy is a remarkably important treatment cisplatin ddp is widely applied in the treatment of several malignan‘cies and it exhibits a broad spectrum of antitumor effects by inducing dna damage and hindering dna damage repair paclitaxel ptx another commonly used chemotherapeutic agent in the clinic targets the microtubule cytoskeleton and impedes cell division the majority of patients have a good initial response to chemotherapy agents however subse‘quent relapse is common and largely due to the emergence of drug resistance thus chemoresistance is considered one of the main factors of poor prognosis in patients with advanced nsclc therefore there is an urgent need to investigate the target and mechanism of chemoresistance in nsclcclose homolog of l1 chl1 is a member of the l1 family of nerve cell adhesion molecules and is located on the 3q26 locus as a nerve cell adhesion molecule chl1 serves an important role in the development regeneration and plasticity of the nervous system the absence or mutation of chl1 can trigger 3p syndrome and schizophrenia the abnormal expression of chl1 may lead to reduced working memory and social behavior mental damage and abnormal behavior chl1 has been reported to be involved in carcinogenesis and progression in a variety of human cancers in esophageal squamous cell carcinoma escc chl1 downregulation is associated with invasion lymph node metastasis and poor overall survival functional studies revealed that chl1 has anti‘proliferation and anti‘metastasis abilities the expression of chl1 is downregulated by hypermethylation in human breast cancer and its negative expression contributes to breast tumorigenesis and progression in thyroid cancer and colonic adenocarcinoma chl1 impedes cell proliferation and invasion and acts as a tumor suppressor in lung cancer hÓ§tzel evaluated chl1 expression 0ccai chl1 enhances the chemosensitivity of lung cancer cellsin nsclc cases based on a tissue microarray and it was reported that chl1 expression is associated with t stage in adenocarcinomas as well as with metastatic lymph node status and improved survival additionally by analyzing the gene expression omnibus dataset gse21656 submitted by sun microarray results demonstrated that chl1 expression in ddp‘resistant h460 cells is significantly lower compared with that in parental cells suggesting that chl1 may be involved in nsclc chemoresistance however to the best of our knowledge the underlying mechanism remains unknownin the present study the expression of chl1 in ddp‘ and ptx‘resistant a549 cells and the parental cells was assessed functional studies of chl1 were performed to investigate its potential role in chemoresistancematerials and methodsdata processing the human gse21656 microarray dataset was downloaded from the ncbi gene expression omnibus geo database wwwncbinlmnihgovgeo the available dataset gse21656 was based on the gpl6244 platform affymetrix human gene st array affymetrix thermo fisher scientific inc this data includes h460 cells and ddp‘resistant h460 cells sample and each cell has three repeats samples the online tool geo2r httpwwwncbinlmnihgovgeogeo2r was used to determine the differen‘tially expressed genes in h460 and ddp‘resistant h460 cells p005 and log2fold‘change‰¥ were set as cut‘off standardscell culture the human nsclc cell line a549 the ptx‘resistant cell line a549ptx and the ddp‘resistant cells a549ddp were purchased from procell life science technology co ltd the cells were cultured in ham's f‘12k medium supplemented with fetal bovine serum both purchased from thermo fisher scientific inc uml penicillin and uml strep‘tomycin cat no thermo fisher scientific inc in a ˚c humidified incubator with co2cell transfection the resistant cells a549ptx and a549ddp cells were transfected with µg chl1 recombinant expres‘sion plasmid cat no hg10143‘ny sino biological inc empty vector pcmv3‘sp‘n‘ha was used as the control a549 cells were transfected with pmol small interfering sirnas the sirna sequence for chl1 guangzhou ribobio co ltd were sirna‘ '‘gga gcu aau uug acc aua utt‘' sirna‘ '‘cag caa uau uag cga gua utt‘' and scrambled control '‘uuc ucc gaa cgu guc acg utt‘' plasmids and sirnas were transfected into cells using lipofectamine® thermo fisher scientific inc following the manufacturer's instructions the time interval between transfection and subsequent experimentation was h for the rescue experiments the chl1 silenced a549 cells were treated with the akt inhibitor sc66 cat no s5313 selleck chemicals along with ddp µgml or ptx ngml both purchased from selleck chemicals for h at ˚crna extraction and reverse transcription‘quantitative pcr rt‘qpcr assay total rnas were isolated using trizol reagent thermo fisher scientific inc according to the manufacturer's instructions and the mixed dnas were elim‘inated by dnase i new england biolabs inc first‘strand cdna synthesis was conducted using the goscripttm kit promega corporation according to the manufacturer's instructions the reaction conditions for reverse transcription were ˚c for min ˚c for min and ˚c for min the sybr green real‘time pcr master mix thermo fisher scientific inc was used to perform rt‘qpcr using a lightcycler480 system roche diagnostics gmbh the chl1 primer sequences were as follows forward '‘ggc ttg gtc tct tgc ttt cc‘' and reverse '‘atc ttc cct ccc ttt gca cg‘' and ‘actin forward '‘ttc ctt cct ggg cat gga gtc ‘' and reverse '‘tct tca ttg tgc tgg gtg cc‘' the following thermocycling conditions were used for qpcr min at ˚c followed by cycles at ˚c for sec sec at ˚c and a final extension at ˚c for sec each reaction was conducted in triplicate relative expression levels were calculated using the ‘δδcq method cell viability cell viability was detected by mtt assay a cell suspension µl was seeded into ‘well plates at a density of 1x104 cellswell and incubated overnight at ˚c the concentrations of ddp used to treat a549 cells were and µgml while the concentrations of ptx used to treat a549 cells were and ngml the concentrations of ddp used to treat a549ddp cells were and µgml while the concentrations of ptx used to treat a549ptx cells were and ngml after treating with different concentrations of ddp or ptx for h at ˚c µl mtt mgml solution was added to each well and incubated for h at ˚c subsequently µl dmso was added to each well to dissolve the blue formazan crystals and the absorbance was measured using a microplate reader biotek instruments inc at nmclone formation assay a total of 1x103 cells were seeded into a ‘mm dish in triplicate and maintained in f‘12k medium with or without ddp or ptx at ˚c for h a total of weeks later cells were fixed in paraformalde‘hyde for min at room temperature and stained with crystal violet dye at room temperature for min the rate of colony formation was calculated using the following equation number of coloniesnumber of seeded cells x100flow cytometry apoptosis was detected using a fitc annexin v apoptosis kit bd pharmingen bd biosciences according to the manufacturer's protocol cells 1x105 were collected and washed twice with pbs prior to being suspended in µl binding buffer subsequently cells were incubated with µl annexin v‘fitc and µl propidium iodide in the dark for min at room temperature and apoptosis was analyzed using a cytoflex flow cytometer beckman coulter inc data were analyzed using cytexpert software beckman coulter inc the ratio between early and late apoptosis was calculatedwestern blotting cells were collected washed twice with pbs and lysed with ripa lysis buffer thermo fisher scientific inc proteins were isolated from the cell lysis buffer and 0concology letters quantified using the piercetm„¢ bca protein assay kit cat no thermo fisher scientific inc with bovine serum album as a standard equal amount of protein µg proteins were separated by sds‘page gel next the proteins were transferred onto a polyvinylidene membrane thermo fisher scientific inc blocked with bsa thermo fisher scientific inc for h at ˚c and incubated overnight at ˚c with primary antibodies against chl1 cat no ‘‘ap proteintech inc multi‘drug resistance gene mdr1 cat no ‘‘ap proteintech inc multidrug resistance‘associated protein mrp cat no ‘‘ig proteintech inc low‘density lipoprotein receptor‘related protein lrp cat no ‘‘ap proteintech inc phosphorylated p‘akt cat no ab38449 abcam and akt cat no ab227385 abcam after washing three times with pbs the membrane was incubated with horseradish peroxide‘conjugated goat anti‘rabbit cat no ab6271 abcam_or rabbit anti‘mouse cat no ab6728 abcam secondary antibodies for h at room temperature and the blots were detected with enhanced chemiluminescence reagent thermo fisher scientific inc protein expression was quantified using image‘pro plus software media cybernetics incanimal experiments the animal experiments were approved by the medical ethics committee of xiangya changde hospital approval no and were performed in compliance with all regulatory institutional guidelines for animal welfare the national institutes of health publications no ‘ a total of male balbc‘nu mice ‘week‘old ± g hunan sja laboratory animals center of the chinese academy of sciences were used in this study all animals were kept at the spf level laboratory at ‘Ëšc a relative humidity of ‘ a h lightdark cycle and timesh of fresh air exchange all mice were given free access to food and water the bedding materials drinking water feeding cages and other items in contact with the animals were all autoclaved prior to use a549ddp cells 1x107 transfected with empty vector and chl1 overexpression vector using lipofectamine® reagent thermo fisher scientific inc were subcu‘taneously injected into the nude mice to establish xenograft models following anaesthesia with chloral hydrate mgkg xenografts were allowed to grow to mm3 over weeks and the mice were randomly divided into four groups n3group as follows i vector group a549ddp cells transfected with empty vector and treated with µl saline solution ii vector‘ddp group a549ddp cells trans‘fected with empty vector and treated with mgkg ddp iii chl1 group a549ddp cells transfected with chl1 overexpression vector and treated with µl saline solu‘tion and iv chl1‘ddp group a549ddp cells transfected with chl1 overexpression vector and treated with mgkg ddp ddp was administered by intraperitoneal injection every days for weeks the mice were observed daily and the tumors were measured by a vernier caliper every days the tumor volumes were calculated as length x width22 a total of weeks post‘injection mice were euthanized with co2 at volume displacement rate vdr per min using a programmable logic controller barry‘wehmiller design group inc mice were monitored continuously and once the mice were immobile except for breathing for min the vdr was provided at for min the animals remained in the euthanasia chamber for min and were then observed for an additional min breathing and heart rate were monitored to determine deathstatistical analysis all experiments were performed in tripli‘cate and data are presented as the mean ± standard deviation all experiments were performed at least three times paired student's t‘test was performed for comparisons between two groups and one‘way analysis of variance followed by tukey's multiple comparison post‘hoc analysis was performed for comparisons between multiple groups spss ibm corp was used to perform the analysis p005 was considered to indicate a statistically significant differenceresultschl1 is downregulated in a549ddp and a549ptx‘resistant cells in order to investigate the mechanism of chemore‘sistance in lung cancer the lung adenocarcinoma cell line a549 the ddp‘resistant cells a549ddp and ptx‘resistant cells a549ptx were used in the present study cells were exposed to different concentrations of ddp ‘ µgml and ptx ‘ ngml and mtt assay was used to detect the cell survival rate a549ddp and a549ptx cells demonstrated higher resistance to ddp and ptx compared with a549 cells fig 1a the half maximal inhibitory concentration ic50 of ddp was significantly higher in a549ddp cells ± µgml compared with a549 cells ± µgml and the ic50 of ptx was significantly higher in a549ptx cells ± ngml compared with a549 cells ± ngml fig 1b in addition the expression levels of the drug‘resistant markers mdr1 mrp and lrp were significantly higher in a549ddp and a549ptx cells compared with a549 cells fig 1c additionally the mrna and protein expression levels of chl1 were significantly lower in a549ddp and a549ptx cells compared with those in a549 cells fig 1d and e and this was also observed in h460 ddp‘resistant cells obtained from the geo dataset gse21656 fig 1f these results suggested that chl1 may be involved in regulating ddp and ptx resistance in nsclcknockdown of chl1 enhances resistance to ddp and ptx in a549 cells as chl1 was upregulated in a549 cells chl1 was silenced in a549 cells using sirnas chl1 expres‘sion was significantly reduced in the chl1 sirna groups compared with that of the scrambled control group fig 2a as sirna‘ demonstrated the greatest interference efficiency it was selected for use in the following experiments notably chl1‘knockdown enhanced the resistance to ddp and ptx in a549 cells fig 2b and c colony formation assay revealed that compared with the control group chl1‘knockdown significantly increased the rate of colony formation in the absence of chemotherapeutics and enhanced the resistance to ddp and ptx fig 2d flow cytometry results demon‘strated significantly reduced apoptosis in chl1‘knockdown cells after ddp and ptx treatment compared with that of the control group fig 2e 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure chl1 is downregulated in ddp and ptx‘resistant a549 cells a cell survival of a549 and a549‘resistant cells a549ddp and a549ptx treated with increasing concentrations of ddp and ptx as assessed by mtt assay b the ic50 values of ddp in a549ddp and a549 cells and the ic50 values of ptx in a549ptx and a549 cells p005 vs a549 cells c western blotting demonstrated the expression of drug resistance‘related proteins mdr1 mrp and lrp in a549 cells and a549‘resistant cells a549ddp and a549ptx p005 vs a549 cells the protein and mrna expression levels of chl1 in a549 cells and a549‘resistant cells a549ddp and a549ptx were analysed by d western blotting and e reverse transcription‘quantitative pcr respectively p005 vs a549 cells f the mrna expression of chl1 in h460 and h460ddp cells in the gse21656 dataset p005 vs h460 cells chl1 close homolog of l1 ddp cisplatin ptx paclitaxel mdr1 multi‘drug resistance gene mrp multidrug resistance‘associated protein lrp low‘density lipoprotein receptor‘related protein ic50 half maximal inhibitory concentration chl1 overexpression enhances the sensitivity of a549 resistant cells to ddp and ptx as chl1 is downregu‘lated in a549ddp and a549ptx cells the present study successfully overexpressed chl1 in these cells using chl1 recombinant expression plasmids fig 3a the results demonstrated that chl1 overexpression alleviated the resis‘tance to ddp and ptx compared with that of the control group fig 3b and c in addition chl1 overexpression inhibited colony formation in the absence or presence of ddp and ptx fig 3d additionally flow cytometry results demonstrated that restoration of chl1 expression promoted apoptosis in resistant cells following ddp and ptx treat‘ment fig 3eto further validate the effects of chl1 overexpression on ddp or ptx sensitivity xenograft mice model experi‘ments were performed the results demonstrated that chl1 overexpression or ddp treatment significantly impeded the tumor growth fig 3f and decreased the tumor weight fig 3g in addition chl1 overexpression further aggra‘vated ddp‘mediated repression on tumor growth fig 3f and g these data suggested that chl1 overexpression suppressed tumor growth and enhanced the chemosensitivity in nsclcchl1 mediates chemosensitivity by inhibiting akt activity recently studies have confirmed that chl1 inhibits akt activity in escc and neuroblastoma cell lines thus the present study investigated whether chl1 mediates chemoresistance via the akt pathway in nsclc in a549 cells compared with the scrambled group chl1‘knockdown elevated the expression of p‘aktser473 fig 4a by contrast restoring chl1 expression in a549ddp and a549ptx cells inhibited the akt phosphorylation compared with the control group fig 4a suggesting chl1 mediates chemosensitivity via the akt pathway subsequently chl1‘silenced a549 cells were treated with the akt inhibitor sc66 and it was demon‘strated that inhibiting akt activity significantly reduced the promotive effects on cell survival fig 4b and clone forma‘tion fig 4c and the inhibitory effects on apoptosis fig 4d induced by chl1‘depletion these results confirmed that chl1 mediates chemosensitivity in nsclc by inhibiting the akt pathway 0concology letters figure chl1‘knockdown increases a549 cell resistance to ddp and ptx a western blotting was performed to validate the efficiency of transfection with chl1 sirnas p005 vs scramble mtt assays were performed to determine the survival rate of chl1‘knockdown a549 cells treated with b ‘ µgml ddp or c ‘ ngml ddp d colony formation assay of a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was used to detect apoptosis in a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx p005 p0001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering discussionthe results of the present study demonstrated that chl1 was significantly downregulated in a549ddp and a549ptx cells compared with a549 cells the knockdown of chl1 in a549 cells facilitated the cell survival and clone formation and decreased apoptosis when treated with or without ddp and ptx whereas chl1 overexpression in a549ddp and a549ptx cells inhibited cell survival and clone formation and increased apoptosis the results of the present study also demonstrated that chl1 enhances nsclc chemosensitivity through inhibition of the akt pathway these data suggested that chl1 may be a promising target to improve the efficacy of chemosensitivity in nsclcchl1 belongs to the l1 family of nerve cell adhesion molecules it was initially cloned in mice and its expression in mouse development was analyzed by senchenko through cell‘cell interactions and mediating cell‘cell and cell‘matrix interactions chl1 has an important effect on the development regeneration and plasticity of the nervous system previous reports have demonstrated that chl1 also participates in carcinogenesis ‘ chl1 was observed to be significantly downregulated in up to types of tumor tissues compared with their adjacent normal tissues in most tumors chl1 is a potential tumor suppressor gene whose silencing is associated with tumor growth invasion and metastasis ‘ for example knockdown of chl1 expression results in enhanced cervical cancer cell invasion and migration a low expres‘sion of chl1 in patients with neuroblastoma predicts a poor prognosis and enhancing chl1 expression suppresses tumor progression in contrast chl1 has been reported to promote cell proliferation metastasis and migration in human gliomas however to the best of our knowledge research on chl1 and tumor chemoresistance has rarely been reported 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure overexpression of chl1 increases the sensitivity of resistant a549 cells to ddp and ptx a western blotting was performed to detect chl1 expression in a549ddp and a549ptx cells transfected with chl1 expression plasmids p005 vs vector effect of chl1 overexpression on resistant a549 cell survival rate when treated with b ‘ µgml ddp or c ‘ ngml ptx as determined by mtt assay d colony formation assays demon‘strated the number of colonies of resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was performed to assess apoptosis in resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx chl1 overexpression enhanced chemosensitivity of a549ddp cells to ddp in vivo which was demonstrated by the effect of ddp treatment or chl1 overexpression on the f growth and g weight of xenografts derived from a549ddp cells p005 p001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel the present study examined the differentially expressed genes in nsclc ddp‘resistant cells in a geo dataset chl1 was demonstrated to be upregulated in ddp‘resistant cells compared with parental cells suggesting that chl1 may be involved in nsclc chemotherapy resistance similarly a study that compared and analyzed the differentially expressed genes in chemosensitive tumors and chemoresistant ovarian adenocarcinomas tissues reported that the expression of chl1 in chemotherapy‘sensitive tumor tissues is higher compared with that in drug‘resistant tissues suggesting that chl1 may help to predict the efficacy of chemotherapy for ovarian cancer in addition aberrant methylation of chl1 may be associated with the recurrence of colorectal cancer crc following chemotherapy ‘azadc treatment restores ‘fluro‘uracil sensitivity in vitro which also suggests that chl1 may be involved in crc chemotherapy resistance the results of the present study demonstrated that chl1 was down‘regulated in a549ddp cells additionally as multiple drug resistance is a common characteristic another type of resistant cells a549tax cells were also used in the current study the results also demonstrated that chl1 was downregulated in a549ptx cells compared with control cells overexpression of chl1 significantly increased the sensitivity of cells resistant to ddp and ptx whereas knockdown of chl1 expression in 0concology letters figure chl1 mediates ddp and ptx sensitivity by inhibiting akt activity a western blotting was performed to detect the expression of p‘akt and total akt in chl‘silenced and ‘restored cell models p005 vs scramble or vector b mtt assays were performed to detect cell survival rates of a549 cells treated with chl1 sirna and akt inhibitor sc66 p005 c colony formation assays were performed in a549 cells treated with chl1 sirna and the akt inhibitor sc66 in the presence of ddp µgml or ptx ngml p005 vs si‘chl1 d apoptosis were measured in a549 cells treated with chl1 sirna and akt inhibitor sc66 in the presence of ddp µgml and ptx ngml p005 vs si‘chl1 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering p‘ phosphorylated parent a549 cells displayed the opposite results to the best of our knowledge this study is the first study to suggest that chl1 may be involved in chemosensitivity in lung cancer the concentration of ddp used in vivo is mgkg however this may not be in line with the concentrations that would be used in a clinical setting in a clinical trial the human initial dose was calculated from the no observed adverse effect levels noaels verified in animal experiments noael is the maximum dose level without significant adverse reactions the noael verified in animal experiments can be converted to a human equivalent dose according to the body surface area conversion which is based on the area standardization mgm2 proportional among different species in the present study the concentration of ddp used in vivo was not the noael thus it was not consistent with the concentrations used in clinical settingsakt is a serinethreonine protein kinase that is activated by phosphorylation as a key molecule of the pi3kakt signaling pathway p‘akt regulates cell survival cell growth cell motility and angiogenesis and prevents apoptosis additionally akt activation is associated with tumor chemore‘sistance the results of the present study demonstrated that compared with the control groups the expression of p‘akt was increased in chl1‘knockdown a549 cells and its expres‘sion was reduced in chl1 overexpressed a549ddp and a549ptx cells when akt activity was inhibited by the akt inhibitor the sensitivity to ddp and ptx in chl1‘knockdown a549 cells was restored this finding suggested that chl1 enhanced the chemosensitivity of nsclc by inhibiting the akt pathway considering numerous studies have confirmed that the akt pathway mediates chemoresistance via regulation of atp binding cassette abc members ‘ the present study didn't further investigate the specific abc members and mechanisms which was a of the limitation to the present study thus this research should be further investigated in vivoin summary the present study demonstrated that chl1 was downregulated in resistant cells a549ddp and a549ptx and upregulation of chl1 enhanced the chemosensitivity of nsclc via inhibiting the akt pathway to the best of our knowledge this was the first study to confirm the function and 0ccai chl1 enhances the chemosensitivity of lung cancer cellsmechanism of chl1 in mediating chemosensitivity in cancer thus the development of chl1‘based therapeutic strategies may improve the efficacy of chemosensitivity in nsclcacknowledgementsthe authors of the present study would like to thank mr dingliang li xiangya hospital changsha china for his guidance and assistance in flow cytometric analysisfundingno funding was receivedavailability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author upon reasonable requestauthors' contributionsrh conceived and designed the present study xc bh yh and pl performed experiments and collected the data sl zz and zh analyzed and interpreted the data ml and lz analyzed the data and prepared the figure xc ml and lz drafted the initial manuscript and revised it for intellectual content all authors read and approved the final manuscriptethics approval and consent to participatethe animal experiments were approved by the medical ethics committee of xiangya changde hospital changde china approval no patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences parascandola m and xiao l tobacco and the lung cancer epidemic in china transl lung cancer res suppl s21‘s30 chen w cancer statistics updated cancer burden in china chin j cancer res oser mg niederst mj sequist lv and engelman ja transformation from non‘small‘cell lung cancer to small‘cell lung cancer molecular drivers and cells of origin lancet oncol e165‘e172 thatcher n faivre‘finn c blackhall f anderson h and lorigan p sequential platinum‘based chemotherapy‘thoracic radiotherapy in early stage non‘small cell lung cancer clin cancer res suppl s5051‘s5056 yano t okamoto t fukuyama s and maehara y therapeutic strategy for postoperative recurrence in patients with non‘small cell lung cancer world j clin oncol ‘ fang z chen w yuan z liu x and jiang h lncrna‘malat1 contributes to the cisplatin‘resistance of lung cancer by upregulating mrp1 and mdr1 via stat3 activation biomed pharmacother ‘ cai y dong zy and wang jy lncrna nnt‘as1 is a major mediator of cisplatin chemoresistance in non‘small cell lung cancer through mapkslug pathway eur rev med pharmacol sci ‘ han ml zhao yf tan ch xiong yj wang wj wu f fei y wang l and liang zq cathepsin l upregulation‘induced emt phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in a549 cells acta pharmacol sin ‘ liu j meisner d kwong e wu xy and johnston mr translymphatic chemotherapy by intrapleural placement of gelatin sponge containing biodegradable paclitaxel colloids controls lymphatic metastasis in lung cancer cancer res ‘ hassan wa yoshida r kudoh s kameyama h hasegawa k niimori‘kita k and ito t notch1 controls cell chemoresistance in small cell lung carcinoma cells thorac cancer ‘ tang h jiang l zhu c liu r wu y yan q liu m jia y chen j qin y loss of cell adhesion molecule l1 like promotes tumor growth and metastasis in esophageal squamous cell carcinoma oncogene ‘ liu h focia pj and he x homophilic adhesion mechanism of neurofascin a member of the l1 family of neural cell adhesion molecules j biol chem ‘ tassano e biancheri r denegri l porta s novara f zuffardi o gimelli g and cuoco c heterozygous deletion of chl1 gene detailed array‘cgh and clinical characterization of a new case and review of the literature eur j med genet ‘ morellini f lepsveridze e kahler b dityatev a and schachner m reduced reactivity to novelty impaired social behavior and enhanced basal synaptic excitatory activity in perforant path projections to the dentate gyrus in young adult mice deficient in the neural cell adhesion molecule chl1 mol cell neurosci ‘ he lh ma q shi yh ge j zhao hm li sf and tong zs chl1 is involved in human breast tumorigenesis and progres‘sion biochem biophys res commun ‘ martín‘sánchez e mendaza s ulazia‘garmendia a monreal‘santesteban i blanco‘luquin i córdoba a vicente‘garcía f pérez‘janices n escors d megías d chl1 hypermethylation as a potential biomarker of poor prog‘nosis in breast cancer oncotarget ‘ zhu h fang j zhang j zhao z liu l wang j xi q and gu m mir‘ targets chl1 and controls tumor growth and invasion in papillary thyroid carcinoma biochem biophys res commun ‘ yu w zhu k wang y yu h and guo j overexpression of mir‘‘5p promotes proliferation and invasion of colon adeno‘carcinoma cells through targeting chl1 mol med hötzel j melling n müller j polonski a wolters‘eisfeld g izbicki jr karstens kf and tachezy m protein expression of close homologue of l1 chl1 is a marker for overall survival in non‘small cell lung cancer nsclc j cancer res clin oncol ‘ sun y zheng s torossian a speirs ck sc Answer:

Colon_Cancer

" colorectal cancer crc is the third leading cause of cancer‘associated mortality the present study aimed to investigate novel biomarkers to predict prognosis and provide a theoretical basis for studies of the pathogenesis and the development of therapies for crc the present study compared mrna expression levels of patients with crc with short‘ and long‘term prognosis and of individuals with and without tumors in the cancer genome atlas tcga data‘base differentially expressed genes degs were identified via volcano plot and venn diagram analysis gene ontology go analysis and gene set enrichment analysis gsea were performed to identify the functions of the degs and the degs were further verified using clinical crc samples a total of degs were identified as candidate genes using the tcga database and four degs [defensin 4a defb4a hyaluronan binding protein habp2 oleoyl‘acp hydrolase and tbc1 domain family member 3g] were associated with poor prognosis of patients with crc two degs defb4a and habp2 were upregulated in tumor tissues of patients with crc in the tcga database go and gsea analyses revealed that defb4a was highly associated with immunosuppression participates in ˜myeloid leukocyte differentiation™ ˜leukocyte proliferation™ and ˜positive regulation of leukocyte‘mediated immunity™ and was positively correlated with cd11b cd14 cd45 cd163 and il17a furthermore defb4a expres‘sion was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis survival analysis revealed that defb4a upregulation was associated with poor prognosis defb4a correspondence to dr yi zhang biotherapy center the first affiliated hospital of zhengzhou university jianshe east road zhengzhou henan pr chinae‘mail yizhangzzueducncontributed equallykey words defensin 4a colorectal cancer prognosis immunity biomarkergene knockdown experiments demonstrated that def4ba promotes cell migration these results indicated that defb4a potentially promotes tumor growth by regulating immu‘nosuppressive activity and provided novel insights into the diagnosis and treatment of crcintroductionaccording to the global cancer statistic of colorectal cancer crc is the third most common cancer and the second leading cause of cancer‘associated mortality worldwide crc was reported as the fourth most common and fatal cancer in china in patients with crc usually have a low survival rate and poor therapeutic responses and are susceptible to progression and recurrence early diagnosis and effective treatment are critical to improve the survival of patients with crc crc studies have focused on innovative ideas to identify molecular markers used to develop high‘precision non‘invasive screening tests for crc to increase population compliance and reduce the poten‘tially harmful side effects associated with more invasive techniques diagnostic markers will give an indication of the likely progression of the disease targeting specific molecules in certain patients has facilitated more personalized treatments that help prevent or decelerate cancer progression the present study aimed to determine prognostic factors and novel therapeutic targets to improve the survival of patients with crcprevious studies have focused on the identification of molecules associated with tumor progression through genetic or mrna profiling and screening of patients with colon cancer ‘ for example the expression profiles of long non‘coding rnas lncrnas were compared at specific tumor stages t0 t1 t2 and t3 in an azoxymethanedextran sodium sulfate‘induced primary colon cancer model and upregulation of the lncrna h19 predicted a poor prog‘nosis other studies analyzed microrna mir expression profiles between tumor tissues and matched non‘tumor tissues obtained from patients with crc for example mir‘ is significantly downregulated in tumor tissues and associated with poor survival of patients with crc and may thus be considered to be a poor prognostic marker of crc furthermore high expression levels of mir‘ 0cwu prognostic role of defb4a in colorectal cancerand mir‘ in serum are associated with poor survival of patients with crc analysis of the cancer genome atlas tcga database revealed that mmp19 is upregulated in patients with crc and is associated with tumor progres‘sion however to the best of our knowledge no study has directly screened mrna profiles based on prognosis the present study divided patients with crc into different groups based on prognosis and screened the mrna profiles of the respective groups defensin 4a defb4a also known as bd‘ sap1 defb2 defb4 hbd‘ defb‘ and defb102 belongs to the defensin family comprising cytotoxic peptides secreted by neutrophils which serve important roles in innate immune defense against microbial infections ‘ defb4a is upregulated in cutaneous squamous cell carcinoma and basal cell carcinoma it serves an important role in esopha‘geal carcinogenesis both in vivo and in vitro the genomic copy number of defb4a has been analyzed in patients with crohn's disease and controls and an elevated defb4a copy number has been identified as a risk factor for crohn's disease regardless of disease origin however it remains unclear whether defb4a expression is associated with the prognosis of crc furthermore the role of defb4a in the immune system remains unclear the tumor microenvironment serves a significant role in tumor progression various immune elements comprise the tumor microenvironment including bone marrow‘derived cells such as macrophages cd4 t cells cd8 t cells b cells natural killer cells and dendritic cells myeloid cells can differentiate into macrophages or myeloid‘derived suppressor cells mdscs which serve a tumorigenic role in the tumor microenvironment mdscs contribute to tumor vascular development by promoting angiogenesis and tumor growth tumor‘associated macrophages tams are important regulators of tumorigenesis by inhibiting the anti‘tumor effects of other cells thus promoting tumor growth however it remains unclear whether defb4a has a regulatory effect on the tumor microenvironment or whether it promotes crc progressionto identify candidate target genes that potentially prolong patient survival mrna expression profiles of tissues from crc samples were compared in the tcga database venn analysis was performed to determine candidate genes upreg‘ulated in tumor tissues among patients with poor prognosis subsequently immune‘associated pathway enrichment was analyzed using gene ontology go and gene set enrichment analysis gsea and the correlations between candidate target genes and certain immune cells were determined finally clinical samples and crc cell lines were obtained to verify the clinical significance of the identified genes the present results may provide insights into targeted therapy for crcmaterials and methodsacquisition of microarray data microarray data were obtained from tcga httpcancergenomenihgov rna‘seq data for samples were included in the dataset project id tcga‘coadread including tumor samples from patients with crc and normal tissues from healthy donorsidentification of differentially expressed genes degs tcga data were divided into two groups based on different categories patient prognosis and gene expression in tumor and normal tissues venn analysis of the two groups was performed and genes associated with crc prognosis were identifiedvenn analysis to identify candidate genes associated with patient survival the gene expression profiles in the two groups were analyzed using the venn diagram web tool httpbioinformaticspsbugentbewebtoolsvenngo analysis functional analysis of the degs was performed using go httpwwwgeneontology based on biological processes gsea gsea was conducted using gsea v403 software wwwgsea‘msigdbgseaindexjsp and the gene used in the present study was downloaded from the molecular signatures database msigdb oftware broadinstitutegseamsigdbindexjsp v40 msigdb curates various gene sets including canonical signaling pathways from biocarta cgapncinihgovcgap_mitelman_retire_noticehtml kyoto encyclopedia of genes and genomes wwwkeggjp pid httppidncinihgov reactome reactome and other pathway databases tcga data were analyzed via gsea and pathways with a false discovery rate fdr were considered significantpatient characteristics tissue samples were obtained from patients with crc at the first affiliated hospital of zhengzhou university zhengzhou china between april and april patients underwent surgical resection or colonoscopy and the samples were verified via pathological analysis the clinical characteristics of the patients are shown in table i a total of men and women were included in the present study the median age was years age range ‘ years crc was diagnosed by two pathologists on the basis of pathological assessment the collection of speci‘mens was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university zhengzhou china approval no science‘‘lw‘ and informed consent was obtained from each patient with available follow‘up informationreverse transcription‘quantitative pcr total rna was extracted from pairs of tumor and normal tissue samples from patients with crc using trizol reagent invitrogen thermo fisher scientific inc total rna samples µg were incubated at ˚c for min followed by incubation at ˚c for min and ˚c for sec according to the reverse transcrip‘tion reaction protocol takara biotechnology co ltd the conditions of pcr were as follows ˚c10 min ˚c10 sec ˚c10 sec ˚c10 sec cycles premix ex taq ii roche target gene expression was simultaneously assessed relative to that of gapdh a housekeeping gene and internal control the following primers were used defb4a forward '‘ctc ctc ttc tcg ttc ctc ttc a‘' and reverse '‘gca ggt aac agg atc gcc tat‘' and gapdh forward '‘gga gcg aga tcc ctc caa aat‘' and reverse '‘ggc tgtt 0concology letters no of cases table i characteristics of patients with colorectal carcinomacharacteristic percentagesex male female age years ‰¥ treatment surgery others tumor size mm ‰¥ pathological type adenocarcinoma others lymph node metastasis yes no tnm stage i ii iii iv liver metastasis negative positive differentiation poor medium‘well ˜others™ include chemotherapy and radiotherapy gtc ata ctt ctc atg g‘' the present study compared the expression levels of the target genes in clinical samples using the ‘δδcq method expression levels of defb4a and gapdh were examined for each sample and the relative expression levels of defb4a were determined using the ‘δcq value of defb4a divided by that of gapdh cell transfection sw480 and hct116 cells were seeded into a ‘well plate sw480 and hct116 cells were purchased from chinese academy of sciences cell bank and cultivated with dmem‘high glucose containing fbs hyclone ge healthcare life sciences and penicillin‘streptomycin at ˚c in co2 the growth status of the cells was closely observed until they reached a fusion rate of and then cells were transfected with nc‘small interfering rna nega‘tive control si‘nc sense '‘uuc ucc gaa cgu guc acg utt‘' and antisense '‘acg uga cac guu cgg aga att‘' and small interfering rna targeting defb4a si‘defb4a si‘defb4a sense '‘ucc ucu uca uau ucc uga utt‘' and antisense '‘auc agg aau aug aag agg att‘' purchased from shanghai genepharma co ltd with jetprime polyplus transfection reagent polyplus‘transfection sa after h the medium was changed to fresh medium and cells were further incubated in co2 for h subsequently cells were collected for the subsequent experimentswound healing assay for the wound healing assay sw480 and hct116 cells were cultured in µl medium with fbs hyclone and the percentage of serum was in line with previous papers sub‘confluent tumor cells ‘ were scraped using a sterile micropipette tip and then serum‘free medium was added next cells were imaged at and h using an inverted fluorescence microscope magnification x200 olympus corporation transwell assay in the migration test the transfected cells 1x105 were inoculated into the top chamber microns with µl serum‘free medium complete medium µl containing fbs was added to the lower chamber corning inc following incubation at ˚c for h the migratory cells located under the insert were fixed and stained with crystal violet staining solution beyotime institute of biotechnology at room temperature for min and observed using an inverted fluorescence microscope magnification x200 olympus corporationstatistical analysis the χ2 test was used to compare clinicopathological factors and continuous variables were analyzed via unpaired student's t‘test or one‘way anova kaplan‘meier analysis and the log rank test were performed for survival analysis univariate and multivariate logistic regression models confirmed the associations between defb4a expression and clinical features prism graphpad software inc was used for statistical analysis of all clinical samples anova was followed by tukey's post‘hoc test and performed using spss for windows spss inc r software version r foundation for statistical computing was used for bioinformatics analysis p005 was considered to indicate a statistically significant difference all experi‘ments were performed in triplicate and data are presented as the mean ± standard deviation resultsgenes associated with poor prognosis to identify genes associated with poor survival in the crc cohort patients were divided into two groups based on os short ‰¤ days os patients with a survival time of days would be included in short os and long days os fig 1a in total degs fold change were identified using a volcano plot including upregulated and downregulated genes fig 1b and c hierarchical cluster analysis revealed the expression profiles of the degs fig 1d subsequently gene expression profiles in tumor and normal tissues fold change were analyzed and it was observed that genes were upregulated and were downregulated in tumor tissues compared with in normal tissues p005 fdr fold change fig 1e‘g the venn diagram revealed that 0cwu prognostic role of defb4a in colorectal cancerfigure screening of degs based on tcga a patients with crc were divided into two groups based on whether or not they survived for days in accordance with the rna‘seq data from tcga b volcano plot of rna‘seq data from tcga the red dots and blue dots represent upregulated and downregulated degs based on a fold change of the volcano plot displays different genes when comparing patients with a prolonged os and those with a short os c a total of upregulated and downregulated genes were identified d hierarchical clustering analysis of the rna‘seq data of different genes in short os and long os samples e hierarchical clustering analysis of the rna‘seq data of different genes in ca and n samples f using a threshold of p005 false discovery rate and fold change degs were selected using a volcano plot when comparing ca samples with normal colon mucosa samples from tcga g a total of upregulated and downregulated genes were identified h venn diagram representing the distribution of degs in different groups a total of degs were expressed in both patients with ca and patients with a prolonged os crc colorectal carcinoma degs differentially expressed genes os overall survival tcga the cancer genome atlas ca cancer n normal degs were identified in both screening methods fig 1h details are shown in table iivalidated degs are associated with poor prognosis in tcga to determine the prognostic significance of the identified degs their expression levels were determined in cases included in tcga kaplan‘meier survival analysis revealed that defb4a hyaluronan binding protein habp2 oleoyl‘acp hydrolase olah and tbc1 domain family member 3g tbc1d3g upregulation was significantly associated with poor survival in patients with crc fig 2a prognostic significance was not observed for kiss1r or5m11 chrnb3 otx2 s100a7a flj43860 and frmd7 in the patients with crc data not shown furthermore defb4a and habp2 were upregulated in tumor tissues fig 2b a previous study have reported low serum expression levels of habp2 in patients with crc therefore defb4a was selected as a candidate marker of poor prognosis in patients with crcgo and gsea to evaluate the biological role of defb4a in crc progression go enrichment and gsea analyses were 0concology letters descriptiontable ii upregulated genes n10 associated with a poor colorectal carcinoma prognosis in the cancer genome atlas databasegene symbol defb4a habp2 olah tbc1d3g kiss1r frmd7 s100a7a otx2 or5m11 chrnb3 defensin beta 4ahyaluronan binding protein oleoyl‘acp hydrolasetbc1 domain family member 3gkiss1 receptorferm domain containing s100 calcium binding protein a7aorthodenticlehomeobox olfactory receptor family subfamily m member cholinergic receptor nicotinic beta subunitgene id performed defb4a was demonstrated to be involved in various biological processes associated functional pathways are shown in fig 3a and b and closely associated with ˜myeloid leukocyte differentiation™ ˜leukocyte proliferation™ and ˜leukocyte mediated immunity™ implying that defb4a potentially regulates the immune system finally the data‘base was searched for expression profiles of defb4a and immune‘related genes and a positive correlation between defb4a expression and the expression of immune markers such as cd11b cd14 cd45 cd163 and il17a was observed fig 3c these results suggest that defb4a is associated with poor prognosis in patients with crc potentially in an immu‘nosuppressive myeloid leukocyte‘ and cytokine‘dependent mannervalidation in patient samples and clinical relevance of defb4a to further clarify the clinical significance of defb4a expression the present study analyzed tissue samples from patients with crc the associations between their mrna expression levels and clinicopathological vari‘ables were observed detailed information of the patients is provided in table iii defb4a expression was significantly upregulated in the crc tumor tissues fig 4a additionally an association between defb4a upregulation and advanced crc stage stage i cases stage ii cases stage iii cases stage iv cases and metastasis m0 cases m1 cases was observed fig 4b and c furthermore defb4a upregulation in the tumor tissues was associated with poor prognosis p00313 fig 4d additionally defb4a upregulation was significantly associated with advanced liver metastasis p0039 stage p0005 high ca72‘ value p0003 tumor size p0009 and lymph node metastasis p0044 table iii therefore defb4a was considered to be a prognostic marker associated with tumor progres‘sion in patients with crc logistic regression analysis was performed to determine whether defb4a can help predict the prognosis of crc univariate analyses revealed that advanced tnm stage [odds ratio or p001] liver metastasis or p003 lymph node metastasis or p004 high ca199 level or1324 p002 a high ca ‘ level or p001 and high defb4a level or p002 were associated with the survival of patients with crc furthermore multivariate analyses revealed that advanced tnm stage or p004 histological differentiation or p001 liver metastasis or p001 ca199 level or p001 high ca ‘ level or p005 and high defb4a level or p001 were independent prognostic predictors table iv overall these results suggest that defb4a serves an important role in predicting the prognosis of patients with crcdefb4a promotes proliferation and metastasis in crc to explore the biological roles of defb4a in crc defb4a expression was knocked down in hct116 and sw480 cells fig 5a and b a wound healing assay revealed that defb4a knockdown inhibited the migration of hct116 and sw480 cells fig 5c transwell assays demonstrated that the migration of cells was decreased following knockdown of defb4a in sw480 cells compared with that in the nc group fig 5d the number of migratory cells decreased following knockdown of defb4a in sw480 cells fig 5e overall these results suggested that defb4a serves an important role in crc development discussionwith the increasing availability of high‘throughput technolo‘gies numerous novel biomarkers and therapeutic targets have been identified through transcriptomic analysis of various types of tumor however such studies on biomarkers in crc have not been extensively performed the identification of crc biomarkers may help predict and prolong the survival of patients with crc in the present study mrna profiling of microarray analysis data from the tcga database was performed to identify numerous novel genes associated with poor prog‘nosis in crc a critical role of defb4a in patients with crc was identified the mrna profiles of patients were first compared between the long os and short os groups and between the tumor and normal tissue groups in the tcga database subsequently the present study investigated the association between mrna expression and prognosis defb4a habp2 olah and tbc1d3g were identified as potential predicators of poor prognosis defb4a and habp2 0cwu prognostic role of defb4a in colorectal cancerfigure defb4a is upregulated based on data from tcga and predicts poor prognosis a kaplan‘meier curve of four genes defb4a habp2 olah and tbc1d3g derived from data of patients included in the tcga dataset b mrna expression levels of defb4a habp2 olah and tbc1d3g in cancer vs control samples from patients in tcga p005 ns not significant tcga the cancer genome atlas defb4a defensin 4a habp2 hyaluronan binding protein olah oleoyl‘acp hydrolase tbc1d3g tbc1 domain family member 3g ca cancer n normalwere upregulated in crc tissues of patients in the database however habp2 has been reported to be downregulated in the sera of patients with crc p00137 therefore defb4a was considered as a candidate gene for further analysis go and gsea were used to assess the function of defb4a in promoting disease progression and to highlight the role of defb4a in the tumor microenvironment defb4a was involved in ˜myeloid leukocyte differentiation™ ˜leukocyte proliferation™ and ˜leukocyte mediated immunity™ correlation analysis revealed that defb4a expression was positively correlated with immune markers including cd11b cd14 cd45 cd163 and il17a cd11b is expressed on the surface of a number of leukocytes including monocytes granulocytes and macrophages cd14 is expressed on both monocytes 0concology letters figure defb4a is positively correlated with inhibitory immune cells a gene ontology analysis revealed that defb4a is involved in ˜leukocyte proliferation™ ˜lymphocyte differentiation™ ˜leukocyte mediated immunity™ ˜myeloid cell differentiation™ ˜negative regulation of type i interferon production™ and ˜interleukin‘ production™ b gene set enrichment analysis verified the results c correlation between defb4a and cd11b cd14 cd45 cd163 and il17a r and p‘values are indicated defb4a defensin 4a r pearson's correlation coefficientfigure defb4a predicts poor prognosis in colorectal cancer a defb4a mrna expression in groups of cancer tissues and normal tissues are displayed b defb4a mrna levels were compared with respect to tnm stage c defb4a mrna expression levels of patients with different cancer stages d effect of defb4a expression on overall survival in patients with colorectal carcinoma n52 p005 p001 p00001 defb4a defensin 4aand macrophages and cd45 is expressed on leukocytes m2 macrophages may be marked with cd163 and m2 macro‘phages serve a role in promoting tumor growth the os of patients with non‘small‘cell lung cancer and those with esophageal cancer with high m2 macrophage infil‘tration rates is shorter than those with low m2 macrophage infiltration rates patients with high expression levels of il‘17a had a poor prognosis in a crc cohort previous studies have suggested that increased il‘17a promotes crc in various animal models ‘ analysis of clinical specimens of patients with crc demonstrated that defb4a expression was associated with 0cwu prognostic role of defb4a in colorectal cancer 0330a total n χ2 p‘valuedefb4a expressiontable iii association between defb4a expression and clinicopathological characteristics of patients with colorectal carcinoma characteristic sex male female age years ‰¥ site of lesion colon rectum differentiation poor well tumor size cm ‰¥ pathological type adenocarcinoma others lymph node metastasis no yes liver metastasis no yes stage iii iiiiv cea normal high ca ‘ normal high ca ‘ normal high afisher's exact test all others were assessed using a χ2 test defb4a defensin 4a‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘high n low n 0009a0003a 0575a0039a poor survival furthermore defb4a expression was upregu‘lated in patients with crc with advanced and metastatic cancer patients with crc with high defb4a expression had poor survival in addition knockdown of defb4a affected the migration ability of crc cells tcga data of patients with crc were used to identify the degs between the long os days and short os days groups in addition mrna expression was compared between tumor tissues and normal tissues in the same database defb4a was highly expressed in tumors and associated with a poor prognosis defb4a upregulation was associated with poor prognosis and defb4a expression was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis another study used the gene expression omnibus database to screen genes that are increased in patients with recurrence hierarchical clustering and pathway analyses revealed that thrombospondin thbs2 and cartilage 0concology letters table iv logistic regression model analysis of liver metastasis predictors in patients with colorectal carcinoma characteristics sex male vs female age vs ‰¥ years tumor size vs ‰¥ mm pathological type adenocarcinoma vs others tnm stage iii vs iiiiv differentiation medium vs poor liver metastasis no vs yes lymph node metastasis no vs yes cea vs ‰¥ ca199 vs ‰¥ ca724 vs ‰¥ defb4a high vs low or odds ratio‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘ or p‘value univariate ci ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘or p‘value multivariate ci ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ figure defb4a promotes colorectal cancer cell migration a expression levels of defb4a were detected using rt‘qpcr following transfection of hct116 cells with si‘nc and si‘defb4a b expression levels of defb4a were detected using rt‘qpcr following transfection of sw480 cells with si‘nc and si‘defb4a c migration ability of cells was examined using a wounding healing assay d representative images were obtained for the transwell assay magnification x200 e proportions of migrated cells after h were quantified defb4a defensin 4a nc negative control rt‘qpcr reverse transcription‘quantitative pcr si small interfering rna p001 p0001 0cwu prognostic role of defb4a in colorectal canceroligomeric matrix protein comp are associated with the ecm‘receptor interaction focal adhesion and tgf‘ signaling pathways the hypergeometric distribution test demonstrated that the association between thbs2 and crc is stronger than that of comp pearson test results indicated that thbs2 might be considered to be a prognostic biomarker for crc to the best of our knowledge this screening method and the hypothesis that defb4a may serve a pro‘tumor role through immunosuppression have not been seen in other studies defb4a stimulates keratinocytes to release il‘ and il‘ pro‘inflammatory cytokines serving as deciding factors in the pathogenesis of psoriasis furthermore defb4a induction is required for toll‘like receptor tlr activation in monocytes through the convergence of il‘ and vitamin d receptor signaling and exerts direct bactericidal effects against m tuberculosiss the antimicrobial peptides defb4a and camp are inhibited by hsa‘mir‘ leading to suppression of the tlr21‘induced vitamin d antimicrobial signaling pathway defb4a has been suggested as a biomarker for psoriasis because the clinical efficacy of targeted antibody therapy in psoriasis is associated with the inhibition of defb4a expression defb4a expression can directly be inhibited by anthralin in vitro and in vivo thus benefiting patients with psoriasis however it has remained unclear whether defb4a is involved in the immunoregulation in crc go analysis revealed that defb4a is involved in ˜myeloid leukocyte differentiation™ ˜leukocyte proliferation™ and ˜positive regulation of leukocyte‘mediated immunity™ therefore defb4a may be associated with immunity in crc to the best of our knowledge the present study was the first to report defb4a as a prognostic marker for crc and as an immunoregulatory factor in the tumor microenvironment in patients with crc however a limitation of the present study was that the research cohort was not large enough which may affect the statistical results in addition the specific role of defb4a and immune factors in colon cancer and the underlying molecular mechanism need to be further exploredin conclusion to the best of our knowledge defb4a is upregulated in patients with crc and is closely associated with poor prognosis defb4a regulates immune function and potentially promotes immunosuppression therefore defb4a may be considered as a prognostic marker and immunotherapeutic target for crcacknowledgementsnot applicablefundingthe present study was supported by grants from the national natural science foundation of china grant nos u1804281 and and funding from state's key project of research and development plan grant no 2016yfc1303500availability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author on reasonable requestauthors' contributionsyz qw and dw participated in the design and conception of the present study yz qw dw zs jl and wty were involved in data acquisition and analysis of certain clinical data qw dw zz and yw performed the clinical experiments and analysis of the data the manuscript was written by qw and critically reviewed by yz dw zz yw wny and nrm wny ks and nrm were involved in performing and analyzing the cell experiments all authors read and approved the final manuscriptethics approval and consent to participatethe present study was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university approval no science‘‘lw‘ and informed consent was obtained from each patient with available follow‘up informationpatient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences siegel rl miller kd and jemal a cancer statistics ca cancer j clin ‘ cidon eu the challenge of metastatic colorectal cancer clin med insights oncol ‘ vreeland tj clifton gt herbert gs hale df jackson do berry js and peoples ge gaining ground on a cure through synergy combining checkpoint inhibitors with cancer vaccines expert rev clin immuno ‘ siegel rl miller kd fedewa sa ahnen dj meester rgs barzi a and jemal a colorectal cancer statistics ca cancer j clin ‘ dickinson bt kisiel j ahlquist da and grady wm molecular markers for colorectal cancer screening gut ‘ nikolouzakis tk vassilopoulou l fragkiadaki p mari

cancer7543

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " colorectal cancer crc is the third leading cause of cancer‘associated mortality the present study aimed to investigate novel biomarkers to predict prognosis and provide a theoretical basis for studies of the pathogenesis and the development of therapies for crc the present study compared mrna expression levels of patients with crc with short‘ and long‘term prognosis and of individuals with and without tumors in the cancer genome atlas tcga data‘base differentially expressed genes degs were identified via volcano plot and venn diagram analysis gene ontology go analysis and gene set enrichment analysis gsea were performed to identify the functions of the degs and the degs were further verified using clinical crc samples a total of degs were identified as candidate genes using the tcga database and four degs [defensin 4a defb4a hyaluronan binding protein habp2 oleoyl‘acp hydrolase and tbc1 domain family member 3g] were associated with poor prognosis of patients with crc two degs defb4a and habp2 were upregulated in tumor tissues of patients with crc in the tcga database go and gsea analyses revealed that defb4a was highly associated with immunosuppression participates in ˜myeloid leukocyte differentiation™ ˜leukocyte proliferation™ and ˜positive regulation of leukocyte‘mediated immunity™ and was positively correlated with cd11b cd14 cd45 cd163 and il17a furthermore defb4a expres‘sion was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis survival analysis revealed that defb4a upregulation was associated with poor prognosis defb4a correspondence to dr yi zhang biotherapy center the first affiliated hospital of zhengzhou university jianshe east road zhengzhou henan pr chinae‘mail yizhangzzueducncontributed equallykey words defensin 4a colorectal cancer prognosis immunity biomarkergene knockdown experiments demonstrated that def4ba promotes cell migration these results indicated that defb4a potentially promotes tumor growth by regulating immu‘nosuppressive activity and provided novel insights into the diagnosis and treatment of crcintroductionaccording to the global cancer statistic of colorectal cancer crc is the third most common cancer and the second leading cause of cancer‘associated mortality worldwide crc was reported as the fourth most common and fatal cancer in china in patients with crc usually have a low survival rate and poor therapeutic responses and are susceptible to progression and recurrence early diagnosis and effective treatment are critical to improve the survival of patients with crc crc studies have focused on innovative ideas to identify molecular markers used to develop high‘precision non‘invasive screening tests for crc to increase population compliance and reduce the poten‘tially harmful side effects associated with more invasive techniques diagnostic markers will give an indication of the likely progression of the disease targeting specific molecules in certain patients has facilitated more personalized treatments that help prevent or decelerate cancer progression the present study aimed to determine prognostic factors and novel therapeutic targets to improve the survival of patients with crcprevious studies have focused on the identification of molecules associated with tumor progression through genetic or mrna profiling and screening of patients with colon cancer ‘ for example the expression profiles of long non‘coding rnas lncrnas were compared at specific tumor stages t0 t1 t2 and t3 in an azoxymethanedextran sodium sulfate‘induced primary colon cancer model and upregulation of the lncrna h19 predicted a poor prog‘nosis other studies analyzed microrna mir expression profiles between tumor tissues and matched non‘tumor tissues obtained from patients with crc for example mir‘ is significantly downregulated in tumor tissues and associated with poor survival of patients with crc and may thus be considered to be a poor prognostic marker of crc furthermore high expression levels of mir‘ 0cwu prognostic role of defb4a in colorectal cancerand mir‘ in serum are associated with poor survival of patients with crc analysis of the cancer genome atlas tcga database revealed that mmp19 is upregulated in patients with crc and is associated with tumor progres‘sion however to the best of our knowledge no study has directly screened mrna profiles based on prognosis the present study divided patients with crc into different groups based on prognosis and screened the mrna profiles of the respective groups defensin 4a defb4a also known as bd‘ sap1 defb2 defb4 hbd‘ defb‘ and defb102 belongs to the defensin family comprising cytotoxic peptides secreted by neutrophils which serve important roles in innate immune defense against microbial infections ‘ defb4a is upregulated in cutaneous squamous cell carcinoma and basal cell carcinoma it serves an important role in esopha‘geal carcinogenesis both in vivo and in vitro the genomic copy number of defb4a has been analyzed in patients with crohn's disease and controls and an elevated defb4a copy number has been identified as a risk factor for crohn's disease regardless of disease origin however it remains unclear whether defb4a expression is associated with the prognosis of crc furthermore the role of defb4a in the immune system remains unclear the tumor microenvironment serves a significant role in tumor progression various immune elements comprise the tumor microenvironment including bone marrow‘derived cells such as macrophages cd4 t cells cd8 t cells b cells natural killer cells and dendritic cells myeloid cells can differentiate into macrophages or myeloid‘derived suppressor cells mdscs which serve a tumorigenic role in the tumor microenvironment mdscs contribute to tumor vascular development by promoting angiogenesis and tumor growth tumor‘associated macrophages tams are important regulators of tumorigenesis by inhibiting the anti‘tumor effects of other cells thus promoting tumor growth however it remains unclear whether defb4a has a regulatory effect on the tumor microenvironment or whether it promotes crc progressionto identify candidate target genes that potentially prolong patient survival mrna expression profiles of tissues from crc samples were compared in the tcga database venn analysis was performed to determine candidate genes upreg‘ulated in tumor tissues among patients with poor prognosis subsequently immune‘associated pathway enrichment was analyzed using gene ontology go and gene set enrichment analysis gsea and the correlations between candidate target genes and certain immune cells were determined finally clinical samples and crc cell lines were obtained to verify the clinical significance of the identified genes the present results may provide insights into targeted therapy for crcmaterials and methodsacquisition of microarray data microarray data were obtained from tcga httpcancergenomenihgov rna‘seq data for samples were included in the dataset project id tcga‘coadread including tumor samples from patients with crc and normal tissues from healthy donorsidentification of differentially expressed genes degs tcga data were divided into two groups based on different categories patient prognosis and gene expression in tumor and normal tissues venn analysis of the two groups was performed and genes associated with crc prognosis were identifiedvenn analysis to identify candidate genes associated with patient survival the gene expression profiles in the two groups were analyzed using the venn diagram web tool httpbioinformaticspsbugentbewebtoolsvenngo analysis functional analysis of the degs was performed using go httpwwwgeneontology based on biological processes gsea gsea was conducted using gsea v403 software wwwgsea‘msigdbgseaindexjsp and the gene used in the present study was downloaded from the molecular signatures database msigdb oftware broadinstitutegseamsigdbindexjsp v40 msigdb curates various gene sets including canonical signaling pathways from biocarta cgapncinihgovcgap_mitelman_retire_noticehtml kyoto encyclopedia of genes and genomes wwwkeggjp pid httppidncinihgov reactome reactome and other pathway databases tcga data were analyzed via gsea and pathways with a false discovery rate fdr were considered significantpatient characteristics tissue samples were obtained from patients with crc at the first affiliated hospital of zhengzhou university zhengzhou china between april and april patients underwent surgical resection or colonoscopy and the samples were verified via pathological analysis the clinical characteristics of the patients are shown in table i a total of men and women were included in the present study the median age was years age range ‘ years crc was diagnosed by two pathologists on the basis of pathological assessment the collection of speci‘mens was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university zhengzhou china approval no science‘‘lw‘ and informed consent was obtained from each patient with available follow‘up informationreverse transcription‘quantitative pcr total rna was extracted from pairs of tumor and normal tissue samples from patients with crc using trizol reagent invitrogen thermo fisher scientific inc total rna samples µg were incubated at ˚c for min followed by incubation at ˚c for min and ˚c for sec according to the reverse transcrip‘tion reaction protocol takara biotechnology co ltd the conditions of pcr were as follows ˚c10 min ˚c10 sec ˚c10 sec ˚c10 sec cycles premix ex taq ii roche target gene expression was simultaneously assessed relative to that of gapdh a housekeeping gene and internal control the following primers were used defb4a forward '‘ctc ctc ttc tcg ttc ctc ttc a‘' and reverse '‘gca ggt aac agg atc gcc tat‘' and gapdh forward '‘gga gcg aga tcc ctc caa aat‘' and reverse '‘ggc tgtt 0concology letters no of cases table i characteristics of patients with colorectal carcinomacharacteristic percentagesex male female age years ‰¥ treatment surgery others tumor size mm ‰¥ pathological type adenocarcinoma others lymph node metastasis yes no tnm stage i ii iii iv liver metastasis negative positive differentiation poor medium‘well ˜others™ include chemotherapy and radiotherapy gtc ata ctt ctc atg g‘' the present study compared the expression levels of the target genes in clinical samples using the ‘δδcq method expression levels of defb4a and gapdh were examined for each sample and the relative expression levels of defb4a were determined using the ‘δcq value of defb4a divided by that of gapdh cell transfection sw480 and hct116 cells were seeded into a ‘well plate sw480 and hct116 cells were purchased from chinese academy of sciences cell bank and cultivated with dmem‘high glucose containing fbs hyclone ge healthcare life sciences and penicillin‘streptomycin at ˚c in co2 the growth status of the cells was closely observed until they reached a fusion rate of and then cells were transfected with nc‘small interfering rna nega‘tive control si‘nc sense '‘uuc ucc gaa cgu guc acg utt‘' and antisense '‘acg uga cac guu cgg aga att‘' and small interfering rna targeting defb4a si‘defb4a si‘defb4a sense '‘ucc ucu uca uau ucc uga utt‘' and antisense '‘auc agg aau aug aag agg att‘' purchased from shanghai genepharma co ltd with jetprime polyplus transfection reagent polyplus‘transfection sa after h the medium was changed to fresh medium and cells were further incubated in co2 for h subsequently cells were collected for the subsequent experimentswound healing assay for the wound healing assay sw480 and hct116 cells were cultured in µl medium with fbs hyclone and the percentage of serum was in line with previous papers sub‘confluent tumor cells ‘ were scraped using a sterile micropipette tip and then serum‘free medium was added next cells were imaged at and h using an inverted fluorescence microscope magnification x200 olympus corporation transwell assay in the migration test the transfected cells 1x105 were inoculated into the top chamber microns with µl serum‘free medium complete medium µl containing fbs was added to the lower chamber corning inc following incubation at ˚c for h the migratory cells located under the insert were fixed and stained with crystal violet staining solution beyotime institute of biotechnology at room temperature for min and observed using an inverted fluorescence microscope magnification x200 olympus corporationstatistical analysis the χ2 test was used to compare clinicopathological factors and continuous variables were analyzed via unpaired student's t‘test or one‘way anova kaplan‘meier analysis and the log rank test were performed for survival analysis univariate and multivariate logistic regression models confirmed the associations between defb4a expression and clinical features prism graphpad software inc was used for statistical analysis of all clinical samples anova was followed by tukey's post‘hoc test and performed using spss for windows spss inc r software version r foundation for statistical computing was used for bioinformatics analysis p005 was considered to indicate a statistically significant difference all experi‘ments were performed in triplicate and data are presented as the mean ± standard deviation resultsgenes associated with poor prognosis to identify genes associated with poor survival in the crc cohort patients were divided into two groups based on os short ‰¤ days os patients with a survival time of days would be included in short os and long days os fig 1a in total degs fold change were identified using a volcano plot including upregulated and downregulated genes fig 1b and c hierarchical cluster analysis revealed the expression profiles of the degs fig 1d subsequently gene expression profiles in tumor and normal tissues fold change were analyzed and it was observed that genes were upregulated and were downregulated in tumor tissues compared with in normal tissues p005 fdr fold change fig 1e‘g the venn diagram revealed that 0cwu prognostic role of defb4a in colorectal cancerfigure screening of degs based on tcga a patients with crc were divided into two groups based on whether or not they survived for days in accordance with the rna‘seq data from tcga b volcano plot of rna‘seq data from tcga the red dots and blue dots represent upregulated and downregulated degs based on a fold change of the volcano plot displays different genes when comparing patients with a prolonged os and those with a short os c a total of upregulated and downregulated genes were identified d hierarchical clustering analysis of the rna‘seq data of different genes in short os and long os samples e hierarchical clustering analysis of the rna‘seq data of different genes in ca and n samples f using a threshold of p005 false discovery rate and fold change degs were selected using a volcano plot when comparing ca samples with normal colon mucosa samples from tcga g a total of upregulated and downregulated genes were identified h venn diagram representing the distribution of degs in different groups a total of degs were expressed in both patients with ca and patients with a prolonged os crc colorectal carcinoma degs differentially expressed genes os overall survival tcga the cancer genome atlas ca cancer n normal degs were identified in both screening methods fig 1h details are shown in table iivalidated degs are associated with poor prognosis in tcga to determine the prognostic significance of the identified degs their expression levels were determined in cases included in tcga kaplan‘meier survival analysis revealed that defb4a hyaluronan binding protein habp2 oleoyl‘acp hydrolase olah and tbc1 domain family member 3g tbc1d3g upregulation was significantly associated with poor survival in patients with crc fig 2a prognostic significance was not observed for kiss1r or5m11 chrnb3 otx2 s100a7a flj43860 and frmd7 in the patients with crc data not shown furthermore defb4a and habp2 were upregulated in tumor tissues fig 2b a previous study have reported low serum expression levels of habp2 in patients with crc therefore defb4a was selected as a candidate marker of poor prognosis in patients with crcgo and gsea to evaluate the biological role of defb4a in crc progression go enrichment and gsea analyses were 0concology letters descriptiontable ii upregulated genes n10 associated with a poor colorectal carcinoma prognosis in the cancer genome atlas databasegene symbol defb4a habp2 olah tbc1d3g kiss1r frmd7 s100a7a otx2 or5m11 chrnb3 defensin beta 4ahyaluronan binding protein oleoyl‘acp hydrolasetbc1 domain family member 3gkiss1 receptorferm domain containing s100 calcium binding protein a7aorthodenticlehomeobox olfactory receptor family subfamily m member cholinergic receptor nicotinic beta subunitgene id performed defb4a was demonstrated to be involved in various biological processes associated functional pathways are shown in fig 3a and b and closely associated with ˜myeloid leukocyte differentiation™ ˜leukocyte proliferation™ and ˜leukocyte mediated immunity™ implying that defb4a potentially regulates the immune system finally the data‘base was searched for expression profiles of defb4a and immune‘related genes and a positive correlation between defb4a expression and the expression of immune markers such as cd11b cd14 cd45 cd163 and il17a was observed fig 3c these results suggest that defb4a is associated with poor prognosis in patients with crc potentially in an immu‘nosuppressive myeloid leukocyte‘ and cytokine‘dependent mannervalidation in patient samples and clinical relevance of defb4a to further clarify the clinical significance of defb4a expression the present study analyzed tissue samples from patients with crc the associations between their mrna expression levels and clinicopathological vari‘ables were observed detailed information of the patients is provided in table iii defb4a expression was significantly upregulated in the crc tumor tissues fig 4a additionally an association between defb4a upregulation and advanced crc stage stage i cases stage ii cases stage iii cases stage iv cases and metastasis m0 cases m1 cases was observed fig 4b and c furthermore defb4a upregulation in the tumor tissues was associated with poor prognosis p00313 fig 4d additionally defb4a upregulation was significantly associated with advanced liver metastasis p0039 stage p0005 high ca72‘ value p0003 tumor size p0009 and lymph node metastasis p0044 table iii therefore defb4a was considered to be a prognostic marker associated with tumor progres‘sion in patients with crc logistic regression analysis was performed to determine whether defb4a can help predict the prognosis of crc univariate analyses revealed that advanced tnm stage [odds ratio or p001] liver metastasis or p003 lymph node metastasis or p004 high ca199 level or1324 p002 a high ca ‘ level or p001 and high defb4a level or p002 were associated with the survival of patients with crc furthermore multivariate analyses revealed that advanced tnm stage or p004 histological differentiation or p001 liver metastasis or p001 ca199 level or p001 high ca ‘ level or p005 and high defb4a level or p001 were independent prognostic predictors table iv overall these results suggest that defb4a serves an important role in predicting the prognosis of patients with crcdefb4a promotes proliferation and metastasis in crc to explore the biological roles of defb4a in crc defb4a expression was knocked down in hct116 and sw480 cells fig 5a and b a wound healing assay revealed that defb4a knockdown inhibited the migration of hct116 and sw480 cells fig 5c transwell assays demonstrated that the migration of cells was decreased following knockdown of defb4a in sw480 cells compared with that in the nc group fig 5d the number of migratory cells decreased following knockdown of defb4a in sw480 cells fig 5e overall these results suggested that defb4a serves an important role in crc development discussionwith the increasing availability of high‘throughput technolo‘gies numerous novel biomarkers and therapeutic targets have been identified through transcriptomic analysis of various types of tumor however such studies on biomarkers in crc have not been extensively performed the identification of crc biomarkers may help predict and prolong the survival of patients with crc in the present study mrna profiling of microarray analysis data from the tcga database was performed to identify numerous novel genes associated with poor prog‘nosis in crc a critical role of defb4a in patients with crc was identified the mrna profiles of patients were first compared between the long os and short os groups and between the tumor and normal tissue groups in the tcga database subsequently the present study investigated the association between mrna expression and prognosis defb4a habp2 olah and tbc1d3g were identified as potential predicators of poor prognosis defb4a and habp2 0cwu prognostic role of defb4a in colorectal cancerfigure defb4a is upregulated based on data from tcga and predicts poor prognosis a kaplan‘meier curve of four genes defb4a habp2 olah and tbc1d3g derived from data of patients included in the tcga dataset b mrna expression levels of defb4a habp2 olah and tbc1d3g in cancer vs control samples from patients in tcga p005 ns not significant tcga the cancer genome atlas defb4a defensin 4a habp2 hyaluronan binding protein olah oleoyl‘acp hydrolase tbc1d3g tbc1 domain family member 3g ca cancer n normalwere upregulated in crc tissues of patients in the database however habp2 has been reported to be downregulated in the sera of patients with crc p00137 therefore defb4a was considered as a candidate gene for further analysis go and gsea were used to assess the function of defb4a in promoting disease progression and to highlight the role of defb4a in the tumor microenvironment defb4a was involved in ˜myeloid leukocyte differentiation™ ˜leukocyte proliferation™ and ˜leukocyte mediated immunity™ correlation analysis revealed that defb4a expression was positively correlated with immune markers including cd11b cd14 cd45 cd163 and il17a cd11b is expressed on the surface of a number of leukocytes including monocytes granulocytes and macrophages cd14 is expressed on both monocytes 0concology letters figure defb4a is positively correlated with inhibitory immune cells a gene ontology analysis revealed that defb4a is involved in ˜leukocyte proliferation™ ˜lymphocyte differentiation™ ˜leukocyte mediated immunity™ ˜myeloid cell differentiation™ ˜negative regulation of type i interferon production™ and ˜interleukin‘ production™ b gene set enrichment analysis verified the results c correlation between defb4a and cd11b cd14 cd45 cd163 and il17a r and p‘values are indicated defb4a defensin 4a r pearson's correlation coefficientfigure defb4a predicts poor prognosis in colorectal cancer a defb4a mrna expression in groups of cancer tissues and normal tissues are displayed b defb4a mrna levels were compared with respect to tnm stage c defb4a mrna expression levels of patients with different cancer stages d effect of defb4a expression on overall survival in patients with colorectal carcinoma n52 p005 p001 p00001 defb4a defensin 4aand macrophages and cd45 is expressed on leukocytes m2 macrophages may be marked with cd163 and m2 macro‘phages serve a role in promoting tumor growth the os of patients with non‘small‘cell lung cancer and those with esophageal cancer with high m2 macrophage infil‘tration rates is shorter than those with low m2 macrophage infiltration rates patients with high expression levels of il‘17a had a poor prognosis in a crc cohort previous studies have suggested that increased il‘17a promotes crc in various animal models ‘ analysis of clinical specimens of patients with crc demonstrated that defb4a expression was associated with 0cwu prognostic role of defb4a in colorectal cancer 0330a total n χ2 p‘valuedefb4a expressiontable iii association between defb4a expression and clinicopathological characteristics of patients with colorectal carcinoma characteristic sex male female age years ‰¥ site of lesion colon rectum differentiation poor well tumor size cm ‰¥ pathological type adenocarcinoma others lymph node metastasis no yes liver metastasis no yes stage iii iiiiv cea normal high ca ‘ normal high ca ‘ normal high afisher's exact test all others were assessed using a χ2 test defb4a defensin 4a‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘high n low n 0009a0003a 0575a0039a poor survival furthermore defb4a expression was upregu‘lated in patients with crc with advanced and metastatic cancer patients with crc with high defb4a expression had poor survival in addition knockdown of defb4a affected the migration ability of crc cells tcga data of patients with crc were used to identify the degs between the long os days and short os days groups in addition mrna expression was compared between tumor tissues and normal tissues in the same database defb4a was highly expressed in tumors and associated with a poor prognosis defb4a upregulation was associated with poor prognosis and defb4a expression was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis another study used the gene expression omnibus database to screen genes that are increased in patients with recurrence hierarchical clustering and pathway analyses revealed that thrombospondin thbs2 and cartilage 0concology letters table iv logistic regression model analysis of liver metastasis predictors in patients with colorectal carcinoma characteristics sex male vs female age vs ‰¥ years tumor size vs ‰¥ mm pathological type adenocarcinoma vs others tnm stage iii vs iiiiv differentiation medium vs poor liver metastasis no vs yes lymph node metastasis no vs yes cea vs ‰¥ ca199 vs ‰¥ ca724 vs ‰¥ defb4a high vs low or odds ratio‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘ or p‘value univariate ci ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘or p‘value multivariate ci ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ figure defb4a promotes colorectal cancer cell migration a expression levels of defb4a were detected using rt‘qpcr following transfection of hct116 cells with si‘nc and si‘defb4a b expression levels of defb4a were detected using rt‘qpcr following transfection of sw480 cells with si‘nc and si‘defb4a c migration ability of cells was examined using a wounding healing assay d representative images were obtained for the transwell assay magnification x200 e proportions of migrated cells after h were quantified defb4a defensin 4a nc negative control rt‘qpcr reverse transcription‘quantitative pcr si small interfering rna p001 p0001 0cwu prognostic role of defb4a in colorectal canceroligomeric matrix protein comp are associated with the ecm‘receptor interaction focal adhesion and tgf‘ signaling pathways the hypergeometric distribution test demonstrated that the association between thbs2 and crc is stronger than that of comp pearson test results indicated that thbs2 might be considered to be a prognostic biomarker for crc to the best of our knowledge this screening method and the hypothesis that defb4a may serve a pro‘tumor role through immunosuppression have not been seen in other studies defb4a stimulates keratinocytes to release il‘ and il‘ pro‘inflammatory cytokines serving as deciding factors in the pathogenesis of psoriasis furthermore defb4a induction is required for toll‘like receptor tlr activation in monocytes through the convergence of il‘ and vitamin d receptor signaling and exerts direct bactericidal effects against m tuberculosiss the antimicrobial peptides defb4a and camp are inhibited by hsa‘mir‘ leading to suppression of the tlr21‘induced vitamin d antimicrobial signaling pathway defb4a has been suggested as a biomarker for psoriasis because the clinical efficacy of targeted antibody therapy in psoriasis is associated with the inhibition of defb4a expression defb4a expression can directly be inhibited by anthralin in vitro and in vivo thus benefiting patients with psoriasis however it has remained unclear whether defb4a is involved in the immunoregulation in crc go analysis revealed that defb4a is involved in ˜myeloid leukocyte differentiation™ ˜leukocyte proliferation™ and ˜positive regulation of leukocyte‘mediated immunity™ therefore defb4a may be associated with immunity in crc to the best of our knowledge the present study was the first to report defb4a as a prognostic marker for crc and as an immunoregulatory factor in the tumor microenvironment in patients with crc however a limitation of the present study was that the research cohort was not large enough which may affect the statistical results in addition the specific role of defb4a and immune factors in colon cancer and the underlying molecular mechanism need to be further exploredin conclusion to the best of our knowledge defb4a is upregulated in patients with crc and is closely associated with poor prognosis defb4a regulates immune function and potentially promotes immunosuppression therefore defb4a may be considered as a prognostic marker and immunotherapeutic target for crcacknowledgementsnot applicablefundingthe present study was supported by grants from the national natural science foundation of china grant nos u1804281 and and funding from state's key project of research and development plan grant no 2016yfc1303500availability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author on reasonable requestauthors' contributionsyz qw and dw participated in the design and conception of the present study yz qw dw zs jl and wty were involved in data acquisition and analysis of certain clinical data qw dw zz and yw performed the clinical experiments and analysis of the data the manuscript was written by qw and critically reviewed by yz dw zz yw wny and nrm wny ks and nrm were involved in performing and analyzing the cell experiments all authors read and approved the final manuscriptethics approval and consent to participatethe present study was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university approval no science‘‘lw‘ and informed consent was obtained from each patient with available follow‘up informationpatient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences siegel rl miller kd and jemal a cancer statistics ca cancer j clin ‘ cidon eu the challenge of metastatic colorectal cancer clin med insights oncol ‘ vreeland tj clifton gt herbert gs hale df jackson do berry js and peoples ge gaining ground on a cure through synergy combining checkpoint inhibitors with cancer vaccines expert rev clin immuno ‘ siegel rl miller kd fedewa sa ahnen dj meester rgs barzi a and jemal a colorectal cancer statistics ca cancer j clin ‘ dickinson bt kisiel j ahlquist da and grady wm molecular markers for colorectal cancer screening gut ‘ nikolouzakis tk vassilopoulou l fragkiadaki p mari Answer:

Colon_Cancer

coronaviruses covs belong to a family of envelopedviruses with a positive sense singlestranded rna genomecovs cause illness ranging from upper respiratory tractinfections urtis resembling the common cold to lowerrespiratory tract infections lrtis such as bronchitis pneumonia and even severe acute respiratory syndrome sarswith most serious disease outcomes in the elderly immunocompromised patients and infants [ ] hcovoc43oc43 hcov229e 229e hcovnl63 nl63 andhcovhku1 hku1 were the first documented humancovs hcovs which usually cause urtis and less frequently are associated with ltri diseases in the lastdecades two human coronaviruses created great concernfor the world medical community due to significant diseaseand mortality [ ] in severe acute respiratorysyndromecoronavirus sarscov was characterized byacute atypical pneumonia and diï¬use alveolar damagedad in roughly patients and with almost deathsrepresenting a nearly mortality rate more recentlyin a new human coronavirus designated as middle eastrespiratory syndromecoronavirus merscov was identified and the global ongoing outbreak of mers with over official cases and deaths represented approximately case fatality rate to date in humans over the last few months a new strain of human coronavirus sarscov2 also known as 2019ncov hascaught the world™s seven continents™ attention with its rapidglobal spread aï¬ecting at least countries and territoriesinfecting more than and claiming more than lives worldwide the coronavirus pandemichas promoted isolation and uncertainly fear and panicworldwide in additionlikely lead to changes inpolitical and economic power in ways that can be determined only later it willit is important to note that there are many similaritiesamong diï¬erent coronavirus species but not in all aspects 0coxidative medicine and cellular longevitydepending on the molecular mechanism of viral inhibitionpromoted by an antiviral agent the analysis of the data andcomparison between animal and human covs must be donevery carefully in fact it is important to note that there arediï¬erences between human and animal cov receptorswhich will likely result in diï¬erent affinities or unlikely interactions of an antiviral agent with the diï¬erent cov receptors howeverif the antiviral agent interferes with thereplication andor assembly of the covs there is a higherprobability of obtaining similar antiviral activity results inhuman cov tests [ ] following this line our searchin specialized literature was focused mainly on studies thatinvestigated the anticoronavirus eï¬ects of natural antioxidants by inhibiting proteases for viral replication materials and methodsthe present study was carried out based on a search of the literature of natural antioxidants and coronavirus the searchperformed in the pubmed database included studies published until march and used the following keywordscoronavirusstressmerscov sarscov 229e nl63 oc43 hku1merscov virus infection and middle east respiratorysyndrome virus the scientific publications were selectedfrom studies published in the english languageantioxidants flavonoids oxidative pathogenic mechanism of coronavirusinduced cell damagethe high mortality rate associated with the three pathogenichcovs has been mainly attributed to the development ofdigestive and respiratory tract injuries observed followinginfection acute atypical pneumonia and diï¬use alveolardamage that progress to deposition of fibrous tissue denudedairways haemorrhage and elevated macrophage ltrationare sometimes accompanied by watery diarrhoea dehydration and vomiting [ ]despite the molecular mechanisms of coronavirusinduced intestine and lung pathogenesis not fully elucidatedand still unclear studies have suggested that lateterm diseaseprogression is unrelated to viremia it is now believed morelikely to be associated with the immunopathological mechanism [ ] viral clearance and subsequent recovery frominfection require activation of an eï¬ective host immuneresponse however many immune eï¬ector cells may alsocause injury to host tissues together with ‚ammatoryand immune response signaling the presence of oxidativecompounds such as reactive oxygen species ros playsimportant roles in the pathogenic mechanism of cell damageinduced by covs through oxidative stress oxidative stress is defined as an interruption andorderegulation of the signaling and redox system that can becaused by an imbalance in the production of oxidant andantioxidant species among the main oxidant agentsros and reactive nitrogen species rns stand out in orderto counterbalance the oxidant species there is an antioxidantsystem formed by enzymes and nonenzymatic molecules however during pathological events such as viral infections there may be an increase in the production of oxidantspecies not neutralized by the antioxidant system resultingin oxidative stress that promotes cellular damage throughprotein denaturation changes in the functions of nucleicacids lipid peroxidation and cell death [“]in addition during viral infection oxidative stress contributes to viral pathogenesis through stimulating ‚ammation loss of immune function and increased viral replicationthat may occur due to the activation of the nuclear factorkappa b nfκb transcription pathway [“] currentevidence suggests that cytokine dysregulation”also calledcytokine storm”contributes to severe disease caused by thepathogenic covs [ ] the exact mechanisms are notclear yet but research on ‚uenza a virus shows that infection causes a rapid ‚ux of ‚ammatory cells this isfollowed by an increase in reactive oxygen species productionand cytokine expression and release which ultimately leadsto acute lung injury in general rna viruses promotechanges in the body™s antioxidant defense system aï¬ectingenzymes such as superoxide dismutase sod and catalasecat in addition to reducing the levels of antioxidantmolecules such as ascorbic acid carotenoids and reducedglutathione gsh [“] wu reported that glucose6phosphate dehydrogenasean important antioxidantenzyme that produces nadph knockdown cells were moresusceptible to infection by hcov229e than normal cells interestingly ye and colleagues have reported that theinhibition of ros production alleviates ‚ammation causedby ‚uenza a virus infections in an experimental model of sarsinduced acute lunginjury in mice it was noted that phospholipid oxidationdue to oxidative stress is one of the main triggering factorsof acute lung injury this happens through the activation ofthe innate immune response culminating in the activationof pulmonary macrophages via tlr4triftraf6nfκbsignaling furthermore hypoxia caused by acute lunginjury can cause myocardial injury due to the production ofros aggravating infections caused by coronavirus disease covid19 mitochondria have an essential function in energy generation and for this reason their function and integrity arestrictly regulated in order to respond to varying energyrequirements and environmental conditions mitochondria are known to function as the control point in apoptoticpathways releasing proapoptotic factors mainly ros whichfunction as a signaling molecule that may result in cell death[ ] some studies have shown a relationship betweencoronavirus infection and dysfunctional or damaged mitochondria leading to the release of ros and other proapoptotic substances [ ] in a recent study xu reportedthat ros and p53 play key roles in regulating many kindsof the cell process during coronavirus infection in vero cellsaccording to the authors coronavirus infection appears toinduce a timedependent ros accumulation which in turnis linked to regulatory mechanisms of p53 activation andapoptosis in infected cells antioxidant substances promote improvement in casesof disease caused by coronaviruses such as apolipoproteind”a lipocalin that promoted a neuroprotective eï¬ect against 0coxidative medicine and cellular longevityencephalitis induced by human coronavirus oc43”in micethis protective eï¬ect occurred through the reduction of oxidative stress cerebral lipid peroxidation and regulation of‚ammation [ ] also the treatment with antioxidantssuch as pyrrolidine dithiocarbamate or nacetylcysteine significantly inhibits moreover melatonin promotes downregulation of acute lungoxidative injury due to its anti‚ammatory and antioxidantactions making it a possible compound in the treatment ofcovid19 based on these studies compounds thathave antioxidant actions can be helpful in the treatment ofinfections promoted by coronaviruscoronavirusinduced apoptosisin general antioxidant properties of polyphenolic compounds such as some flavonoids have been associated withthe presence of aromatic phenolic rings that promote theelectron donation and hydrogen atom transfer to free radicals acting as free radical scavengers reducing agents andquenchers of single oxygen formation thus the aimof this study was to investigate the antioxidant capacity andantiviral activity of natural antioxidants against coronavirusthe compounds are illustrated in figure occurrence and antioxidant properties ofanticoronavirus compoundsquercetin can be found in plants such as rubus fruticosus land lagerstroemia speciosa l pers [ ] also quercetinshows antioxidant activity at a concentration of μmoll inhepg2 cells inhibiting oxidative stress promoted by h2o2 promotes an increase in sod cat and glutathioneperoxidase gpx and reduces lipid peroxidation in rats withchronic prostatitischronic pelvic pain syndrome moreover quercetin improves sepsisinduced acute lung injury inrats by reducing lipid peroxidation and ‚ammation andincreasing sod and cat levels in addition quercetin glycosides with antioxidant activity such as quercetin 3βglucoside have already been isolated from plants such as passiflora subpeltata ortega andchamomilla suaveolens pursh rydb [ ] the administration of quercetin 3βglucoside mgkg poinstreptozotocininduced diabetic rats promotes an increasein the levels of antioxidant enzymes sod cat andgpx and nonenzymatic antioxidants vitamins c and eand gsh and a reduction of lipid peroxidation quercetin 3βgalactoside hyperoside is found mainly in plantsof the hypericum genus such as hypericum perforatum l[ ] moreover it showed cardioprotective activity inhigh glucoseinduced injury of myocardial cells throughdecreased apoptosis and ros production and increasedsod levels quercetin 7ramnoside is also found inplants of the hypericum genus such as hypericum japonicum thunb ex murray this flavonoid shows hepatoprotective activity against carbon tetrachloride in mice bydecreasing lipid peroxidation and increasing cat andgsh levels in addition to presenting values of μmand22diphenyl1picrylhydrazyldpph and ²azinobis3ethylbenzthiazoline6sulphonic acid abts assays respectively μm intheepigallocatechin gallate is present in parkia roxburghii gdon and is one of the main metabolites found in green teaand liubao tea camellia sinensis lo kuntze also gallocatechin gallate can be found in this plant [“] literaturedata reveal that the administration ip of mg100 g ofepigallocatechin gallate in rats with streptozotocininduceddiabetes mellitus promotes a reduction in oxidative stressthrough reductions in parameters such as indirect nitricoxide synthesis and status total oxidative as well as anincrease in levels of cat and total antioxidant capacity ofplasma furthermore it promotes cardioprotection byantioxidant mechanisms green tea has a high antioxidant capacity due to the highlevels of catechins present he and collaborators compared the antioxidant activities of catechins and reported thatepigallocatechin gallate has greater antioxidant activity viaradical scavenging activity μm with values of ± ± and ± compared to its epimergallocatechin gallate with values of ± ± and ± in the dpph abts and ferric reducingantioxidant power frap respectively amentoflavone is a biflavonoid present in leaves ofginkgo biloba l garcinia brasiliensis l and nandinadomestica l [“] this biflavonoid has a high antioxidantcapacity “ demonstrated in scavenging tests ofdpph abts superoxide and hydroxyl radicals moreover amentoflavone prevents acute lung injury induced bysepsis in rats by decreasing thiobarbituric acid reactive substance tbars levels and by increasing levels of sod andgsh apigenin is mainly present in flowers and leaves beingabundantly found in apium graveolens l petroselinum crispum mill fuss and matricaria chamomilla l sánchezmarzo and collaborators evaluated the antioxidantcapacity of apigenin using the trolox equivalent antioxidantcapacity teac oxygen radical absorbance capacityorac and frap assays the results show that apigeninhas good antioxidant activity with values of ± μmol teammol ± μmol teammol and ± μmol fe2mmol respectively in addition oraladministration of apigenin mgkgday for days in anexperimental model of cardiotoxicity induced by doxorubicin in rats promoted cardioprotection by reducing levels ofmalondialdehyde mda increasing sod levels and preventing cardiomyocyte apoptosis luteolin is present in foods such as carrot cabbage teaand apple and is found in ugni molinae turcz [ ] datashow that luteolin μgml increases the levels of gsh theexpression of gsh synthetase and the activity of sod andcat in human colon cancer cells ht29 furthermoreluteolin attenuates the sepsisinduced acute lunginjury in mice by reducing lipid peroxidation and increasingsod and cat activity in addition to suppressing the nfκbpathway herbacetin is ubiquitous in plants of the genus rhodiolasuch as rhodiola rosea l herbacetin glycosides are alsopresent in the roots of r sachalinensis a bor and show antioxidant activity veeramani reported that theadministration of herbacetin mgkg po in mice with 0coxidative medicine and cellular longevityohohohohhoohooohohhooohoh oquercetinohohoooohohohquercetin 3𝛽galactosideohohooohoohoohohhoohohohoh oquercetin 7ramnosideohooohohoohoohoohohohquercetin 3𝛽glucosidehooohooohohohohohohepigallocatechin gallateohgallocatechin gallateohhooohoh ohooluteolinhooohoooohoohohoohrhoifolinhohohoohohohohohohoohoohooh oamentoflavoneohhooohohohoherbacetinhohoooohooohohohoapigeninooohooohhopectolinarinooopsoralidinhooohohohohcatechinohhoohoooh ohelichrysetinohohomyricetinohohohhohoohoohoisobavachalconeohhooohscutellareinohresveratrolfigure chemical structures of bioactive antioxidants against coronavirusobesityassociated insulin resistance promotes an increasein the activity of the enzyme glucose6phosphate dehydrogenase which is directly related to the production ofnadph pectolinarin is present in plants of the genus cirsiumsuch as cirsium setidens nakai and cirsium japonicum dcthe administration of pectolinarin and mgkg pofor two weeks in rats promotes antioxidant eï¬ects in hepatic 0coxidative medicine and cellular longevitytable antioxidant properties of natural inhibitors of coronaviruscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencetype of cells μmoll mgkg po mgkg poinhibiting oxidative stress promoted byh2o2promoted an increase in sod cat andgpx and reduced lipid peroxidationreduces lipid peroxidation and increasessod and cat levelsincreases levels of sod cat gpx mgkg povitamins c and e and gsh and reduces nmoll mgkgic50 μm dpphec50 μm abtsrats with streptozotocininduced diabetes mellitus mg100 g iprats with streptozotocinnicotinamideinduceddiabetes mellitus mgkg po μm mgkg μgml ± μmol teammol ± μmol teammol and ± μmol fe2mmolrespectively mgkg pomodelshepg2 cellsrats with chronicprostatitischronic pelvicpain syndromesepsisinduced acute lunginjury in ratsstreptozotocininduceddiabetic ratshigh glucoseinducedinjury of myocardial cellsccl4induced liver damagemodel in micedpphabtsquercetinquercetin 3βglucosidequercetin 3βgalactosidequercetin ramnosideepigallocatechingallateepigallocatechingallatedpphabtsfrapgallocatechingallateamentoflavoneacute lung injury inducedby sepsis in ratsdpph abts superoxideand hydroxyl radicalsteacoracfrapcardiotoxicity induced bydoxorubicin in ratshuman colon cancer cellsht29acute lung injury inducedby sepsis in micemice with obesityassociated insulinresistance inductionhepatic injury induced bydgalactosamine in ratsoracapigeninluteolinherbacetinpectolinarinrhoifolincatechinlipid peroxidationdecreases apoptosis and ros productionand increases sod levelsdecreases lipid peroxidation and increasescat and gsh levelsscavenging of free radicalsreduces indirect nitric oxide synthesis andtotal oxidative statusincreased levels of cat and totalantioxidant capacity of plasmaincreased levels of cat sod and gshreduced levels of superoxide and proteincarbonyl pco and prevented dnadamage ± ± and ± ± and ± respectively ± respectivelydecreases tbars levels and increaseslevels of sod and gshscavenging of free radicalsscavenging of free radicalsreduces levels of mda increases sodlevels and prevents cardiomyocyteapoptosis μgmlincreases levels of gsh expression of gshsynthetase and the activity of sod and mgkg ip mgkg po and mgkg poapproximately troloxequivalents μmcatreduces lipid peroxidation increases theactivity of sod and cat and suppressesthe nfκb pathwayincreases the activity of glucose6phosphate dehydrogenaseincreases levels of sod gsh glutathionereductase and glutathione stransferasescavenging of free radicalsscavenging of free radicals 0coxidative medicine and cellular longevitytable continuedtype of cellscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencemodelsabtsfrap ± mol troloxequivalentsmol ± mol trolox equivalentsmoldihydrorhodamine oxidation assayandic50 ± μmisobavachalconedpph sc50frapabts sc50psoralidinelectron spin resonancemyricetinhelichrysetinscutellareinresveratroldpphchinese hamster lungfibroblast cells v794treated with h2o2oracdpphabtssuperoxide radicalsdpph sc50r2rats with obstructive lungdiseasehypercholesterolemicapoeko mouserespectively μm ± mmic50 μmequivalent to feso4 mm respectively·7h2o and μgml and μgml μgml ± trolox equivalents ± μm ± μm and ± μm respectivelyscavenging of free radicalsscavenging of free radicalsscavenging of free radicals and respectivelyprevents dna damage and lipidperoxidationincreases the activity of sod cat andgpxscavenging of free radicalsscavenging of free radicals μmoldmscavenging of free radicals mgkg mgkgincreases sod activity and reduces mdalevelsinhibits the activity and expression ofnadph oxidasesincreases sod gpx and cat levels injury induced by dgalactosamine by increasing levels ofsod gsh glutathione reductase and glutathione stransferase [ ]rhoifolin is found in citrus fruits such as citrus limettarisso studies have indicated that its radical peroxyl scavenging capacity is higher than trolox in orac assays approximately trolox equivalents μm [ ]meanwhile the catechin is a flavonoid present inleaves of green tea wine and fruits [ ] grzesik investigated the antioxidant action of catechinthrough the abts scavenging activity and frap teststhe results show values of ± mol troloxequivalentsmol and ± mol trolox equivalentsmol respectively in addition catechin shows greaterprotective properties in the dihydrorhodamine oxida ± μm than gsh and ascorbiction assay ic50acid and μm respectively psoralidin is a prenylated coumestan which is found inplants of the fabaceae family such as psoralea corylifolia lxiao and collaborators investigating the antioxidant potential of compounds isolated from p corylifolia observed thatpsoralidin shows the best antioxidant activity by the methodof electron spin resonance spectroscopy with an ic50 value of μm the compound isobavachalcone has been isolated fromplants of the fabaceae and moraceae families [ ] isobavachalcone shows a strong antioxidant activity in dpphsc50 frap and abts sc50 assays with values of μm ± mm equivalent to feso4·7h2o and mm respectively in addition the compound has beenreported to inhibit the nfκb pathway in sephadexinducedlung injury in rats [ ]helichrysetin is a chalcone that is found in plants of thehelichrysum genus such as helichrysum odoratissimum l in a study investigating the antioxidant activity of natural and prenylated chalcones vogel found that helichrysetin is the substance that shows the highest antioxidantactivity in the orac test with values of ± troloxequivalents myricetin is widely found in the plant families myricaceae and anacardiaceae and is widely used as health foodsupplement due to its antioxidant properties [ ]bennett demonstrated that myricetin reacts withoxygencentered galvinoxyl radicals more than timeshigher than vitamin e dalphatocopherol furthermoremyricetin was able to scavenge and on the dpphassay μgml and μgml respectively interestinglythe compound prevents dna damage by lipid 0coxidative medicine and cellular longevity2h2o2 2gsh2o2h2h2o2vit evit cgshnadphnonenzymatic antioxidantsgpxsodcatsemyzne tnadixoitna2h2o gssgo2 h2o2o2 h2omdapcotbarsbio m arkers of oxidative stressgeneration of rosoxidative stresscell damagenaturalantioxidants sesadixohpdan2o2 nadphnadp 2o2 hfigure the main antioxidant mechanisms of natural compounds reported in this review dashed line inhibition full line activationperoxidation and increasing the activity of sod cat andgpx in chinese hamster lung fibroblast cells v794treated with h2o2 scutellarein is found in scutellaria barbata d don andpolygonum viscosum buchham [ ] liu investigated the antioxidant activity of scutellarein through thedpph abts and superoxide scavenging assays they notedthat the compound shows good antioxidant activity withvalues of ± μm ± μm and ± μmrespectively while the trolox a standard antioxidant compound presented ± μm ± μm and ± μm respectively resveratrol is found in grapes peanuts and blueberriesand can be isolated from veratrum grandiflorum o loes literature shows that resveratrol has good antioxidantvalues of μmoldmactivity with dpph sc50r2moreover it is able to reduce the production of ros by inhibiting the activity and expression of nadph oxidases byeliminating oxidant agentsincluding radical hydroxylsuperoxide hydrogen peroxide and peroxynitrite the treatment of resveratrol mgkg po in ratsreduces oxidative stress in obstructive lung disease byincreasing sod activity and reducing mda levels indicatinga decrease in lipid peroxidation table shows themain actions of natural antioxidants discussed in this studyand figure illustrates these activities effect of natural antioxidants incoronavirus infectionsthis review focused on studies reporting on the anticoronavirus activity of natural antioxidants based on exclusioncriteria data from nineteen compounds were discussedthe oxidative stress pathway could potentially be a keyelement in coronavirusinduced apoptosis and pathogenesis for this reason it is interesting to investigate the useof antioxidants as potential therapeutic tools”either as analternative or as an adjuvant to conventional therapies”inthe treatment of coronavirus infections among the antioxidant compounds evaluated as for coronavirus infectionsare the flavonoids which are compounds widely found infruits vegetables and certain beverages in fact researchgroups have reported that antioxidant flavonoids includingcatechin luteolin apigenin quercetin and quercetin rhamnosideinhibit ros accumulation and apoptosis ofcells infected with diï¬erent coronavirus including porcineepidemic diarrhoea coronavirus pedv and transmissiblegastroenteritis coronavirus tgev [“]as shown with the recent covid19 pandemic thesearch for alternative or new antiviral therapies for theremainstreatment of coronavirus diseasesimportantbased on the literature antioxidanttherapies oï¬er anattractive option 0coxidative medicine and cellular longevitytable natural antioxidants tested in in vitro coronavirus infection models and their main results and mechanism of actionantioxidanttype of cells testedconcentrationic50antiviral eï¬ectmechanism of actionreferencecatechintgevinfected st cellscatechin“ μminhibition of tgevinduced apoptosissuppression of the tgevinducedbcl2 reduction baxredistribution cytochrome crelease and caspase3 activation resveratrolmersinfected vero e6cellsresveratrol μmquercetinepigallocatechingallategallocatechingallate gcgquercetin rhamnosideq7rrecombinant 3clprowas expressed in pichiapastoris gs115quercetin μmepigallocatechingallate μmgallocatechingallate μmpedvinfectedvero cellsq7r μminhibition ofmersinducedreduction of the expression ofinfectionapoptosis andnucleocapsid n protein essential prolonged cellular survivalfor merscov replicationafter virus infectioninhibition of coronavirusreplicationreduction of the formationof a visible cytopathiceï¬ect cpe without dnafragmentationgcg displayed a binding energyof kcal mol1 to the active siteof 3clpro and the galloyl moietyat 3oh position was required for3clpro inhibition activitynot specificity amentoflavoneapigeninluteolinquercetinquercetin3βgalactosideherbacetinrhoifolinpectolinarinsarscov 3clproinhibition usingfluorescence resonanceenergy transfer analysismolecular dockingsprfretbasedbioassays andmutagenesistryptophanbasedfluorescence methodherbacetinisobavachalconequercetin3βdglucosidehelichrysetintryptophanbasedfluorescence methodamentoflavone3βgalactoside μmapigenin μmluteolin μmquercetin μmquercetin μmherbacetin μmrhoifolin μmpectolinarin μmherbacetin μmisobavachalcone μmquercetin3βdglucoside μmhelichrysetin μminhibition of sarscovreplicationflavonoids exhibited sarscov3clpro inhibitory activity[ ]inhibition of merscovreplicationflavonoids exhibited merscov3clpro inhibitory activity isobavachalconepsoralidinlineweaver“burk anddixon plotsmyricetinscutellareinsprfretbasedbioassaysisobavachalcone μmpsoralidin μmmyricetin μmscutellarein μminhibition of sarscovreplicationisobavachalcone and psoralidinexhibited sarscov papainlikeprotease inhibitory activitymyricetin and scutellareininhibition of sarscovpotently inhibit the sarscovreplicationhelicase protein in vitro byaï¬ecting the atpase activity the high number of deaths and clinical complicationsobserved in sars and merscov epidemics motivatedthe search for eï¬ective therapeutic agents this was necessitated when many of the tested conventional drugs andtherapies proved ineï¬ective in treating sarsantiviralcov infections for exampletreatment ofthe initial 0coxidative medicine and cellular longevitycell culture coronavirusnaturalantioxidants suppress bcl2 reductionsuppress bax redistributionsuppress cytochorome c releasesuppress caspase3 activationreduce formation of a visiblecytopathic effect without dnafragmentationreduce nucleocapsid n protein expressioninhibit 3clike proteaseinhibit 3clike proteaseinhibit papainlike proteaseinhibit helicase protein by affectedatpase activitytgevpedvmers“covsars“covfigure inhibitory actions of natural antioxidants against coronavirussarscov with antiviral agents such as ribavirin and corticosteroids did not achieve very satisfactory resultsmainly because corticosteroids exertimmunosuppressoreï¬ects on the humoral and cellular immune systems[ ] other drugs such as pentoxifylline were considered for the treatment of sars due to its interestingtherapeutic propertiesanti‚ammatoryantiviral immunomodulatory and bronchodilatory eï¬ectshowever it too was not successful in the clinical treatment of sarscov infection includethatinhibition ofmany antioxidant compounds show antiviral activityagainst sarscov the antiviral activity has been mainlyattributed to thethe 3clike protease3clpro of sarscov a vital enzyme for sarscov replication as an example multiples studies havereported that quercetin and quercetinderived compoundssuch as quercetin 3βgalactoside display potent 3clproinhibitory e5ï¬ect and consequent reduction of sarscovreplication other antioxidants such as epigallocatechin gallate gallocatechin gallate amentoflavone apigeninluteolin herbacetin rhoifolin and pectolinarin are alsofound to efficiently block the enzymatic activity of sarscov 3clpro [ ]moreover some natural antioxidants exhibit promisingantiviral activity against sarscov infection by interferingwith diï¬erent targets involved in sarscov replication inparticular the sarscov papainlike protease plpro andsarscov helicase protein kim reported that isobavachalcone and psoralidin inhibit plpro in a dosedependentmanner with ic50 ranging between and μm previously yu reported that myricetin and scutellareinpotently inhibit the sarscov helicase protein in vitro byaï¬ecting the atpase activity merscov is another zoonotic coronavirus transmitted between animals and human beings that causes severemorbidity and mortality no antiviral medicines with satisfactory efficacy for the treatment of merscovinfectedpatients have been identified to date similar to sarscov natural antioxidant libraries have been probed forpotentialinhibitory compounds against merscov 3clike protease jo showed that herbacetin isobavachalcone quercetin 3βdglucoside and helichrysetinfourcompounds with recognized antioxidant activity can blockthe enzymatic activity of merscov 3clpro using atryptophanbased fluorescence method furthermore theexperimental and computational studies show that flavonoland chalcone are favourite scaï¬olds to bind with the catalytic site of merscov 3clpro in a study performed by lin the antiviral activitiesof resveratrol were investigated in mersinfected vero e6cells the authors reported a significantinhibition ofmerscov infection and prolonged host cell survival aftervirus infection which they speculate was promoted by resveratrol in addition they also found that the expression ofthe nucleocapsid n protein which is essential for merscov replicationis decreased after resveratrol treatment it is important to mention that in vitro models of coronavirus infection also show antiviral activity of flavonoidsextracted from flowering cherry cultivars and black tea[ ] finally antioxidants such as resveratrol alsoare able to block infection produced by herpesvirus the discovery of antiviral compounds from a bioactivecompound against other viruses is an interesting strategy forobtaining new antiviral drugs table shows the mainactions of the natural antioxidants against the coronavirusand figure summarizes these activities 0c conclusionsin conclusion this review shows that antioxidant compounds prominently flavonoids exhibit antiviral action inmodels of coronavirus infections in general the antiviralactivity might be attributed at least in part to the inhibitoryeï¬ect on the enzymatic activity of targets involved in coronavirus replication including sarscov 3clpro sarscovpapainlike protease plpro sarscov helicase proteinand merscov 3clpro in addition some studies provideevidence that the reduction of ros accumulation retardsthe coronavirusactivated apoptotic signaling thereforethe mechanisms of oxidative stress could be the key elementto be studied in coronavirus infectionsincluding thoserelated to ‚ammatory processes arising from the action ofthis virus obviously further investigations are needed toelucidate other pharmacological mechanisms by which natural antioxidants play an antiviral eï¬ect despite the findingsreported in this reviewthey cannot be generalized tocovid19 however the data provided support to the investigation of natural antioxidants as a potential therapeuticapproach in the treatment for covid19 and its severe clinical complications either as an alternative or as an adjuvantto conventional therapies and contribute to the search fornew prototypes in the development of drugs against coronavirus infectionsabbreviations229e3clproabtshuman coronavirus229e3clike protease²azinobis3ethylbenzthiazoline6sulphonic acidcoronavirusescatalasediï¬use alveolar damage22diphenyl1picrylhydrazylferric reducing antioxidant powerreduced glutathioneglutathione peroxidasehuman coronaviruseshuman coronavirushku1lower respiratory tract infectionsmalondialdehydecovscovid19 coronavirus disease catdaddpphfrapgshgpxhcovshku1lrtismdamerscov middle east respiratory syndromecoronavirusnfκbnuclear factor kappa bhuman coronavirusnl63nl63human coronavirusoc43oc43oxygen radical absorbance capacityoracprotein carbonylpcoporcine epidemic diarrhoea coronaviruspedvplpropapainlike proteasereactive nitrogen speciesrnsreactive oxygenated speciesrossarssevere acute respiratory syndromesarscov severe acute respirator

cancer7544

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: coronaviruses covs belong to a family of envelopedviruses with a positive sense singlestranded rna genomecovs cause illness ranging from upper respiratory tractinfections urtis resembling the common cold to lowerrespiratory tract infections lrtis such as bronchitis pneumonia and even severe acute respiratory syndrome sarswith most serious disease outcomes in the elderly immunocompromised patients and infants [ ] hcovoc43oc43 hcov229e 229e hcovnl63 nl63 andhcovhku1 hku1 were the first documented humancovs hcovs which usually cause urtis and less frequently are associated with ltri diseases in the lastdecades two human coronaviruses created great concernfor the world medical community due to significant diseaseand mortality [ ] in severe acute respiratorysyndromecoronavirus sarscov was characterized byacute atypical pneumonia and diï¬use alveolar damagedad in roughly patients and with almost deathsrepresenting a nearly mortality rate more recentlyin a new human coronavirus designated as middle eastrespiratory syndromecoronavirus merscov was identified and the global ongoing outbreak of mers with over official cases and deaths represented approximately case fatality rate to date in humans over the last few months a new strain of human coronavirus sarscov2 also known as 2019ncov hascaught the world™s seven continents™ attention with its rapidglobal spread aï¬ecting at least countries and territoriesinfecting more than and claiming more than lives worldwide the coronavirus pandemichas promoted isolation and uncertainly fear and panicworldwide in additionlikely lead to changes inpolitical and economic power in ways that can be determined only later it willit is important to note that there are many similaritiesamong diï¬erent coronavirus species but not in all aspects 0coxidative medicine and cellular longevitydepending on the molecular mechanism of viral inhibitionpromoted by an antiviral agent the analysis of the data andcomparison between animal and human covs must be donevery carefully in fact it is important to note that there arediï¬erences between human and animal cov receptorswhich will likely result in diï¬erent affinities or unlikely interactions of an antiviral agent with the diï¬erent cov receptors howeverif the antiviral agent interferes with thereplication andor assembly of the covs there is a higherprobability of obtaining similar antiviral activity results inhuman cov tests [ ] following this line our searchin specialized literature was focused mainly on studies thatinvestigated the anticoronavirus eï¬ects of natural antioxidants by inhibiting proteases for viral replication materials and methodsthe present study was carried out based on a search of the literature of natural antioxidants and coronavirus the searchperformed in the pubmed database included studies published until march and used the following keywordscoronavirusstressmerscov sarscov 229e nl63 oc43 hku1merscov virus infection and middle east respiratorysyndrome virus the scientific publications were selectedfrom studies published in the english languageantioxidants flavonoids oxidative pathogenic mechanism of coronavirusinduced cell damagethe high mortality rate associated with the three pathogenichcovs has been mainly attributed to the development ofdigestive and respiratory tract injuries observed followinginfection acute atypical pneumonia and diï¬use alveolardamage that progress to deposition of fibrous tissue denudedairways haemorrhage and elevated macrophage ltrationare sometimes accompanied by watery diarrhoea dehydration and vomiting [ ]despite the molecular mechanisms of coronavirusinduced intestine and lung pathogenesis not fully elucidatedand still unclear studies have suggested that lateterm diseaseprogression is unrelated to viremia it is now believed morelikely to be associated with the immunopathological mechanism [ ] viral clearance and subsequent recovery frominfection require activation of an eï¬ective host immuneresponse however many immune eï¬ector cells may alsocause injury to host tissues together with ‚ammatoryand immune response signaling the presence of oxidativecompounds such as reactive oxygen species ros playsimportant roles in the pathogenic mechanism of cell damageinduced by covs through oxidative stress oxidative stress is defined as an interruption andorderegulation of the signaling and redox system that can becaused by an imbalance in the production of oxidant andantioxidant species among the main oxidant agentsros and reactive nitrogen species rns stand out in orderto counterbalance the oxidant species there is an antioxidantsystem formed by enzymes and nonenzymatic molecules however during pathological events such as viral infections there may be an increase in the production of oxidantspecies not neutralized by the antioxidant system resultingin oxidative stress that promotes cellular damage throughprotein denaturation changes in the functions of nucleicacids lipid peroxidation and cell death [“]in addition during viral infection oxidative stress contributes to viral pathogenesis through stimulating ‚ammation loss of immune function and increased viral replicationthat may occur due to the activation of the nuclear factorkappa b nfκb transcription pathway [“] currentevidence suggests that cytokine dysregulation”also calledcytokine storm”contributes to severe disease caused by thepathogenic covs [ ] the exact mechanisms are notclear yet but research on ‚uenza a virus shows that infection causes a rapid ‚ux of ‚ammatory cells this isfollowed by an increase in reactive oxygen species productionand cytokine expression and release which ultimately leadsto acute lung injury in general rna viruses promotechanges in the body™s antioxidant defense system aï¬ectingenzymes such as superoxide dismutase sod and catalasecat in addition to reducing the levels of antioxidantmolecules such as ascorbic acid carotenoids and reducedglutathione gsh [“] wu reported that glucose6phosphate dehydrogenasean important antioxidantenzyme that produces nadph knockdown cells were moresusceptible to infection by hcov229e than normal cells interestingly ye and colleagues have reported that theinhibition of ros production alleviates ‚ammation causedby ‚uenza a virus infections in an experimental model of sarsinduced acute lunginjury in mice it was noted that phospholipid oxidationdue to oxidative stress is one of the main triggering factorsof acute lung injury this happens through the activation ofthe innate immune response culminating in the activationof pulmonary macrophages via tlr4triftraf6nfκbsignaling furthermore hypoxia caused by acute lunginjury can cause myocardial injury due to the production ofros aggravating infections caused by coronavirus disease covid19 mitochondria have an essential function in energy generation and for this reason their function and integrity arestrictly regulated in order to respond to varying energyrequirements and environmental conditions mitochondria are known to function as the control point in apoptoticpathways releasing proapoptotic factors mainly ros whichfunction as a signaling molecule that may result in cell death[ ] some studies have shown a relationship betweencoronavirus infection and dysfunctional or damaged mitochondria leading to the release of ros and other proapoptotic substances [ ] in a recent study xu reportedthat ros and p53 play key roles in regulating many kindsof the cell process during coronavirus infection in vero cellsaccording to the authors coronavirus infection appears toinduce a timedependent ros accumulation which in turnis linked to regulatory mechanisms of p53 activation andapoptosis in infected cells antioxidant substances promote improvement in casesof disease caused by coronaviruses such as apolipoproteind”a lipocalin that promoted a neuroprotective eï¬ect against 0coxidative medicine and cellular longevityencephalitis induced by human coronavirus oc43”in micethis protective eï¬ect occurred through the reduction of oxidative stress cerebral lipid peroxidation and regulation of‚ammation [ ] also the treatment with antioxidantssuch as pyrrolidine dithiocarbamate or nacetylcysteine significantly inhibits moreover melatonin promotes downregulation of acute lungoxidative injury due to its anti‚ammatory and antioxidantactions making it a possible compound in the treatment ofcovid19 based on these studies compounds thathave antioxidant actions can be helpful in the treatment ofinfections promoted by coronaviruscoronavirusinduced apoptosisin general antioxidant properties of polyphenolic compounds such as some flavonoids have been associated withthe presence of aromatic phenolic rings that promote theelectron donation and hydrogen atom transfer to free radicals acting as free radical scavengers reducing agents andquenchers of single oxygen formation thus the aimof this study was to investigate the antioxidant capacity andantiviral activity of natural antioxidants against coronavirusthe compounds are illustrated in figure occurrence and antioxidant properties ofanticoronavirus compoundsquercetin can be found in plants such as rubus fruticosus land lagerstroemia speciosa l pers [ ] also quercetinshows antioxidant activity at a concentration of μmoll inhepg2 cells inhibiting oxidative stress promoted by h2o2 promotes an increase in sod cat and glutathioneperoxidase gpx and reduces lipid peroxidation in rats withchronic prostatitischronic pelvic pain syndrome moreover quercetin improves sepsisinduced acute lung injury inrats by reducing lipid peroxidation and ‚ammation andincreasing sod and cat levels in addition quercetin glycosides with antioxidant activity such as quercetin 3βglucoside have already been isolated from plants such as passiflora subpeltata ortega andchamomilla suaveolens pursh rydb [ ] the administration of quercetin 3βglucoside mgkg poinstreptozotocininduced diabetic rats promotes an increasein the levels of antioxidant enzymes sod cat andgpx and nonenzymatic antioxidants vitamins c and eand gsh and a reduction of lipid peroxidation quercetin 3βgalactoside hyperoside is found mainly in plantsof the hypericum genus such as hypericum perforatum l[ ] moreover it showed cardioprotective activity inhigh glucoseinduced injury of myocardial cells throughdecreased apoptosis and ros production and increasedsod levels quercetin 7ramnoside is also found inplants of the hypericum genus such as hypericum japonicum thunb ex murray this flavonoid shows hepatoprotective activity against carbon tetrachloride in mice bydecreasing lipid peroxidation and increasing cat andgsh levels in addition to presenting values of μmand22diphenyl1picrylhydrazyldpph and ²azinobis3ethylbenzthiazoline6sulphonic acid abts assays respectively μm intheepigallocatechin gallate is present in parkia roxburghii gdon and is one of the main metabolites found in green teaand liubao tea camellia sinensis lo kuntze also gallocatechin gallate can be found in this plant [“] literaturedata reveal that the administration ip of mg100 g ofepigallocatechin gallate in rats with streptozotocininduceddiabetes mellitus promotes a reduction in oxidative stressthrough reductions in parameters such as indirect nitricoxide synthesis and status total oxidative as well as anincrease in levels of cat and total antioxidant capacity ofplasma furthermore it promotes cardioprotection byantioxidant mechanisms green tea has a high antioxidant capacity due to the highlevels of catechins present he and collaborators compared the antioxidant activities of catechins and reported thatepigallocatechin gallate has greater antioxidant activity viaradical scavenging activity μm with values of ± ± and ± compared to its epimergallocatechin gallate with values of ± ± and ± in the dpph abts and ferric reducingantioxidant power frap respectively amentoflavone is a biflavonoid present in leaves ofginkgo biloba l garcinia brasiliensis l and nandinadomestica l [“] this biflavonoid has a high antioxidantcapacity “ demonstrated in scavenging tests ofdpph abts superoxide and hydroxyl radicals moreover amentoflavone prevents acute lung injury induced bysepsis in rats by decreasing thiobarbituric acid reactive substance tbars levels and by increasing levels of sod andgsh apigenin is mainly present in flowers and leaves beingabundantly found in apium graveolens l petroselinum crispum mill fuss and matricaria chamomilla l sánchezmarzo and collaborators evaluated the antioxidantcapacity of apigenin using the trolox equivalent antioxidantcapacity teac oxygen radical absorbance capacityorac and frap assays the results show that apigeninhas good antioxidant activity with values of ± μmol teammol ± μmol teammol and ± μmol fe2mmol respectively in addition oraladministration of apigenin mgkgday for days in anexperimental model of cardiotoxicity induced by doxorubicin in rats promoted cardioprotection by reducing levels ofmalondialdehyde mda increasing sod levels and preventing cardiomyocyte apoptosis luteolin is present in foods such as carrot cabbage teaand apple and is found in ugni molinae turcz [ ] datashow that luteolin μgml increases the levels of gsh theexpression of gsh synthetase and the activity of sod andcat in human colon cancer cells ht29 furthermoreluteolin attenuates the sepsisinduced acute lunginjury in mice by reducing lipid peroxidation and increasingsod and cat activity in addition to suppressing the nfκbpathway herbacetin is ubiquitous in plants of the genus rhodiolasuch as rhodiola rosea l herbacetin glycosides are alsopresent in the roots of r sachalinensis a bor and show antioxidant activity veeramani reported that theadministration of herbacetin mgkg po in mice with 0coxidative medicine and cellular longevityohohohohhoohooohohhooohoh oquercetinohohoooohohohquercetin 3𝛽galactosideohohooohoohoohohhoohohohoh oquercetin 7ramnosideohooohohoohoohoohohohquercetin 3𝛽glucosidehooohooohohohohohohepigallocatechin gallateohgallocatechin gallateohhooohoh ohooluteolinhooohoooohoohohoohrhoifolinhohohoohohohohohohoohoohooh oamentoflavoneohhooohohohoherbacetinhohoooohooohohohoapigeninooohooohhopectolinarinooopsoralidinhooohohohohcatechinohhoohoooh ohelichrysetinohohomyricetinohohohhohoohoohoisobavachalconeohhooohscutellareinohresveratrolfigure chemical structures of bioactive antioxidants against coronavirusobesityassociated insulin resistance promotes an increasein the activity of the enzyme glucose6phosphate dehydrogenase which is directly related to the production ofnadph pectolinarin is present in plants of the genus cirsiumsuch as cirsium setidens nakai and cirsium japonicum dcthe administration of pectolinarin and mgkg pofor two weeks in rats promotes antioxidant eï¬ects in hepatic 0coxidative medicine and cellular longevitytable antioxidant properties of natural inhibitors of coronaviruscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencetype of cells μmoll mgkg po mgkg poinhibiting oxidative stress promoted byh2o2promoted an increase in sod cat andgpx and reduced lipid peroxidationreduces lipid peroxidation and increasessod and cat levelsincreases levels of sod cat gpx mgkg povitamins c and e and gsh and reduces nmoll mgkgic50 μm dpphec50 μm abtsrats with streptozotocininduced diabetes mellitus mg100 g iprats with streptozotocinnicotinamideinduceddiabetes mellitus mgkg po μm mgkg μgml ± μmol teammol ± μmol teammol and ± μmol fe2mmolrespectively mgkg pomodelshepg2 cellsrats with chronicprostatitischronic pelvicpain syndromesepsisinduced acute lunginjury in ratsstreptozotocininduceddiabetic ratshigh glucoseinducedinjury of myocardial cellsccl4induced liver damagemodel in micedpphabtsquercetinquercetin 3βglucosidequercetin 3βgalactosidequercetin ramnosideepigallocatechingallateepigallocatechingallatedpphabtsfrapgallocatechingallateamentoflavoneacute lung injury inducedby sepsis in ratsdpph abts superoxideand hydroxyl radicalsteacoracfrapcardiotoxicity induced bydoxorubicin in ratshuman colon cancer cellsht29acute lung injury inducedby sepsis in micemice with obesityassociated insulinresistance inductionhepatic injury induced bydgalactosamine in ratsoracapigeninluteolinherbacetinpectolinarinrhoifolincatechinlipid peroxidationdecreases apoptosis and ros productionand increases sod levelsdecreases lipid peroxidation and increasescat and gsh levelsscavenging of free radicalsreduces indirect nitric oxide synthesis andtotal oxidative statusincreased levels of cat and totalantioxidant capacity of plasmaincreased levels of cat sod and gshreduced levels of superoxide and proteincarbonyl pco and prevented dnadamage ± ± and ± ± and ± respectively ± respectivelydecreases tbars levels and increaseslevels of sod and gshscavenging of free radicalsscavenging of free radicalsreduces levels of mda increases sodlevels and prevents cardiomyocyteapoptosis μgmlincreases levels of gsh expression of gshsynthetase and the activity of sod and mgkg ip mgkg po and mgkg poapproximately troloxequivalents μmcatreduces lipid peroxidation increases theactivity of sod and cat and suppressesthe nfκb pathwayincreases the activity of glucose6phosphate dehydrogenaseincreases levels of sod gsh glutathionereductase and glutathione stransferasescavenging of free radicalsscavenging of free radicals 0coxidative medicine and cellular longevitytable continuedtype of cellscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencemodelsabtsfrap ± mol troloxequivalentsmol ± mol trolox equivalentsmoldihydrorhodamine oxidation assayandic50 ± μmisobavachalconedpph sc50frapabts sc50psoralidinelectron spin resonancemyricetinhelichrysetinscutellareinresveratroldpphchinese hamster lungfibroblast cells v794treated with h2o2oracdpphabtssuperoxide radicalsdpph sc50r2rats with obstructive lungdiseasehypercholesterolemicapoeko mouserespectively μm ± mmic50 μmequivalent to feso4 mm respectively·7h2o and μgml and μgml μgml ± trolox equivalents ± μm ± μm and ± μm respectivelyscavenging of free radicalsscavenging of free radicalsscavenging of free radicals and respectivelyprevents dna damage and lipidperoxidationincreases the activity of sod cat andgpxscavenging of free radicalsscavenging of free radicals μmoldmscavenging of free radicals mgkg mgkgincreases sod activity and reduces mdalevelsinhibits the activity and expression ofnadph oxidasesincreases sod gpx and cat levels injury induced by dgalactosamine by increasing levels ofsod gsh glutathione reductase and glutathione stransferase [ ]rhoifolin is found in citrus fruits such as citrus limettarisso studies have indicated that its radical peroxyl scavenging capacity is higher than trolox in orac assays approximately trolox equivalents μm [ ]meanwhile the catechin is a flavonoid present inleaves of green tea wine and fruits [ ] grzesik investigated the antioxidant action of catechinthrough the abts scavenging activity and frap teststhe results show values of ± mol troloxequivalentsmol and ± mol trolox equivalentsmol respectively in addition catechin shows greaterprotective properties in the dihydrorhodamine oxida ± μm than gsh and ascorbiction assay ic50acid and μm respectively psoralidin is a prenylated coumestan which is found inplants of the fabaceae family such as psoralea corylifolia lxiao and collaborators investigating the antioxidant potential of compounds isolated from p corylifolia observed thatpsoralidin shows the best antioxidant activity by the methodof electron spin resonance spectroscopy with an ic50 value of μm the compound isobavachalcone has been isolated fromplants of the fabaceae and moraceae families [ ] isobavachalcone shows a strong antioxidant activity in dpphsc50 frap and abts sc50 assays with values of μm ± mm equivalent to feso4·7h2o and mm respectively in addition the compound has beenreported to inhibit the nfκb pathway in sephadexinducedlung injury in rats [ ]helichrysetin is a chalcone that is found in plants of thehelichrysum genus such as helichrysum odoratissimum l in a study investigating the antioxidant activity of natural and prenylated chalcones vogel found that helichrysetin is the substance that shows the highest antioxidantactivity in the orac test with values of ± troloxequivalents myricetin is widely found in the plant families myricaceae and anacardiaceae and is widely used as health foodsupplement due to its antioxidant properties [ ]bennett demonstrated that myricetin reacts withoxygencentered galvinoxyl radicals more than timeshigher than vitamin e dalphatocopherol furthermoremyricetin was able to scavenge and on the dpphassay μgml and μgml respectively interestinglythe compound prevents dna damage by lipid 0coxidative medicine and cellular longevity2h2o2 2gsh2o2h2h2o2vit evit cgshnadphnonenzymatic antioxidantsgpxsodcatsemyzne tnadixoitna2h2o gssgo2 h2o2o2 h2omdapcotbarsbio m arkers of oxidative stressgeneration of rosoxidative stresscell damagenaturalantioxidants sesadixohpdan2o2 nadphnadp 2o2 hfigure the main antioxidant mechanisms of natural compounds reported in this review dashed line inhibition full line activationperoxidation and increasing the activity of sod cat andgpx in chinese hamster lung fibroblast cells v794treated with h2o2 scutellarein is found in scutellaria barbata d don andpolygonum viscosum buchham [ ] liu investigated the antioxidant activity of scutellarein through thedpph abts and superoxide scavenging assays they notedthat the compound shows good antioxidant activity withvalues of ± μm ± μm and ± μmrespectively while the trolox a standard antioxidant compound presented ± μm ± μm and ± μm respectively resveratrol is found in grapes peanuts and blueberriesand can be isolated from veratrum grandiflorum o loes literature shows that resveratrol has good antioxidantvalues of μmoldmactivity with dpph sc50r2moreover it is able to reduce the production of ros by inhibiting the activity and expression of nadph oxidases byeliminating oxidant agentsincluding radical hydroxylsuperoxide hydrogen peroxide and peroxynitrite the treatment of resveratrol mgkg po in ratsreduces oxidative stress in obstructive lung disease byincreasing sod activity and reducing mda levels indicatinga decrease in lipid peroxidation table shows themain actions of natural antioxidants discussed in this studyand figure illustrates these activities effect of natural antioxidants incoronavirus infectionsthis review focused on studies reporting on the anticoronavirus activity of natural antioxidants based on exclusioncriteria data from nineteen compounds were discussedthe oxidative stress pathway could potentially be a keyelement in coronavirusinduced apoptosis and pathogenesis for this reason it is interesting to investigate the useof antioxidants as potential therapeutic tools”either as analternative or as an adjuvant to conventional therapies”inthe treatment of coronavirus infections among the antioxidant compounds evaluated as for coronavirus infectionsare the flavonoids which are compounds widely found infruits vegetables and certain beverages in fact researchgroups have reported that antioxidant flavonoids includingcatechin luteolin apigenin quercetin and quercetin rhamnosideinhibit ros accumulation and apoptosis ofcells infected with diï¬erent coronavirus including porcineepidemic diarrhoea coronavirus pedv and transmissiblegastroenteritis coronavirus tgev [“]as shown with the recent covid19 pandemic thesearch for alternative or new antiviral therapies for theremainstreatment of coronavirus diseasesimportantbased on the literature antioxidanttherapies oï¬er anattractive option 0coxidative medicine and cellular longevitytable natural antioxidants tested in in vitro coronavirus infection models and their main results and mechanism of actionantioxidanttype of cells testedconcentrationic50antiviral eï¬ectmechanism of actionreferencecatechintgevinfected st cellscatechin“ μminhibition of tgevinduced apoptosissuppression of the tgevinducedbcl2 reduction baxredistribution cytochrome crelease and caspase3 activation resveratrolmersinfected vero e6cellsresveratrol μmquercetinepigallocatechingallategallocatechingallate gcgquercetin rhamnosideq7rrecombinant 3clprowas expressed in pichiapastoris gs115quercetin μmepigallocatechingallate μmgallocatechingallate μmpedvinfectedvero cellsq7r μminhibition ofmersinducedreduction of the expression ofinfectionapoptosis andnucleocapsid n protein essential prolonged cellular survivalfor merscov replicationafter virus infectioninhibition of coronavirusreplicationreduction of the formationof a visible cytopathiceï¬ect cpe without dnafragmentationgcg displayed a binding energyof kcal mol1 to the active siteof 3clpro and the galloyl moietyat 3oh position was required for3clpro inhibition activitynot specificity amentoflavoneapigeninluteolinquercetinquercetin3βgalactosideherbacetinrhoifolinpectolinarinsarscov 3clproinhibition usingfluorescence resonanceenergy transfer analysismolecular dockingsprfretbasedbioassays andmutagenesistryptophanbasedfluorescence methodherbacetinisobavachalconequercetin3βdglucosidehelichrysetintryptophanbasedfluorescence methodamentoflavone3βgalactoside μmapigenin μmluteolin μmquercetin μmquercetin μmherbacetin μmrhoifolin μmpectolinarin μmherbacetin μmisobavachalcone μmquercetin3βdglucoside μmhelichrysetin μminhibition of sarscovreplicationflavonoids exhibited sarscov3clpro inhibitory activity[ ]inhibition of merscovreplicationflavonoids exhibited merscov3clpro inhibitory activity isobavachalconepsoralidinlineweaver“burk anddixon plotsmyricetinscutellareinsprfretbasedbioassaysisobavachalcone μmpsoralidin μmmyricetin μmscutellarein μminhibition of sarscovreplicationisobavachalcone and psoralidinexhibited sarscov papainlikeprotease inhibitory activitymyricetin and scutellareininhibition of sarscovpotently inhibit the sarscovreplicationhelicase protein in vitro byaï¬ecting the atpase activity the high number of deaths and clinical complicationsobserved in sars and merscov epidemics motivatedthe search for eï¬ective therapeutic agents this was necessitated when many of the tested conventional drugs andtherapies proved ineï¬ective in treating sarsantiviralcov infections for exampletreatment ofthe initial 0coxidative medicine and cellular longevitycell culture coronavirusnaturalantioxidants suppress bcl2 reductionsuppress bax redistributionsuppress cytochorome c releasesuppress caspase3 activationreduce formation of a visiblecytopathic effect without dnafragmentationreduce nucleocapsid n protein expressioninhibit 3clike proteaseinhibit 3clike proteaseinhibit papainlike proteaseinhibit helicase protein by affectedatpase activitytgevpedvmers“covsars“covfigure inhibitory actions of natural antioxidants against coronavirussarscov with antiviral agents such as ribavirin and corticosteroids did not achieve very satisfactory resultsmainly because corticosteroids exertimmunosuppressoreï¬ects on the humoral and cellular immune systems[ ] other drugs such as pentoxifylline were considered for the treatment of sars due to its interestingtherapeutic propertiesanti‚ammatoryantiviral immunomodulatory and bronchodilatory eï¬ectshowever it too was not successful in the clinical treatment of sarscov infection includethatinhibition ofmany antioxidant compounds show antiviral activityagainst sarscov the antiviral activity has been mainlyattributed to thethe 3clike protease3clpro of sarscov a vital enzyme for sarscov replication as an example multiples studies havereported that quercetin and quercetinderived compoundssuch as quercetin 3βgalactoside display potent 3clproinhibitory e5ï¬ect and consequent reduction of sarscovreplication other antioxidants such as epigallocatechin gallate gallocatechin gallate amentoflavone apigeninluteolin herbacetin rhoifolin and pectolinarin are alsofound to efficiently block the enzymatic activity of sarscov 3clpro [ ]moreover some natural antioxidants exhibit promisingantiviral activity against sarscov infection by interferingwith diï¬erent targets involved in sarscov replication inparticular the sarscov papainlike protease plpro andsarscov helicase protein kim reported that isobavachalcone and psoralidin inhibit plpro in a dosedependentmanner with ic50 ranging between and μm previously yu reported that myricetin and scutellareinpotently inhibit the sarscov helicase protein in vitro byaï¬ecting the atpase activity merscov is another zoonotic coronavirus transmitted between animals and human beings that causes severemorbidity and mortality no antiviral medicines with satisfactory efficacy for the treatment of merscovinfectedpatients have been identified to date similar to sarscov natural antioxidant libraries have been probed forpotentialinhibitory compounds against merscov 3clike protease jo showed that herbacetin isobavachalcone quercetin 3βdglucoside and helichrysetinfourcompounds with recognized antioxidant activity can blockthe enzymatic activity of merscov 3clpro using atryptophanbased fluorescence method furthermore theexperimental and computational studies show that flavonoland chalcone are favourite scaï¬olds to bind with the catalytic site of merscov 3clpro in a study performed by lin the antiviral activitiesof resveratrol were investigated in mersinfected vero e6cells the authors reported a significantinhibition ofmerscov infection and prolonged host cell survival aftervirus infection which they speculate was promoted by resveratrol in addition they also found that the expression ofthe nucleocapsid n protein which is essential for merscov replicationis decreased after resveratrol treatment it is important to mention that in vitro models of coronavirus infection also show antiviral activity of flavonoidsextracted from flowering cherry cultivars and black tea[ ] finally antioxidants such as resveratrol alsoare able to block infection produced by herpesvirus the discovery of antiviral compounds from a bioactivecompound against other viruses is an interesting strategy forobtaining new antiviral drugs table shows the mainactions of the natural antioxidants against the coronavirusand figure summarizes these activities 0c conclusionsin conclusion this review shows that antioxidant compounds prominently flavonoids exhibit antiviral action inmodels of coronavirus infections in general the antiviralactivity might be attributed at least in part to the inhibitoryeï¬ect on the enzymatic activity of targets involved in coronavirus replication including sarscov 3clpro sarscovpapainlike protease plpro sarscov helicase proteinand merscov 3clpro in addition some studies provideevidence that the reduction of ros accumulation retardsthe coronavirusactivated apoptotic signaling thereforethe mechanisms of oxidative stress could be the key elementto be studied in coronavirus infectionsincluding thoserelated to ‚ammatory processes arising from the action ofthis virus obviously further investigations are needed toelucidate other pharmacological mechanisms by which natural antioxidants play an antiviral eï¬ect despite the findingsreported in this reviewthey cannot be generalized tocovid19 however the data provided support to the investigation of natural antioxidants as a potential therapeuticapproach in the treatment for covid19 and its severe clinical complications either as an alternative or as an adjuvantto conventional therapies and contribute to the search fornew prototypes in the development of drugs against coronavirus infectionsabbreviations229e3clproabtshuman coronavirus229e3clike protease²azinobis3ethylbenzthiazoline6sulphonic acidcoronavirusescatalasediï¬use alveolar damage22diphenyl1picrylhydrazylferric reducing antioxidant powerreduced glutathioneglutathione peroxidasehuman coronaviruseshuman coronavirushku1lower respiratory tract infectionsmalondialdehydecovscovid19 coronavirus disease catdaddpphfrapgshgpxhcovshku1lrtismdamerscov middle east respiratory syndromecoronavirusnfκbnuclear factor kappa bhuman coronavirusnl63nl63human coronavirusoc43oc43oxygen radical absorbance capacityoracprotein carbonylpcoporcine epidemic diarrhoea coronaviruspedvplpropapainlike proteasereactive nitrogen speciesrnsreactive oxygenated speciesrossarssevere acute respiratory syndromesarscov severe acute respirator Answer:

Colon_Cancer

"tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh flavor and health benefits and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves™ drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneficial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneficial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health benefits and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2‹…egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg finally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals ‹…egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe field of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientific community and the general public indeedegcg has shown both prophylactic and therapeutic efficacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table [“]apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoh“o2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site specificity of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpsppar𝛾 cebp𝛼aco ppar𝛼 cpt1acad pgc1𝛼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis [“]accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent “ μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by flow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form biofilms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibiofilm efficacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results verified the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the efficacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microflorawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ “] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are final products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for five days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these findings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o

cancer7545

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh flavor and health benefits and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves™ drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneficial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneficial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health benefits and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2‹…egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg finally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals ‹…egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe field of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientific community and the general public indeedegcg has shown both prophylactic and therapeutic efficacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table [“]apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoh“o2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site specificity of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpsppar𝛾 cebp𝛼aco ppar𝛼 cpt1acad pgc1𝛼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis [“]accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent “ μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by flow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form biofilms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibiofilm efficacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results verified the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the efficacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microflorawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ “] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are final products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for five days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these findings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o Answer:

Colon_Cancer

" chemotherapy is one of the most commonly used treatments for patients with advanced colon cancer yet the toxicity of chemotherapy agents such as ‘fluorouracil 5fu limits the effectiveness of chemotherapy ginsenoside rg3 rg3 is an active ingredient isolated from ginseng rg3 has been shown to display anticancer effects on a variety of malignancies yet whether rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer remains unknown the present study was designed to ascertain whether rg3 is able to enhance the anticolon cancer effect of 5fu the results revealed that combined treatment of rg3 and ‘fu significantly enhanced the inhibition of the proliferation colony formation invasion and migration of human colon cancer cells sw620 and lovo in vitro we also found that combined treatment of rg3 and ‘fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway and arrested the cell cycle of the colon cancer cells in g0g1 by promoting the expression of cyclin d1 cdk2 and cdk4 in addition the pi3kakt signaling pathway in colon cancer cells was suppressed by rg3 and 5fu in vivo rg3 synergized the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude mice similarly combined treatment of rg3 and 5fu altered the expression of colon cancer protein in vivo and in vitro collectively the present study demonstrated that ginsenoside rg3 enhances the anticancer effect of 5fu in colon cancer cells via the pi3kakt pathwaycorrespondence to dr xiangbo chen endoscopy center the quanzhou first hospital affiliated to fujian medical university east street licheng quanzhou fujian pr chinaemail coloboyeahnetkey words ginsenoside rg3 colon cancer 5fluorouracil pi3kaktintroductioncolon cancer is a common malignant tumor of the digestive tract located in the colon which mainly occurs at the junction of the rectum and the sigmoid colon statistics show that the highest incidence of colon cancer is in the age group of years the ratio of male to female is and the incidence of colon cancer ranks third among all cases of gastrointestinal tumors the 5year survival rate of patients with colon cancer is approximately yet the 5year survival rate of patients with advanced stage disease is as low as since the early symptoms of patients with colon cancer are not obvious only about of patients can be diagnosed at the early stage of the disease chemotherapy is one of the most important treatments for patients with advanced colon cancer of which ‘fluorouracil ‘fu is the most widely used 5fu inhibits the proliferation invasion and migration of tumor cells by interfering with the nucleic acid metabolism of tumor cells but it is also toxic to normal cells causing serious adverse reactions even endangering the life safety of patients severely limiting its clinical application previous research has shown that 5fu combined with other agents may reduce the required dosage of 5fu consequently reducing the adverse reactions caused by 5fu without affecting or even improving the efficacy of chemotherapy compared with chemical drugs and biopharmaceuticals multicomponent multitarget and less adverse reactions are unique advantages of traditional chinese medicine for the treatment of diseases in patients with colon cancer chinese medicine can improve patient immunity reduce the side effects of radiotherapy and chemotherapy or enhance drug sensitivity inhibiting the expression of oncogenes helps to inhibit the migration of cancer cells and has a good effect on the treatment of colon cancer ginsenoside rg3 rg3 an active ingredient isolated from ginseng is a tetracyclic triterpenoid saponin that inhibits neovascularization induces tumor cell apoptosis and selectively inhibits tumor cell metastasis and enhances immune function previous studies have shown that rg3 exhibits an inhibitory effect on proliferation invasion and migration of human tumor cells such as lung cancer gastric carcinoma and prostate cancer in colon cancer rg3 was found to activate the ampk signaling pathway to accelerate apoptosis in colon 0chong effects of ginsenoside rg3 on colon cancercancer cell line ht29 in vitro and also to block colon cancer progression by targeting inhibition of cancer stem cells and tumor angiogenesis in vivo although numerous studies have shown that rg3 increases the efficacy and decreases the toxicity of chemotherapeutic drugs and suppresses the chemotherapeutic resistance in cancer its combination with chemotherapeutic agent ‘fu to achieve extra benefits in anticolon cancer treatment warrants detailed investigationin the present study the effects of a combined treatment of rg3 and 5fu on the biological properties of sw620 and lovo cells were investigated in vivo and in vitro we found that a combined treatment of rg3 and 5fu not only enhanced the inhibition of colon cancer cell proliferation migration and invasion but also promoted apoptosis of colon cancer cells and arrested the cells in the g0g1 phase in addition it was also found that rg3 could synergize the capacity of 5fu to inhibit the growth of human colon cancer xenografts in nude mouse and the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt pathway in colon cancer cellsmaterials and methodscell lines and agents sw620 ccl‘ atcc american type culture collection manassas va usa and lovo ccl229 atcc cell lines were cultured with dmem medium cat no thermo fisher scientific inc supplemented with fetal bovine serum fbs cat no ‘ thermo fisher scientific inc and penicillinstreptomycin cat no thermo fisher scientific inc the cell lines used in the present study were cultured at ˚c with co2rg3 cat no sigmaaldrich merck kgaa and 5fu cat no sigmaaldrich merck kgaa were dissolved in dmso for the cell experiments the diluted culture solution of rg3 or 5fu was dissolved in dmso to achieve the experimental concentration and was administered to the cells for h for animal experiments pbs diluted rg3 or 5fu was dissolved in dmso to the experimental concentration the experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinamtt assay a total of 2x103 cellswell were inoculated in a 96well culture plate containing the indicated medium dmem plus fbs we evaluated the viability of the sw620 and lovo cells by mtt assay in short after h of culture mtt µl mgml which was dissolved in dmso was added to the cells and incubated the cell supernatant was removed and then µl dmso was added after min the optical density od570 was determined using a plate reader elx808 bio‘tek instrumentscell colony formation assay a total of 2x103 cellsml were seeded in 6well plates with ml mediumwell and medium was exchanged once every days cells were routinely cultured for about weeks when visible clones appeared in the well the culturing was stopped the supernatant culture medium was drawn washed with pbs times and fixed with formaldehyde for min the supernatant was drawn stained with crystal violet for min and slowly rinsed with sterile water plates were placed in a sterile purification table and images were captured after drying the relative proliferation was measured by measuring the absorbance at nm using a plate reader elx808 bio‘tek instrumentswestern blot analysis ripa lysate buffer cat no p0013c beyotime institute of biotechnology shanghai china was used to extract total cellular protein and the bca kit cat no p0009 beyotime institute of biotechnology was used to determine the protein concentration then cell lysates of sw20 and lovo cells were separated by sdspage with µg total protein and transferred to a pvdf membrane the following primary antibodies were selected as follows anti‘n‘cadherin antibody ab18203 dilution antiecadherin antibody ab1416 dilution anti‘mmp‘ ab38898 dilution anti‘active‘caspase‘ antibody ab2324 dilution antiactivecaspase3 antibody ab2302 dilution antiapaf1 antibody ab2324 dilution anti‘pi3k‘p85 antibody ab191606 dilution antipi3k110 antibody ab32569 dilution anti‘pan‘akt phospho t308 antibody ab38449 dilution anti‘pan‘akt antibody ab8805 dilution anti‘pdk1 antibody ab52893 dilution and anti‘gapdh ab9484 dilution the secondary antibody was selected as follows goat anti‘rabbit ab150077 dilution or goat anti‘rat ab150117 dilution the blocking protocol was with skim milk for h at room temperature the primary antibody was incubated overnight at ˚c and the secondary antibody was incubated for h at room temperature the beyoecl plus kit cat no p0018s beyotime was used for the chromogenic protein bands with beckman coulter immunoassay system unicel dxi beckman coulter and imagej v2147 national institutes of health was used for the densitometric analysis of protein bands all antibodies were purchased from abcam unless otherwise statedtranswell invasion experiment the cell density was adjusted to 05x106 cellsml and then the cells were added to a 24well transwell upper chamber corning corning ny usa medium containing fbs gibco thermo fisher scientific inc was added into the lower transwell chamber and the transwell was incubated at ˚c for h the transwell was taken out and the medium was removed it was washed twice with pbs methanol was added and dried after being fixed for min after the membrane was dried it was stained with crystal violet for min and the relative migration was determined by measuring the absorbance at nm using a plate reader elx808 bio‘tek instruments inccell scratch test a total of 5x105 cells were placed in a 6well plate mlwell a scratch was made as far as possible perpendicular to the back of a horizontal line by using tips against a ruler tips should be vertical and cannot be tilted the cells were washed with pbs for three times and the scratched cells were removed and serumfree dmem was added cells were cultured at ˚c in a co2 incubator for h and images were captured in and h using an ckx41 olympus inverted microscope magnification x100 olympus corp 0concology reports figure effect of the combined treatment of rg3 and 5fu on proliferation of colon cancer cells in vitro sw620 and lovo cells were treated with different doses of a rg3 mmoll or b 5fu µmoll and then the mtt assay was used to detect cell viability c and d after treatment with rg3 mmoll or 5fu µmoll or their combination the colony formation of the colon cancer cells was photographed wt group was used as a baseline for cell viability and cell colony formation three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the wt group ‘fu ‘fluorouracil rg3 ginsenoside rg3 flow cytometric analysis cells that had been treated in different manners were collected and pre‘cooled ethanol pre‘chilled pbs and water‘free configuration was added at ˚c overnight then the cells were washed with pbs and stained with propidium iodine pi for cell cycle macsquant® analyzer flow cytometer miltenyi biotec was used to detect the cell cycle and the annexin v fitcpi kit invitrogen thermo fisher scientific inc was used for flow cytometry to detect apoptosisanimal experiment human colon cancer cells 5x10602 ml in the logarithmic phase were selected a total of female nude mice ‘ weeks of age ‘ g shanghai lingchang biological technology co ltd that were adaptive for feeding [room temperature of ‘Ëšc half day light and night dark cycle air humidity of ] for one week were selected mice were anesthetized [ sodium pentobarbital mgkg intraperitoneal ip] and then the lateral skin of the nude mice was selected as a cell inoculation site to inoculate 5x10602 ml human colon cancer cells at the logarithmic phase of growth when the tumor tissue grew to a volume of approximately mm3 then the mouse were randomly assigned to the solvent group equal amount of pbs dmso rg3 group mgkg gavage administration once every two days 5fu group mgkg ip injection once every two days and rg35fu group combined rg3 and 5fu group administration after weeks of treatment the mice were sacrificed using cervical dislocation and breathing and heartbeat for min were observed to determine death and tumor tissues were extracted and weighed with an analytical balance bsa124s beijing sartorius instruments ltd beijing china all animal experiments were approved by the ethics committee of quanzhou first hospital affiliated to fujian medical universitystatistical analysis all data are expressed as mean ± standard deviation and spss ibm corp was used to analyze the data student's ttest was used to compare differences between two groups and multiple groups were compared with oneway anova followed by duncan test as a post hoc test p005 was assigned to indicate that a difference was statistically significantresultscombined treatment of rg3 and ‘fu enhances inhibition of cell proliferation after treatment with different doses of rg3 or 5fu mtt assay was used to measure the cell viability the results revealed that the cell viability of sw620 and lovo cells was significantly and gradually decreased with an increasing dose of rg3 thus we chose mmoll rg3 for subsequent experiments fig 1a as shown in fig 1b the proliferative activity of the colon cancer cells in the combined treatment group of rg3 and ‘fu was significantly lower than 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on migration and invasion of colon cancer cells in vitro treatment of colon cancer cells with combined treatment of rg3 mmoll and 5fu µmoll inhibited the migration a and invasion b abilities of the sw620 and lovo cells scale bar µm c western blot analysis was used to detect the expression of emtrelated protein ncadherin ecadherin and mmp9 the solvent group was used as a baseline for the migration and invasion of cells three independent repetitions for each experiment were performed p005 p001 and p0001 compared with the rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 emt epithelial‘mesenchymal transition mmp matrix metalloproteinase that of the 5fu treatment alone group in addition the cell viability of sw620 and lovo cells gradually decreased with the increasing dose of 5fu however after treatment with the combination of mmoll rg3 and µmoll ‘fu for h the cell viability of sw620 cells was only which was not conducive to subsequent protein detection experiments thus mmoll rg3 and µmoll 5fu were chosen for subsequent experimentationcell clone formation assays were also used to detect in vitro proliferation of colon cancer cells as shown in fig 1c and d the number of colonies formed by the colon cancer cells treated with rg3 and ‘fu was significantly lower than that of rg3 or ‘fu alone these findings indicated that combined treatment of rg3 and 5fu enhanced the inhibition of colon cancer cell proliferation in vitrocombined treatment of rg3 and ‘fu enhances the inhi‘bition of cell migration and invasion the ability of tumor cells to invade and migrate is the key to tumor progression in the present study we compared the effects of different treatment conditions on the invasion and migration of colon cancer cells it was demonstrated that the invasion and migration ability of the colon cancer cells treated with rg3 combined with ‘fu was significantly lower than that of rg3 or 5fu alone fig 2a and b epithelialmesenchymal transition emt is the source of tumor cell ability to acquire higher invasion and migration capacity thus we determined the levels of three emtrelated proteins ncadherin ecadherin and mmp9 and found that the expression of ncadherin and mmp9 protein in the rg35fu group was significantly lower than that of rg3 or ‘fu alone group but 0concology reports figure effect of the combined treatment of rg3 and 5fu on the apoptosis of colon cancer cells in vitro a the percentage of apoptotic sw620 and lovo cells in the different groups b apoptosisrelated proteins [cleaved clcaspase clcaspase and apaf1] were assessed by western blot analysis in sw620 and lovo cells three independent repetitions for each experiment were carried out p0001 compared with the rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 apaf1 apoptotic protease activating factor the expression of e‘cadherin protein was significantly higher fig 2ccombined treatment of rg3 and ‘fu promotes apoptosis of colon cancer cells fist we found that the apoptosis of the colon cancer cells treated with rg3 combined with 5fu was significantly higher than that of rg3 or ‘fu alone fig 3a the levels of apoptosisrelated proteins in the sw620 and lovo cells were assessed by western blot analysis as shown in fig 3b expression levels of apaf1 cleaved clcaspase and clcaspase protein in colon cancer cells sw620 and lovo treated with rg3 were significantly increased and the expression of these apoptosisrelated protein in colon cancer cells following ‘fu treatment was significantly higher than that treated with rg3 more importantly expression levels of these apoptosisrelated proteins in colon cancer cells treated with the combination of rg2 and 5fu were significantly higher than levels treated with rg3 or 5fu alonewe analyzed the cell cycle distribution of the colon cancer cells after treatment with the different agents as shown in fig 4a the percentages of colon cancer cells in the g0g1 phase treated with the rg3 and 5fu combination were significantly higher than the percentages following rg3 or 5fu alone similarly we also detected cell cycleassociated protein by western blot analysis as shown in fig 4b the expression levels of cyclin d1 cdk2 and cdk4 protein in colon cancer cells which were treated with the rg3 and 5fu combination were significantly lower than levels following treatment with rg3 or 5fu alonecombined treatment of rg3 and ‘fu suppresses pi3kakt signaling in colon cancer cells the pi3kakt signaling 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on cell cycle progression of colon cancer cells in vitro a flow cytometry was used to analysis the cell cycle in colon cancer cells after treatment with rg3 mmoll or 5fu µmoll or the combination b cell cycleassociated protein cyclin d1 cdk2 and cdk4 were assessed by western blot analysis three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 cdk cyclin‘dependent kinase pathway is a signaling pathway involved in cancer cell proliferation invasion and migration and its abnormal activation can confer high proliferation invasion and migration ability of cancer cells in the present study we found that the expression levels of p‘p85 p‘110 ppdk1 and pakt protein in the colon cancer cells which was treated with rg3 and 5fu combination were significantly lower than levels in the cells treated with rg3 or 5fu alone fig these results indicated that the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt signaling pathway in colon cancer cells in vitrocombined treatment of rg3 and ‘fu suppresses tumor growth in nude mice based on the results of in vitro studies we further investigated the effects of the rg3 and 5fu combination on colon cancer cell proliferation and protein expression in nude mice sw620 cells were injected into the armpits of nude mice after weeks of treatment the mice were sacrificed and the weight and volume of tumor tissues were measured it was found that the weight and volume of tumor tissues in the rg35‘fu group were significantly lower than these parameters in the groups treated with rg3 or 5fu alone fig 6a and bmoreover western blot analysis was used to detect the expression of emtrelated proteins cell cyclerelated proteins and key proteins in the pi3kakt signaling pathway it was found that although the effects of the rg3 and 5fu combination were not as obvious as the in vitro results compared with rg3 of 5fu alone the overall trend in protein expression was consistent fig 6ce these results demonstrated that rg3 0concology reports figure effect of the combined treatment of rg3 and 5fu on pi3kakt signaling in colon cancer cells in vitro a and b western blot analysis was used to detect the expression of key proteins in the pi3kakt signaling pathway after treatment with rg3 mmoll or 5fu µmoll or the combination three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude micediscussionthe anticancer effect of 5fluorouracil 5fu is exerted mainly by interfering with tumor cell dna replication and it is a commonly used antitumor agent for the treatment of advanced colon cancer however since 5fu displays nonspecific cytotoxicity it also causes damage to normal cells causing irreversible renal dysfunction and severe gastrointestinal reactions these adverse effects limit its clinical application and further improvements in the efficacy of chemotherapy are needed therefore it is urgent to discover a drug that can enhance the chemotherapeutic effects of 5fu and reduce the 5fu toxicity when used in combination with 5fuginsenoside rg3 rg3 is one of the main active ingredients extracted from ginseng research has shown that ginsenoside rg3 has certain inhibitory effects on lung cancer breast and prostate cancer the antitumor mechanism of rg3 was that rg3 reduced the neovascularization probability of tumor recurrence proliferation and metastasis in tumors by inhibiting kdrvegf protein expression and blocking hif1αcox2vegf pathway in the present study we found that the combined treatment of rg3 and 5fu promoted the inhibition of colon cancer cell proliferation in vivo and in vitro tumor growth development and metastasis are closely related to cell proliferation the previous study found that rg3 inhibits the proliferation of tumor cells such as rg3induced egfrmapk pathway deactivation was found to inhibit melanoma cell proliferation by decreasing fut4ley expression rg3 was found to inhibit the proliferation of multiple myeloma cells by inducing the secretion of igf1 promoting tumor cell apoptosis is also a method of inhibiting tumor cell proliferation in the present study we found that the combined treatment of rg3 and ‘fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway in the mitochondrial pathway of apoptosis apoptosisrelated signals release cytochrome c by stimulating the mitochondrial outer membrane cytochrome c enters the cytoplasm which activates caspase9 by binding with apaf1 activation of caspase9 further activates caspase3 while the activated caspase3 can activate caspase‘ leading to apoptosis in addition we also found that the rg3 and 5fu combination enhanced the number of g0g1 phase colon cancer cells and decreased expression of cyclin d1 cdk2 and cdk4 the cell cycle refers to the whole process that the cell undergoes from the completion of one division to the end of the next division and 0chong effects of ginsenoside rg3 on colon cancerfigure effects of the combined treatment of rg3 and 5fu on tumor growth and protein expression of colon cancer cells in vivo after weeks of treatment the mice were sacrificed tumor tissues were excised and the weight a and volume b of tumor tissues were measured c‘e total protein was extracted from the colon cancer tumor tissues and the expression of proteins was detected by western blot analysis five nude mice in each group and at least tumor tissues were used to evaluate protein expression p005 p001 and p0001 compared with the rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 the regulation of the cell cycle is mainly achieved by the retention of the g1 phase when a cell is in the g1 phase there is an important node regulating the cell cycle the r point when the cell cycle is before the r point the cell needs the external growth factor to achieve the normal operation of the cell cycle after the cell cycle crosses the r point the cell cycle becomes a process that is controlled autonomously by the cell and no longer depends on the presence of external cytokines cyclin d1 is a g1s‘specific cyclin and its main function is to promote the cell cycle from g1 to s by binding and activating the cyclindependent kinase cdk24 a unique cyclindependent kinase of g1 so as to promote cell proliferation invasion and migration of tumor cells are the most important features of malignant tumors and the important causes of death in patients with malignant tumors ncadherin ecadherin and mmp9 are three proteins that play important roles in cell epithelialmesenchymal transition emt whereas emt provides cells the ability to transfer and invade promoting tumor cell emt can inhibit the expression of intercellular junction protein resulting in decreased intercellular connectivity which is beneficial to the invasion and migration of tumor cells to surrounding healthy tissues previous studies have found that rg3 not only inhibits metastasis and invasion of lung cancer cells by inhibiting emt induced by transforming factor 1 but also inhibited the metastasis of prostate pc3m cells by downregulating the expression of aqp1 by downregulating mmp‘ rg3 affected the metastasis and invasion ability of melanoma cells the present study demonstrated that the combined treatment of rg3 and ‘fu significantly suppressed the invasion and migration ability of human colon cancer cell in vitro by altering emtrelated proteinfurthermore we also found that rg3 and 5fu combination inhibited the conduction of the pi3kakt signaling pathway in vivo and in vitro many studies have shown that the occurrence and development of tumors are the result of multifactor multigene and multipathway processes and the cell signal transduction pathway is crucial in the process of tumor development invasion and metastasis the phosphatidylinositol 3kinaseserinethreonine kinase b pi3kakt signaling pathway plays an important role in the regulation of solid tumors [eg liver cancer breast cancer colon cancer gastric cancer neuroblastoma ] and blood tumors [eg leukemia ] pi3k acts as a bridge molecule for the relationship between extracellular signals and cellular responses under the influence of a series of upstream or bypass signaling molecules it acts on the downstream of the effects of a variety of molecules thus promotes cell migration 0concology reports inhibits cell apoptosis accelerates the process of the cell cycle and promotes cell proliferation many previous studies have shown that traditional chinese medicine or traditional chinese medicine monomers can play an antitumor role by inhibiting the pi3kakt signaling pathway in conclusion rg3 enhances 5fu inhibiting proliferation invasion and migration of colorectal cancer cells and helps 5fu block g1 phase induced apoptosis in more colorectal cells all in all our study found that rg3 enhanced the anticancer effect of 5fu on colon cancer cell via pi3kakt pathwayacknowledgementsnot applicablefundingno funding was receivedavailability of data and materialsthe datasets used during the present study are available from the corresponding author upon reasonable requestauthors' contributionsxc made substantial contributions to the conception and design of the study and critically revised it for important intellectual content sh contributed to the acquisition of the data wc zh yw xm yh and zl analyzed and interpreted the data all authors read and approved the final manuscriptethics approval and consent to participateall animal and cell experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinapatient consent for publicationnot applicablecompeting intereststhe authors state that they have no competing interestsreferences siegel rl ward em and jemal a trends in colorectal cancer incidence rates in the united states by tumor location and stage ‘ cancer epidemiol biomarkers prev ‘ siegel rl miller kd and jemal a cancer statistics ca cancer j clin ‘ chen w zheng r baade pd zhang s zeng h bray f jemal a yu xq and he j cancer statistics in china ca cancer j clin chen w zheng r zhang s zeng h zuo t xia c yang z and he j cancer incidence and mortality in china in an analysis based on urbanization level chin j cancer res ‘ li j hou n faried a tsutsumi s and kuwano h inhibition of autophagy augments ‘fluorouracil chemotherapy in human colon cancer in vitro and in vivo model eur j cancer sanoff hk carpenter wr freburger j li l chen k zullig ll goldberg rm schymura mj and schrag d comparison of adverse events during ‘fluorouracil versus ‘fluorouraciloxaliplatin adjuvant chemotherapy for stage iii colon cancer a population‘based analysis cancer ‘ cheah ky howarth gs bindon ka kennedy ja and bastian sep low molecular weight procyanidins from grape seeds enhance the impact of ‘fluorouracil chemotherapy on caco‘ human colon cancer cells plos one e98921 gao y xiao x zhang c yu w guo w zhang z li z feng x hao j zhang k melatonin synergizes the chemotherapeutic effect of ‘fluorouracil in colon cancer by suppressing pi3kakt and nfbinos signaling pathways j pineal res doi 101111jpi12380 wang sf wu my cai cz li m and lu jh autophagy modulators from traditional chinese medicine mechanisms and therapeutic potentials for cancer and neurodegenerative diseases j ethnopharmacol ‘ ernst e traditional chinese medicine for cancer br j cancer sun hy lee jh han ys yoon ym yun cw kim jh song ys and lee sh pivotal roles of ginsenoside rg3 in tumor apoptosis through regulation of reactive oxygen species anticancer res ‘ tang yc zhang y zhou j zhi q wu my gong fr shen m liu l tao m shen b ginsenoside rg3 targets cancer stem cells and tumor angiogenesis to inhibit colorectal cancer progression in vivo int j oncol ‘ wang j tian l khan mn zhang l chen q zhao y yan q fu l and liu j ginsenoside rg3 sensitizes hypoxic lung cancer cells to cisplatin via blocking of nfκb mediated epithelialmesenchymal transition and sternness cancer lett ‘ joo e ha yw and kim ys abstract lb23 molecular mechanisms of ginsenoside rg3 related to apoptosis in human lung and pancreatic adenocarcinomas cancer res lb23 kim bj nah sy jeon jh so i and kim sj transient receptor potential melastatin channels are involved in ginsenoside rg3induced apoptosis in gastric cancer cells basic clin pharmacol kim sm lee sy cho js son sm choi ss yun yp yoo hs yoon dy oh kw han sb and hong jt combination of ginsenoside rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of nfkappa b eur j pharmacol yuan hd quan hy zhang y kim sh and chung sh 20sginsenoside rg3induced apoptosis in ht29 colon cancer cells is associated with ampk signaling pathway mol med rep ‘ liu tg huang y cui dd huang xb mao sh ji ll song hb and yi c inhibitory effect of ginsenoside rg3 combined with gemcitabine on angiogenesis and growth of lung cancer in mice bmc cancer sun my ye y xiao l duan xy zhang ym and zhang h anticancer effects of ginsenoside rg3 review int j mol med ‘ longley db harkin dp and johnston pg fluorouracil mechanisms of action and clinical strategies nat rev cancer ‘ hokmabady l raissi h and khanmohammadi a interactions of the ‘fluorouracil anticancer drug with dna pyrimidine bases a detailed computational approach struct chem ‘ rateesh s luis sa luis cr hughes b and nicolae m myocardial infarction secondary to ‘fluorouracil not an absolute contraindication to rechallenge int j cardiol e331‘e333 shan x aziz f tian ll wang xq yan q and liu jw ginsenoside rg3induced egfrmapk pathway deactivation inhibits melanoma cell proliferation by decreasing fut4ley expression int j oncol ‘ luo y zhang p zeng hq lou sf and wang dx ginsenoside rg3 induces apoptosis in human multiple myeloma cells via the activation of bcl2associated x protein mol med rep ‘ cardone mh roy n stennicke hr salvesen gs franke tf stanbridge e frisch s and reed jc regulation of cell death protease caspase‘ by phosphorylation science ‘ 0chong effects of ginsenoside rg3 on colon can

cancer7546

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " chemotherapy is one of the most commonly used treatments for patients with advanced colon cancer yet the toxicity of chemotherapy agents such as ‘fluorouracil 5fu limits the effectiveness of chemotherapy ginsenoside rg3 rg3 is an active ingredient isolated from ginseng rg3 has been shown to display anticancer effects on a variety of malignancies yet whether rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer remains unknown the present study was designed to ascertain whether rg3 is able to enhance the anticolon cancer effect of 5fu the results revealed that combined treatment of rg3 and ‘fu significantly enhanced the inhibition of the proliferation colony formation invasion and migration of human colon cancer cells sw620 and lovo in vitro we also found that combined treatment of rg3 and ‘fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway and arrested the cell cycle of the colon cancer cells in g0g1 by promoting the expression of cyclin d1 cdk2 and cdk4 in addition the pi3kakt signaling pathway in colon cancer cells was suppressed by rg3 and 5fu in vivo rg3 synergized the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude mice similarly combined treatment of rg3 and 5fu altered the expression of colon cancer protein in vivo and in vitro collectively the present study demonstrated that ginsenoside rg3 enhances the anticancer effect of 5fu in colon cancer cells via the pi3kakt pathwaycorrespondence to dr xiangbo chen endoscopy center the quanzhou first hospital affiliated to fujian medical university east street licheng quanzhou fujian pr chinaemail coloboyeahnetkey words ginsenoside rg3 colon cancer 5fluorouracil pi3kaktintroductioncolon cancer is a common malignant tumor of the digestive tract located in the colon which mainly occurs at the junction of the rectum and the sigmoid colon statistics show that the highest incidence of colon cancer is in the age group of years the ratio of male to female is and the incidence of colon cancer ranks third among all cases of gastrointestinal tumors the 5year survival rate of patients with colon cancer is approximately yet the 5year survival rate of patients with advanced stage disease is as low as since the early symptoms of patients with colon cancer are not obvious only about of patients can be diagnosed at the early stage of the disease chemotherapy is one of the most important treatments for patients with advanced colon cancer of which ‘fluorouracil ‘fu is the most widely used 5fu inhibits the proliferation invasion and migration of tumor cells by interfering with the nucleic acid metabolism of tumor cells but it is also toxic to normal cells causing serious adverse reactions even endangering the life safety of patients severely limiting its clinical application previous research has shown that 5fu combined with other agents may reduce the required dosage of 5fu consequently reducing the adverse reactions caused by 5fu without affecting or even improving the efficacy of chemotherapy compared with chemical drugs and biopharmaceuticals multicomponent multitarget and less adverse reactions are unique advantages of traditional chinese medicine for the treatment of diseases in patients with colon cancer chinese medicine can improve patient immunity reduce the side effects of radiotherapy and chemotherapy or enhance drug sensitivity inhibiting the expression of oncogenes helps to inhibit the migration of cancer cells and has a good effect on the treatment of colon cancer ginsenoside rg3 rg3 an active ingredient isolated from ginseng is a tetracyclic triterpenoid saponin that inhibits neovascularization induces tumor cell apoptosis and selectively inhibits tumor cell metastasis and enhances immune function previous studies have shown that rg3 exhibits an inhibitory effect on proliferation invasion and migration of human tumor cells such as lung cancer gastric carcinoma and prostate cancer in colon cancer rg3 was found to activate the ampk signaling pathway to accelerate apoptosis in colon 0chong effects of ginsenoside rg3 on colon cancercancer cell line ht29 in vitro and also to block colon cancer progression by targeting inhibition of cancer stem cells and tumor angiogenesis in vivo although numerous studies have shown that rg3 increases the efficacy and decreases the toxicity of chemotherapeutic drugs and suppresses the chemotherapeutic resistance in cancer its combination with chemotherapeutic agent ‘fu to achieve extra benefits in anticolon cancer treatment warrants detailed investigationin the present study the effects of a combined treatment of rg3 and 5fu on the biological properties of sw620 and lovo cells were investigated in vivo and in vitro we found that a combined treatment of rg3 and 5fu not only enhanced the inhibition of colon cancer cell proliferation migration and invasion but also promoted apoptosis of colon cancer cells and arrested the cells in the g0g1 phase in addition it was also found that rg3 could synergize the capacity of 5fu to inhibit the growth of human colon cancer xenografts in nude mouse and the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt pathway in colon cancer cellsmaterials and methodscell lines and agents sw620 ccl‘ atcc american type culture collection manassas va usa and lovo ccl229 atcc cell lines were cultured with dmem medium cat no thermo fisher scientific inc supplemented with fetal bovine serum fbs cat no ‘ thermo fisher scientific inc and penicillinstreptomycin cat no thermo fisher scientific inc the cell lines used in the present study were cultured at ˚c with co2rg3 cat no sigmaaldrich merck kgaa and 5fu cat no sigmaaldrich merck kgaa were dissolved in dmso for the cell experiments the diluted culture solution of rg3 or 5fu was dissolved in dmso to achieve the experimental concentration and was administered to the cells for h for animal experiments pbs diluted rg3 or 5fu was dissolved in dmso to the experimental concentration the experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinamtt assay a total of 2x103 cellswell were inoculated in a 96well culture plate containing the indicated medium dmem plus fbs we evaluated the viability of the sw620 and lovo cells by mtt assay in short after h of culture mtt µl mgml which was dissolved in dmso was added to the cells and incubated the cell supernatant was removed and then µl dmso was added after min the optical density od570 was determined using a plate reader elx808 bio‘tek instrumentscell colony formation assay a total of 2x103 cellsml were seeded in 6well plates with ml mediumwell and medium was exchanged once every days cells were routinely cultured for about weeks when visible clones appeared in the well the culturing was stopped the supernatant culture medium was drawn washed with pbs times and fixed with formaldehyde for min the supernatant was drawn stained with crystal violet for min and slowly rinsed with sterile water plates were placed in a sterile purification table and images were captured after drying the relative proliferation was measured by measuring the absorbance at nm using a plate reader elx808 bio‘tek instrumentswestern blot analysis ripa lysate buffer cat no p0013c beyotime institute of biotechnology shanghai china was used to extract total cellular protein and the bca kit cat no p0009 beyotime institute of biotechnology was used to determine the protein concentration then cell lysates of sw20 and lovo cells were separated by sdspage with µg total protein and transferred to a pvdf membrane the following primary antibodies were selected as follows anti‘n‘cadherin antibody ab18203 dilution antiecadherin antibody ab1416 dilution anti‘mmp‘ ab38898 dilution anti‘active‘caspase‘ antibody ab2324 dilution antiactivecaspase3 antibody ab2302 dilution antiapaf1 antibody ab2324 dilution anti‘pi3k‘p85 antibody ab191606 dilution antipi3k110 antibody ab32569 dilution anti‘pan‘akt phospho t308 antibody ab38449 dilution anti‘pan‘akt antibody ab8805 dilution anti‘pdk1 antibody ab52893 dilution and anti‘gapdh ab9484 dilution the secondary antibody was selected as follows goat anti‘rabbit ab150077 dilution or goat anti‘rat ab150117 dilution the blocking protocol was with skim milk for h at room temperature the primary antibody was incubated overnight at ˚c and the secondary antibody was incubated for h at room temperature the beyoecl plus kit cat no p0018s beyotime was used for the chromogenic protein bands with beckman coulter immunoassay system unicel dxi beckman coulter and imagej v2147 national institutes of health was used for the densitometric analysis of protein bands all antibodies were purchased from abcam unless otherwise statedtranswell invasion experiment the cell density was adjusted to 05x106 cellsml and then the cells were added to a 24well transwell upper chamber corning corning ny usa medium containing fbs gibco thermo fisher scientific inc was added into the lower transwell chamber and the transwell was incubated at ˚c for h the transwell was taken out and the medium was removed it was washed twice with pbs methanol was added and dried after being fixed for min after the membrane was dried it was stained with crystal violet for min and the relative migration was determined by measuring the absorbance at nm using a plate reader elx808 bio‘tek instruments inccell scratch test a total of 5x105 cells were placed in a 6well plate mlwell a scratch was made as far as possible perpendicular to the back of a horizontal line by using tips against a ruler tips should be vertical and cannot be tilted the cells were washed with pbs for three times and the scratched cells were removed and serumfree dmem was added cells were cultured at ˚c in a co2 incubator for h and images were captured in and h using an ckx41 olympus inverted microscope magnification x100 olympus corp 0concology reports figure effect of the combined treatment of rg3 and 5fu on proliferation of colon cancer cells in vitro sw620 and lovo cells were treated with different doses of a rg3 mmoll or b 5fu µmoll and then the mtt assay was used to detect cell viability c and d after treatment with rg3 mmoll or 5fu µmoll or their combination the colony formation of the colon cancer cells was photographed wt group was used as a baseline for cell viability and cell colony formation three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the wt group ‘fu ‘fluorouracil rg3 ginsenoside rg3 flow cytometric analysis cells that had been treated in different manners were collected and pre‘cooled ethanol pre‘chilled pbs and water‘free configuration was added at ˚c overnight then the cells were washed with pbs and stained with propidium iodine pi for cell cycle macsquant® analyzer flow cytometer miltenyi biotec was used to detect the cell cycle and the annexin v fitcpi kit invitrogen thermo fisher scientific inc was used for flow cytometry to detect apoptosisanimal experiment human colon cancer cells 5x10602 ml in the logarithmic phase were selected a total of female nude mice ‘ weeks of age ‘ g shanghai lingchang biological technology co ltd that were adaptive for feeding [room temperature of ‘Ëšc half day light and night dark cycle air humidity of ] for one week were selected mice were anesthetized [ sodium pentobarbital mgkg intraperitoneal ip] and then the lateral skin of the nude mice was selected as a cell inoculation site to inoculate 5x10602 ml human colon cancer cells at the logarithmic phase of growth when the tumor tissue grew to a volume of approximately mm3 then the mouse were randomly assigned to the solvent group equal amount of pbs dmso rg3 group mgkg gavage administration once every two days 5fu group mgkg ip injection once every two days and rg35fu group combined rg3 and 5fu group administration after weeks of treatment the mice were sacrificed using cervical dislocation and breathing and heartbeat for min were observed to determine death and tumor tissues were extracted and weighed with an analytical balance bsa124s beijing sartorius instruments ltd beijing china all animal experiments were approved by the ethics committee of quanzhou first hospital affiliated to fujian medical universitystatistical analysis all data are expressed as mean ± standard deviation and spss ibm corp was used to analyze the data student's ttest was used to compare differences between two groups and multiple groups were compared with oneway anova followed by duncan test as a post hoc test p005 was assigned to indicate that a difference was statistically significantresultscombined treatment of rg3 and ‘fu enhances inhibition of cell proliferation after treatment with different doses of rg3 or 5fu mtt assay was used to measure the cell viability the results revealed that the cell viability of sw620 and lovo cells was significantly and gradually decreased with an increasing dose of rg3 thus we chose mmoll rg3 for subsequent experiments fig 1a as shown in fig 1b the proliferative activity of the colon cancer cells in the combined treatment group of rg3 and ‘fu was significantly lower than 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on migration and invasion of colon cancer cells in vitro treatment of colon cancer cells with combined treatment of rg3 mmoll and 5fu µmoll inhibited the migration a and invasion b abilities of the sw620 and lovo cells scale bar µm c western blot analysis was used to detect the expression of emtrelated protein ncadherin ecadherin and mmp9 the solvent group was used as a baseline for the migration and invasion of cells three independent repetitions for each experiment were performed p005 p001 and p0001 compared with the rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 emt epithelial‘mesenchymal transition mmp matrix metalloproteinase that of the 5fu treatment alone group in addition the cell viability of sw620 and lovo cells gradually decreased with the increasing dose of 5fu however after treatment with the combination of mmoll rg3 and µmoll ‘fu for h the cell viability of sw620 cells was only which was not conducive to subsequent protein detection experiments thus mmoll rg3 and µmoll 5fu were chosen for subsequent experimentationcell clone formation assays were also used to detect in vitro proliferation of colon cancer cells as shown in fig 1c and d the number of colonies formed by the colon cancer cells treated with rg3 and ‘fu was significantly lower than that of rg3 or ‘fu alone these findings indicated that combined treatment of rg3 and 5fu enhanced the inhibition of colon cancer cell proliferation in vitrocombined treatment of rg3 and ‘fu enhances the inhi‘bition of cell migration and invasion the ability of tumor cells to invade and migrate is the key to tumor progression in the present study we compared the effects of different treatment conditions on the invasion and migration of colon cancer cells it was demonstrated that the invasion and migration ability of the colon cancer cells treated with rg3 combined with ‘fu was significantly lower than that of rg3 or 5fu alone fig 2a and b epithelialmesenchymal transition emt is the source of tumor cell ability to acquire higher invasion and migration capacity thus we determined the levels of three emtrelated proteins ncadherin ecadherin and mmp9 and found that the expression of ncadherin and mmp9 protein in the rg35fu group was significantly lower than that of rg3 or ‘fu alone group but 0concology reports figure effect of the combined treatment of rg3 and 5fu on the apoptosis of colon cancer cells in vitro a the percentage of apoptotic sw620 and lovo cells in the different groups b apoptosisrelated proteins [cleaved clcaspase clcaspase and apaf1] were assessed by western blot analysis in sw620 and lovo cells three independent repetitions for each experiment were carried out p0001 compared with the rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 apaf1 apoptotic protease activating factor the expression of e‘cadherin protein was significantly higher fig 2ccombined treatment of rg3 and ‘fu promotes apoptosis of colon cancer cells fist we found that the apoptosis of the colon cancer cells treated with rg3 combined with 5fu was significantly higher than that of rg3 or ‘fu alone fig 3a the levels of apoptosisrelated proteins in the sw620 and lovo cells were assessed by western blot analysis as shown in fig 3b expression levels of apaf1 cleaved clcaspase and clcaspase protein in colon cancer cells sw620 and lovo treated with rg3 were significantly increased and the expression of these apoptosisrelated protein in colon cancer cells following ‘fu treatment was significantly higher than that treated with rg3 more importantly expression levels of these apoptosisrelated proteins in colon cancer cells treated with the combination of rg2 and 5fu were significantly higher than levels treated with rg3 or 5fu alonewe analyzed the cell cycle distribution of the colon cancer cells after treatment with the different agents as shown in fig 4a the percentages of colon cancer cells in the g0g1 phase treated with the rg3 and 5fu combination were significantly higher than the percentages following rg3 or 5fu alone similarly we also detected cell cycleassociated protein by western blot analysis as shown in fig 4b the expression levels of cyclin d1 cdk2 and cdk4 protein in colon cancer cells which were treated with the rg3 and 5fu combination were significantly lower than levels following treatment with rg3 or 5fu alonecombined treatment of rg3 and ‘fu suppresses pi3kakt signaling in colon cancer cells the pi3kakt signaling 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on cell cycle progression of colon cancer cells in vitro a flow cytometry was used to analysis the cell cycle in colon cancer cells after treatment with rg3 mmoll or 5fu µmoll or the combination b cell cycleassociated protein cyclin d1 cdk2 and cdk4 were assessed by western blot analysis three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 cdk cyclin‘dependent kinase pathway is a signaling pathway involved in cancer cell proliferation invasion and migration and its abnormal activation can confer high proliferation invasion and migration ability of cancer cells in the present study we found that the expression levels of p‘p85 p‘110 ppdk1 and pakt protein in the colon cancer cells which was treated with rg3 and 5fu combination were significantly lower than levels in the cells treated with rg3 or 5fu alone fig these results indicated that the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt signaling pathway in colon cancer cells in vitrocombined treatment of rg3 and ‘fu suppresses tumor growth in nude mice based on the results of in vitro studies we further investigated the effects of the rg3 and 5fu combination on colon cancer cell proliferation and protein expression in nude mice sw620 cells were injected into the armpits of nude mice after weeks of treatment the mice were sacrificed and the weight and volume of tumor tissues were measured it was found that the weight and volume of tumor tissues in the rg35‘fu group were significantly lower than these parameters in the groups treated with rg3 or 5fu alone fig 6a and bmoreover western blot analysis was used to detect the expression of emtrelated proteins cell cyclerelated proteins and key proteins in the pi3kakt signaling pathway it was found that although the effects of the rg3 and 5fu combination were not as obvious as the in vitro results compared with rg3 of 5fu alone the overall trend in protein expression was consistent fig 6ce these results demonstrated that rg3 0concology reports figure effect of the combined treatment of rg3 and 5fu on pi3kakt signaling in colon cancer cells in vitro a and b western blot analysis was used to detect the expression of key proteins in the pi3kakt signaling pathway after treatment with rg3 mmoll or 5fu µmoll or the combination three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude micediscussionthe anticancer effect of 5fluorouracil 5fu is exerted mainly by interfering with tumor cell dna replication and it is a commonly used antitumor agent for the treatment of advanced colon cancer however since 5fu displays nonspecific cytotoxicity it also causes damage to normal cells causing irreversible renal dysfunction and severe gastrointestinal reactions these adverse effects limit its clinical application and further improvements in the efficacy of chemotherapy are needed therefore it is urgent to discover a drug that can enhance the chemotherapeutic effects of 5fu and reduce the 5fu toxicity when used in combination with 5fuginsenoside rg3 rg3 is one of the main active ingredients extracted from ginseng research has shown that ginsenoside rg3 has certain inhibitory effects on lung cancer breast and prostate cancer the antitumor mechanism of rg3 was that rg3 reduced the neovascularization probability of tumor recurrence proliferation and metastasis in tumors by inhibiting kdrvegf protein expression and blocking hif1αcox2vegf pathway in the present study we found that the combined treatment of rg3 and 5fu promoted the inhibition of colon cancer cell proliferation in vivo and in vitro tumor growth development and metastasis are closely related to cell proliferation the previous study found that rg3 inhibits the proliferation of tumor cells such as rg3induced egfrmapk pathway deactivation was found to inhibit melanoma cell proliferation by decreasing fut4ley expression rg3 was found to inhibit the proliferation of multiple myeloma cells by inducing the secretion of igf1 promoting tumor cell apoptosis is also a method of inhibiting tumor cell proliferation in the present study we found that the combined treatment of rg3 and ‘fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway in the mitochondrial pathway of apoptosis apoptosisrelated signals release cytochrome c by stimulating the mitochondrial outer membrane cytochrome c enters the cytoplasm which activates caspase9 by binding with apaf1 activation of caspase9 further activates caspase3 while the activated caspase3 can activate caspase‘ leading to apoptosis in addition we also found that the rg3 and 5fu combination enhanced the number of g0g1 phase colon cancer cells and decreased expression of cyclin d1 cdk2 and cdk4 the cell cycle refers to the whole process that the cell undergoes from the completion of one division to the end of the next division and 0chong effects of ginsenoside rg3 on colon cancerfigure effects of the combined treatment of rg3 and 5fu on tumor growth and protein expression of colon cancer cells in vivo after weeks of treatment the mice were sacrificed tumor tissues were excised and the weight a and volume b of tumor tissues were measured c‘e total protein was extracted from the colon cancer tumor tissues and the expression of proteins was detected by western blot analysis five nude mice in each group and at least tumor tissues were used to evaluate protein expression p005 p001 and p0001 compared with the rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 the regulation of the cell cycle is mainly achieved by the retention of the g1 phase when a cell is in the g1 phase there is an important node regulating the cell cycle the r point when the cell cycle is before the r point the cell needs the external growth factor to achieve the normal operation of the cell cycle after the cell cycle crosses the r point the cell cycle becomes a process that is controlled autonomously by the cell and no longer depends on the presence of external cytokines cyclin d1 is a g1s‘specific cyclin and its main function is to promote the cell cycle from g1 to s by binding and activating the cyclindependent kinase cdk24 a unique cyclindependent kinase of g1 so as to promote cell proliferation invasion and migration of tumor cells are the most important features of malignant tumors and the important causes of death in patients with malignant tumors ncadherin ecadherin and mmp9 are three proteins that play important roles in cell epithelialmesenchymal transition emt whereas emt provides cells the ability to transfer and invade promoting tumor cell emt can inhibit the expression of intercellular junction protein resulting in decreased intercellular connectivity which is beneficial to the invasion and migration of tumor cells to surrounding healthy tissues previous studies have found that rg3 not only inhibits metastasis and invasion of lung cancer cells by inhibiting emt induced by transforming factor 1 but also inhibited the metastasis of prostate pc3m cells by downregulating the expression of aqp1 by downregulating mmp‘ rg3 affected the metastasis and invasion ability of melanoma cells the present study demonstrated that the combined treatment of rg3 and ‘fu significantly suppressed the invasion and migration ability of human colon cancer cell in vitro by altering emtrelated proteinfurthermore we also found that rg3 and 5fu combination inhibited the conduction of the pi3kakt signaling pathway in vivo and in vitro many studies have shown that the occurrence and development of tumors are the result of multifactor multigene and multipathway processes and the cell signal transduction pathway is crucial in the process of tumor development invasion and metastasis the phosphatidylinositol 3kinaseserinethreonine kinase b pi3kakt signaling pathway plays an important role in the regulation of solid tumors [eg liver cancer breast cancer colon cancer gastric cancer neuroblastoma ] and blood tumors [eg leukemia ] pi3k acts as a bridge molecule for the relationship between extracellular signals and cellular responses under the influence of a series of upstream or bypass signaling molecules it acts on the downstream of the effects of a variety of molecules thus promotes cell migration 0concology reports inhibits cell apoptosis accelerates the process of the cell cycle and promotes cell proliferation many previous studies have shown that traditional chinese medicine or traditional chinese medicine monomers can play an antitumor role by inhibiting the pi3kakt signaling pathway in conclusion rg3 enhances 5fu inhibiting proliferation invasion and migration of colorectal cancer cells and helps 5fu block g1 phase induced apoptosis in more colorectal cells all in all our study found that rg3 enhanced the anticancer effect of 5fu on colon cancer cell via pi3kakt pathwayacknowledgementsnot applicablefundingno funding was receivedavailability of data and materialsthe datasets used during the present study are available from the corresponding author upon reasonable requestauthors' contributionsxc made substantial contributions to the conception and design of the study and critically revised it for important intellectual content sh contributed to the acquisition of the data wc zh yw xm yh and zl analyzed and interpreted the data all authors read and approved the final manuscriptethics approval and consent to participateall animal and cell experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinapatient consent for publicationnot applicablecompeting intereststhe authors state that they have no competing interestsreferences siegel rl ward em and jemal a trends in colorectal cancer incidence rates in the united states by tumor location and stage ‘ cancer epidemiol biomarkers prev ‘ siegel rl miller kd and jemal a cancer statistics ca cancer j clin ‘ chen w zheng r baade pd zhang s zeng h bray f jemal a yu xq and he j cancer statistics in china ca cancer j clin chen w zheng r zhang s zeng h zuo t xia c yang z and he j cancer incidence and mortality in china in an analysis based on urbanization level chin j cancer res ‘ li j hou n faried a tsutsumi s and kuwano h inhibition of autophagy augments ‘fluorouracil chemotherapy in human colon cancer in vitro and in vivo model eur j cancer sanoff hk carpenter wr freburger j li l chen k zullig ll goldberg rm schymura mj and schrag d comparison of adverse events during ‘fluorouracil versus ‘fluorouraciloxaliplatin adjuvant chemotherapy for stage iii colon cancer a population‘based analysis cancer ‘ cheah ky howarth gs bindon ka kennedy ja and bastian sep low molecular weight procyanidins from grape seeds enhance the impact of ‘fluorouracil chemotherapy on caco‘ human colon cancer cells plos one e98921 gao y xiao x zhang c yu w guo w zhang z li z feng x hao j zhang k melatonin synergizes the chemotherapeutic effect of ‘fluorouracil in colon cancer by suppressing pi3kakt and nfbinos signaling pathways j pineal res doi 101111jpi12380 wang sf wu my cai cz li m and lu jh autophagy modulators from traditional chinese medicine mechanisms and therapeutic potentials for cancer and neurodegenerative diseases j ethnopharmacol ‘ ernst e traditional chinese medicine for cancer br j cancer sun hy lee jh han ys yoon ym yun cw kim jh song ys and lee sh pivotal roles of ginsenoside rg3 in tumor apoptosis through regulation of reactive oxygen species anticancer res ‘ tang yc zhang y zhou j zhi q wu my gong fr shen m liu l tao m shen b ginsenoside rg3 targets cancer stem cells and tumor angiogenesis to inhibit colorectal cancer progression in vivo int j oncol ‘ wang j tian l khan mn zhang l chen q zhao y yan q fu l and liu j ginsenoside rg3 sensitizes hypoxic lung cancer cells to cisplatin via blocking of nfκb mediated epithelialmesenchymal transition and sternness cancer lett ‘ joo e ha yw and kim ys abstract lb23 molecular mechanisms of ginsenoside rg3 related to apoptosis in human lung and pancreatic adenocarcinomas cancer res lb23 kim bj nah sy jeon jh so i and kim sj transient receptor potential melastatin channels are involved in ginsenoside rg3induced apoptosis in gastric cancer cells basic clin pharmacol kim sm lee sy cho js son sm choi ss yun yp yoo hs yoon dy oh kw han sb and hong jt combination of ginsenoside rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of nfkappa b eur j pharmacol yuan hd quan hy zhang y kim sh and chung sh 20sginsenoside rg3induced apoptosis in ht29 colon cancer cells is associated with ampk signaling pathway mol med rep ‘ liu tg huang y cui dd huang xb mao sh ji ll song hb and yi c inhibitory effect of ginsenoside rg3 combined with gemcitabine on angiogenesis and growth of lung cancer in mice bmc cancer sun my ye y xiao l duan xy zhang ym and zhang h anticancer effects of ginsenoside rg3 review int j mol med ‘ longley db harkin dp and johnston pg fluorouracil mechanisms of action and clinical strategies nat rev cancer ‘ hokmabady l raissi h and khanmohammadi a interactions of the ‘fluorouracil anticancer drug with dna pyrimidine bases a detailed computational approach struct chem ‘ rateesh s luis sa luis cr hughes b and nicolae m myocardial infarction secondary to ‘fluorouracil not an absolute contraindication to rechallenge int j cardiol e331‘e333 shan x aziz f tian ll wang xq yan q and liu jw ginsenoside rg3induced egfrmapk pathway deactivation inhibits melanoma cell proliferation by decreasing fut4ley expression int j oncol ‘ luo y zhang p zeng hq lou sf and wang dx ginsenoside rg3 induces apoptosis in human multiple myeloma cells via the activation of bcl2associated x protein mol med rep ‘ cardone mh roy n stennicke hr salvesen gs franke tf stanbridge e frisch s and reed jc regulation of cell death protease caspase‘ by phosphorylation science ‘ 0chong effects of ginsenoside rg3 on colon can Answer:

Colon_Cancer

" breast cancer bc is the most commonly diagnosed malignant cancer in women bc is the main cause of cancerrelated death in women and seriously threatens the life and health of women worldwide micrornas mirnasmirs have been reported to regulate the development and progression of different types of cancer however the regulatory functions of mir1885p in bc have not been thoroughly demonstrated in this present research we identified that mir‘‘5p was downregulated in bc tissues and several bc cell lines downregulation of mir‘‘5p was significantly associated with advanced tnm stage moreover we identified that mir‘‘5p mimics significantly inhibited proliferation using cck8 assay colony formation and xenograft animal model suppressed invasion and migration detected by transwell invasion assay and increased the cellular apoptosis of bc cells as determined by cell apoptosis assay moreover mir1885p mimics also reduced the expression of nfκb p65rel to further investigate its regulatory mechanism transcription factor zinc finger protein zfp91 was predicted as the targeted protein of mir1885p by bioinformatic method we confirmed their specific binding by dual luciferase dlr assay we demonstrated that the overexpression of mir1885p significantly inhibited the expression of zfp91 in bc cell lines and reduced the expression of nfκb p65rel an inverse correlation was found between the expression of mir1885p and zfp91 in bc tissues importantly we demonstrated that the restoration of zfp91 was able to block the effect of mir1885p on the progression of mdamb231 cells therefore our study showed that mir1885p may be one of the important indicators and could inhibit the progression of human bc via targeting the zfp91nf‘κb p65rel signaling pathway suggesting that mir1885p may be a promising future target for bc treatmentcorrespondence to dr zhaoyu liu department of radiology shengjing hospital of china medical university sanhao street shenyang liaoning pr chinaemail liuzy1226126comkey words breast cancer mir1885p zinc finger protein zfp91 proliferation apoptosisintroductionbreast cancer bc is one of the most commonly diagnosed malignancy in the world the mortality rate for bc ranks first among all female malignant tumors globally the number of newly diagnosed bc cases reached approximately million in accounting for almost of cancer cases among women bc exhibits a complex pathogenesis and is a clinically heterogeneous disease with a wide range of clinical behaviors and treatment responses although many dysregulated molecular pathways have been discovered in bc the development of effective therapeutic methods has been limited it is urgent to discover novel molecules to suppress bc proliferation induce apoptosis and inhibit invasion and provide potential therapeutic strategies to improve the survival and quality of life of bc patients micrornas mirnasmirs are a class of endogenous noncoding rnas of approximately nucleotides in length which are generally located in unstable regions of the human genome and are usually dysregulated in malignant tumors to regulate gene functions mirnas regulate target genes through binding to the 'untranslated regions 'utrs of the target mrnas subsequently inhibiting gene expression through regulation of the targeted proteins mirnas play an important role in many tumor cellular processes such as proliferation cell cycle apoptosis invasion and metastasis and participate in almost all signaling pathways mir1885p has been reported to be an inhibitor of tumor growth and metastasis in prostate cancer and hepatocellular carcinoma however to the best of our knowledge the functions of mir1885p in bc remain elusivein the present study we detected the expression of mir1885p in tumor tissues of bc patient tissues and several bc cell lines furthermore we investigated its regulatory role in bc proliferation apoptosis and invasion we also predicted and confirmed the targeted protein of mir‘‘5p transcription factor zinc finger protein zfp91 elucidating the regulatory mechanisms of mir1885p in bcmaterials and methodspatients and tissues one hundred paired bc tissue specimens including malignant and normal tissues used in this study were obtained from shengjing hospital of china medical university shenyang liaoning china during the period 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91from january to december with the informed consent of patients the age range of the patients was from to years with a mean age of years all experiments were approved by the ethics committee of shengjing hospital of china medical university no 2016ps18j the samples were snap‘frozen in liquid nitrogen and stored at ‘Ëšc tnm staging system was performed for tumor grading of bc and for evaluating and staging of patients respectively which was carried out according to the 7th edition of the american joint committee on cancer ajcc tnm classification system cell cultivation the bc cell lines mdamb231 atcc crmhtb26 bt549 atcc htb122 and mcf7 atcc crl3435 were cultured in rpmi1640 medium sigma‘aldrich merck kgaa containing heat‘inactivated fetal bovine serum fbs mp biomedicals penicillin‘streptomycin invitrogen thermo fisher scientific inc no the nonmalignant mammary epithelial cell line mcf 10a atcc crl10317 was cultivated in dmemf12 ham's mixture supplemented with equine serum hyclone ge healthcare egf ngml insulin µgml hydrocortisone mgml and cholera toxin ngml all from sigma‘aldrich merck kgaa all cells were incubated at ˚c in a humidified co2 atmosphererna isolation and quantitative qpcr total rnas from tissues or cells were isolated using rnx„¢plus reagent cinnagen and cdna was synthesized using the primescript„¢ rt reagent kit takara according to the manufacturer's instructions qpcr was performed using a sybr premix extaq„¢ kit takara with the following primer sets on the abi qpcr system applied biosystems actin was used to normalize the relative expression of the target genes mirnas were detected through a miscript ii rt kit qiagen in a fluorescence thermal cycler bio‘rad laboratories inc the primers for mir1885p and the reference gene u6 were purchased from novland biopharm the thermocycling condition were ˚c for min followed by cycles of ˚c for sec and ˚c for 1min followed by a hold at ˚c the relative expression ratio of mir‘‘5p was quantified using the 2δδcq method the relative expression of mir1885p was normalized to u6 the primer sequences are listed in table iplasmid preparation the coding region of human zfp91 was amplified from human breast cancer cell mda‘mb‘ cdna library by pcr then we cloned the prepared zfp91 fragment into pcmv‘tag2b stratagene to obtain pcmv‘tag2b‘zfp91 the primer sequences are listed in table icell transfection the mir1885p mimics and mirnc were purchased from thermo fisher scientific inc firstly lipofectamine transfection reagent thermo fisher scientific inc was used to transfect mir1885p mimics mirnc into mdamb231 cells in accordance with the manufacturer's instructionsafter the whole detection the pcmvtag2b vector pc was transfected into mdamb231 cells with mirnc ncpc or mir1885p mimics pcmir1885p pcmv‘tag2b‘zfp91 was transfected into mda‘mb‘ cells with mir‘‘5p mimics mir‘‘5pzfp91 as the cells were grown to mir mimics or vector was transfected into cells according to the manufacturer's instructions then cells were cultivated for up to h finally the total rna and protein were extracted and properly stored for further researchcell proliferation assay cell counting kit8 cck8 kit dojindo was performed to detect cell proliferation based on the reduction of wst‘ to wst‘ formazan briefly the mdamb231 cells were seeded in a 96well plate at a density of 5x103 cellswell on the day of the experiment the cells were transfected with empty vector and mir1885p cck8 reagent was added into the culture medium at the indicated time and incubated for min the absorbance at nm was measured by a microplate readercolony formation assay mdamb231 cells from the different treated groups were seeded in a 60mm dish containing cells followed by a 14day cultivation at ˚c with co2 the supernatant was discarded and cells were washed twice with pbs the colonies were fixed in paraformaldehyde for min and then stained with giemsa staining solution solarbio science technology co ltd beijing china for min colonies were counted and images were captured under an inverted microscope nikon tokyo japan this assay was repeated timescell apoptosis assay mdamb231 cells were stained by annexin valexa fluor488propidium iodide pi staining to identify the apoptotic mdamb231 cells after transfection with mir1885p for h mdamb231 cells were stained with annexin valexa fluor488 for min on ice followed by the addition of pi solution for the secondary staining process all experimental procedures were strictly protected from lights the data were calculated by flowjo software v87 tree star after facs calibur bd biosciences analysiscell migration and invasion assays after the counting mdamb231 cells in the different groups were inoculated equally at a density of 5x105 cellsml in the upper compartments of polycarbonate membrane filters cell migration and invasion assays were performed uncoated for the migration assay and coated with matrigel bd biosciences for the invasion assay after h the migrated and invaded cells in the membrane were fixed with methanol and then stained with crystal violet for min at room temperature cells were observed under a light microscope with magnification x100western blotting protein samples extracted from tissues or cultivated cells were lysed in ripa buffer containing protease and phosphatase inhibitor cocktail and incubated at ˚c followed by the quantified measurement of protein using bca kit fujifilm wako pure chemical corp after protein samples µgeach sample were loaded and separated on sdspage gels for electrophoresis the proteins were then transferred onto a polyvinylidene difluoride pvdf membranes millipore usa the membranes were blocked in wv skim milk for h at room temperature and incubated at ˚c overnight with primary antibodies anti‘zfp91 dilution 0concology reports bidirectional primer sequencetable i primer informationgene name f 'ccctctctcacatcccttgcat3'mir1885p r 'atcctgcaaaccctgcatgtg3' f '‘tgagacctacaaaccccactt‘'zfp91 r 'ccttttgggtaaacgtggacttt3'homoactin f 'ttcctccgcaaggatgacacgc3'r 'ccttttgggtaaacgtggacttt3' f 'cgggtttgttttgcatttct3'u6 snrna f 'agtcccag catgaacagctt3'zfp91 zinc finger protein homolog f forward r reverse abcam ab30970 and antivimentin dilution cell signaling technology inc antiecadherin dilution cell signaling technology inc ncadherin dilution cell signaling technology inc matrix metalloproteinase mmp2 dilution cell signaling technology inc mmp9 dilution cell signaling technology inc nfκb p65 dilution cell signaling technology inc relb dilution cell signaling technology inc and gapdh dilution cell signaling technology inc as internal control on the following day all membranes were incubated with antirabbit igg hrplabeled secondary antibodies dilution cell signaling technology inc finally the signals were detected and analyzed with the application of luminata forte western hrp substrate millipore in the biorad chemidox xrs imaging system biorad laboratoriesluciferase reporter assay to further investigate the specific correlation between mir‘‘5p and zfp91 targetscan wwwtargetscanorgmamm_31 and miranda wwwmicrornaorgmicrornahomedo were performed the zfp91 was selected to be the predicted targeting of mir1885p the fragments of the 'utr of zfp91 containing mir‘‘5p binding sites and its mutants were amplified by pcr and then the pcr products were inserted into pmirglo dualluciferase mirna target expression vector promega corp the reporter and control vector were transfected into 293t cells using lipofectamine thermo fisher scientific inc after cultivation for h the relative luciferase activity was examined by the dualluciferase reporter assay kit thermo fisher scientific inc according to the manufacturer's instructionspreparation of tumor xenograft animal model and treat‘ment with mir‘‘5p mimics thirtysix nude mice female weighing ± g were purchased from huafukang biotech beijing china the experiments were performed in the animal facility at the department of laboratory animal science of china medical university and approved by the animal ethics committee of shengjing hospital approval no nude mice were randomly divided into a control group n12 mir1885p group n12 and nc group n12 a density of 5x106 cells in logarithmic phase were transfected with 1x pbs control group nc or mir1885p then the different groups of cells were resuspended in 1x pbs and injected into the nude mice respectively then tumor size was measured every days using a slide caliper and the tumor volume v was calculated using the formula vlength x width22 after days the mice were euthanasia by cervical dislocation and the tumors were excised imaged weighed and stored properly for further investigationsstatistical analysis graphpad prism graphpad software inc was used to perform statistical analysis the results are represented as mean ± sd of at least independent experiments the comparisons between groups were evaluated by student' t‘test one‘way anova followed by tukey test was used to evaluate the differences for multiple comparisons the statistical significance of correlations between mir‘‘5p and zfp91 expression in bc tissue were analyzed by pearson's correlation coefficient p005 was considered to indicate a statistically significant differenceresultsmir‘‘5p is signif\ufeff\ufefficantly decreased in bc tissue and cell lines firstly we analyzed the expression level of mir1885p in cases of bc tissues and adjacent counterparts by rtqpcr the results showed that the level of mir1885p in bc tissues was significantly lower than that in the normal adjacent counterparts fig 1a p005 we also found that mir1885p was correlated with bc tnm stage fig 1b p005 the expression level of mir‘‘5p in advanced bc tumors was lower than that in early stage tumors suggesting that mir1885p is inversely correlated with the malignancy of bcwe also compared the expression level of mir1885p in the nonmalignant mammary epithelial cell line mcf10a and bc cell lines mdamb231 bt549 and mcf7 our data showed that the levels of mir1885p in the mdamb231 bt549 and mcf7 cells were lower than that in the mcf‘10a cells fig 1c p005 meanwhile the lowest mir1885p expression was detected in mdamb231 therefore mdamb231 cells were selected for further experimentsmir‘‘5p inhibits proliferation induces cell apoptosis and suppresses migration and invasion of bc cells as the expression of mir1885p in both bc cell lines and tumor tissues of bc patients were clearly downregulated we sought to investigate the effects of mir1885p on bc development by using both in vitro bc cell line cultivation and in vivo mouse tumor xenografts as shown in fig 2a transfection of mda‘mb‘ cells with mir‘‘5p mimics significantly elevated the expression level of mir1885p when compared to the control and mir‘nc groups p005 importantly the increased level of mir‘‘5p in mda‘mb‘ cells significantly inhibited the cell proliferation when compared to the control and mir‘nc groups fig 2b and c p005 it was also observed that the apoptotic mdamb231 cell numbers were significantly increased by the upregulation of mir‘‘5p when compared to the control and mirnc groups fig 2d 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure mir1885p is downregulated in bc tissue and bc cell lines a rtqpcr results showed that the level of mir1885p in bc tissues was downregulated compared with counterpart bc tissues the comparisons between groups were evaluated by student's ttest b relative expression level of mir1885p in patients at different clinical stages c relative expression level of mir1885p in bc cell lines mdamb231 bt549 and mcf7 relative to the normal human breast epithelial cell line mcf‘10a one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons in b and c all data are presented as means ± sd n3 bc breast cancerp005 importantly mir‘‘5p mimics significantly inhibited the invasion and migration abilities of the mdamb23 cells under transwell assay detection when compared to the control and mir‘nc groups fig 2e p005 moreover mir‘‘5p mimics significantly enhanced the expression of vimentin and ncadherin and reduced the level of ecadherin when compared to the control and mirnc groups fig 2f p005 the matrix metalloproteinases mmp2 and mmp9 mmp29 possess the ability to hydrolyze components of the basement membrane and stimulate tumor growth metastasis and epithelialmesenchymal transition emt mir‘‘5p mimics were demonstrated to significantly inhibit the expression of mmp2 and mmp9 fig 2f p005 these data provide robust evidence that mir1885p inhibits the tumor proliferation induces apoptosis reduces tumor invasion and migration and inhibits emt of bc which may be through the regulation of mmp29 expressionzfp91 is the downstream target of mir‘‘5p to further investigate the specific correlation between mir1885p and zfp91 targetscan wwwtargetscanorgmamm_31 and miranda wwwmicrornaorgmicrornahomedo were performed the results predicted that mir1885p possesses the binding sites of zfp91 fig 3a hence we sought to discover the regulatory mechanisms of mir1885p on bc development through targeting on zfp91 as hypothesized the upregulation of mir‘‘5p in mda‘mb‘ cells decreased zfp91 mrna and protein levels when compared to the mirnc and control groups fig 3b p005 moreover the luciferase assay confirmed that mir1885p specifically binds to the 'utr of zfp91 fig 3c p005 it was also discovered that the injection of mdamb231 transfected with mir1885p mimics in tumor xenograft mice inhibited zfp91 expression fig 6b p005 these results suggested that zfp91 is the downstream target gene of mir1885p in bcmir‘‘5p regulates bc cell progression through targeting zfp91 to further investigate the biological functions of mir1885p in bc development we established a zfp91‘overexpressing mda‘mb‘ cell line the expression of zfp91 was confirmed by rt‘qpcr fig s1 p005 with this system inhibition of zfp91 by mir‘‘5p mimics was reversed fig 4a p005 then it was found that the co‘transfection of mda‘mb‘ cells mir‘‘5pzfp91 group significantly enhanced the cell proliferation compared to that in pcmir‘‘5p group fig 4b and c p005 significantly suppressed cell apoptosis fig 4d p005 and significantly promoted invasion migration fig 4e p005 and emt fig 4f p005 in contrast with the mono‘transfection of mir1885p mimics in mdamb231 cells moreover the regulatory role of mir1885p on mmp2 and mmp9 was also reversed by overexpression of zfp91mir‘‘5p and zfp91 are correlated in tumor tissues of bc patients furthermore we examined the levels of zfp91 in the tumor tissues and adjacent normal tissues of bc patients the aberrantly high level of zfp91 was observed in the tumor tissues of the bc patients fig 5a p005 spearman's correlation analysis showed a significantly inverse correlation between mir‘‘5p and zfp91 in the bc patient tissues fig 5b p005 taken together these results further confirmed that the proliferation and apoptosis of bc is regulated by mir‘‘5pzfp91mir‘‘5p inhibits the proliferation of mda‘mb‘ cells and reduces the expression of zpf91 in a bc xenograft mouse model moreover to evaluate the regulatory role of mir1885p in a bc xenograft mouse model we injected the mdamb231 cells transfected with mir1885p mimics or mirnc into nude mice the results showed that mir1885p mimics inhibited the tumor volume and weight compared to the mir‘nc group fig 6a p005 protein expression and the mrna level of zpf91 were also suppressed by mir1885p mimics in the tumor tissues of the xenograft mouse model when compared with the mirnc and control groups fig 6b p005mir‘‘5pzfp91 axis regulates nf‘kbp65 and relb expression numerous studies have reported that zinc finger protein zfp91 promotes proliferation and tumorigenesis 0concology reports figure effect of mir1885p on bc cell line mdamb231 mdamb231 cells were transfected with mir1885p mimics and mirnc a the mrna levels of mir1885p in mdamb231 cells were determined by rtqpcr p005 the proliferation of mda‘mb‘ cells was examined by b cck‘ kit post transfection and c colony formation p005 compared to control group p005 compared to mir‘nc group d the apoptotic mda‘mb‘ cells were analyzed using annexin vpi staining and facs p005 e the invasion and migration capability of mda‘mb‘ cells were detected by transwell assay p005 f expression of e‘cadherin n‘cadherin vimentin mmp2 mmp9 and nf‘κb p65rel were detected by western blotting the data are represented as the mean ± sd n3 p005 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer mmp matrix metalloproteinase nc negative controlof different cancer types via regulation of the nfκb p65 pathway therefore to further investigate the regulatory mechanism of the mir‘‘5pzfp91 axis we detected the expression of nfκbp65 and relb in bc cells the results showed that mir‘‘5p mimics significantly reduced the expression of nfκbp65 and relb together fig s2a p005 moreover the co‘transfection of mir‘‘5p mimics and zfp91 also upregulated the expression levels of 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure zfp91 is a target of mir‘‘5p a zfp91 provides binding sites with mir‘‘5p b rt‘qpcr and western blotting were used for analysis of transcription and translation of zfp91 in vitro and in vivo p005 c the binding between mir‘‘5p and zfp91 was confirmed by luciferase assay p005 relative gene expression was normalized by gapdh expression the data are represented as the mean ± sd n3 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons in b the comparisons between two groups were evaluated by t‘test in c zfp91 zinc finger protein nfκbp65 and relb compared to the monotransfection of mir1885p mimics fig s2b p005 in summary these results illustrated that the mir‘‘5pzfp91 axis regulates the progression of bc via the noncanonical nfκb signaling pathwaydiscussionbreast cancer bc is one of the most common types of tumors diagnosed in women worldwide bc is the second leading cause of cancerrelated mortality worldwide in more than women were diagnosed with bc in china and almost percent of all newly diagnosed cancer cases were in women however the molecular mechanisms of bc still await elucidation and effective molecular targets for the diagnosis and treatment of bc are urgently required recently research has reported that mirnas are small noncoding rna molecules which regulate target protein expression to play critical roles as tumorpromotors or suppressors several studies have demonstrated that mir1885p promotes cell proliferation migration and metastasis in gastric cancer and hepatocellular carcinoma moreover iwakawa detected higher expression of mir1885p in stage iii breast cancer and tnbc wang reported that circulating mir1885p was upregulated in bc patients and associated with tnm of bc interestingly mir1885p was downregulated in bc mdamb231 and mcf7 cells moreover using gainof and lossoffunction analyses of mir1885p in breast cancer cells the authors demonstrated that mir1885p inhibited the proliferation and invasion of bc mdamb231 cells via targeting il6st however we demonstrated that the expression of mir1885p was drastically downregulated in bc tissue specimens which was also decreased in bc cell lines mdamb231 bt549 and mcf7 compared to normal breast epithelial cell line mcf10a moreover the downregulation of mir‘‘5p was significantly 0concology reports figure mda‘mb‘ cell proliferation and apoptosis are regulated by mir‘‘5pzfp91 pcmv‘tag2b vector pc was transfected into bc mda‘mb‘ cells with mir‘nc ncpc or mir‘‘5p mimics pcmir‘‘5p pcmv‘tag2b‘zfp91 was transfected into mda‘mb‘ cells with mir‘‘5p mimics mir‘‘5pzfp91 a the mrna levels of mir‘‘5p in the co‘transfected overexpressing zfp91mda‘mb‘ cells were quantified by rtqpcr p005 cell proliferation was measured by b the cck‘ kit and c colony formation assay p005 compared to control group p005 compared to mirnc group d cell apoptosis was detected by annexin vpi staining and facs p005 e the invasion and migration capability of mdamb231 cells were detected by transwell assay p005 f expression of e‘cadherin n‘cadherin vimentin mmp2 mmp9 and nf‘κb p65rel were detected by western blotting relative genes expression was normalized by gapdh expression the data are represented as the mean ± sd n3 p005 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer zfp91 zinc finger protein mmp matrix metalloproteinase nc negative control 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure mrna and protein levels of zpf91 in bc tissues a the mrna and protein levels of zpf91 in bc tissue and corresponding non‘tumor normal tissue were quantified using rt‘qpcr and western blot analysis the comparisons between two groups were evaluated by t‘test n non‘cancer tissue c cancer tissue p005 b correlation between zpf91 mrna and mir‘‘5p was analyzed using pearson's correlation coefficient the data are represented as the mean ± sd n100 bc breast cancer zfp91 zinc finger protein figure the regulatory role of mir1885p in a bc xenograft mouse model bc mdamb231 cells were transfected with mir1885p mimics and mirnc and then injected into nude mice a the tumor volume and weight in the tumor xenograft mouse were compared among the mir1885p mimics group mirnc group and control group p005 compared to control group p005 compared to mir‘nc group b the protein expression and mrna level of zpf91 were detected by c western blotting and rt‘qpcr the data are represented as the mean ± sd n3 p005 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer zfp91 zinc finger protein nc negative controlassociated with advanced tnm stage however to investigate the relationship of mir1885p and bc patient prognosis we found that kaplanmeier analysis of mir1885p was limited due to the small sample size in tcga these results illustrated that the downregulation of mir1885p may be related with bc progression in the clinic suggesting that mir1885p may be a valuable bc diagnostic indicator moreover we confirmed that mir1885p mimics considerably inhibited the proliferation induced the apoptosis and inhibited the invasion of bc cells suggesting that mir1885p plays an inhibitory role in bc cellstranscription factor zinc finger protein zfp91 was firstly identified in the mouse in which was found to be overexpressed in colon liver prostate stomach and breast cancer zfp91 has a molecular mass of kda with amino acids containing five zinc‘finger motives a leucine zipper a coiledcoil structure and nuclear localization sequences zfp91 was confirmed to be a transcription factor located in the cellular nucleus ma reported that zfp91 functions as an oncogene in cancer development by activating hif1α transcription the overexpression of zfp91 0concology reports was also found to result in the promotion of nkκb signaling pathway activation through increasing nkκb inducing kinase nik whose activity and overexpression are related to cancer progression in melanoma pancreatic breast and lung cancer the inhibition of zfp91 was demonstrated to promote apoptosis in bc stomach cancer cells colon cancer and endometrial cancer in addition the overexpression of zfp91 was found to increase the cancer cell growth rate and metastatic capability zfp91 was also reported to interact with cyclin‘dependent kinase inhibitor 2a cdkn2a which is an alternative reading frame arf tumor suppressor inhibiting the induction of p53dependent cell death to illuminate the molecular mechanisms of mir188 we predicted that il6st foxn2 zfp91 may be the targets of mir‘‘5p using targetscan and miranda furthermore peng reported that zfp91 is the target protein of mir‘‘5p in gastric cancer in addition overexpression of mir1885p was confirmed to inhibit the progression of breast cancer thus we chose the reported oncogene zfp91 for further investigation in the present study we confirmed that the 'untranslated region 'utr of zfp91 was bound by mir‘‘5p through dual luciferase assay moreover transfection of mir1885p mimics in mda‘mb‘ cells reduced the zfp91 mrna and protein levels together mirnas usually bind to the 'utrs of target mrnas and do not reduce the level of mrnas however mirnas also were reported to decay the target mrnas and decease mrna level restoration of zfp91 largely reversed the decreased proliferation and induced apoptosis which were both regulated by mir1885p overexpression moreover in the tumor xenograft mouse model we observed that the expression of zfp91 was downregulated by an increased level of mir1885p furthermore the expression of mir‘‘5p and zfp91 were negatively correlated in bc patient tissues therefore our studies confirmed that mir‘‘5p can inhibit the progression of human bc via targeting zfp91zfp91 has been reported to promote proliferation in colon cancer prostate cancer and gastric cancer ma reported that zfp91 activates nf‘kappabp65 to promote proliferation and tumorigenesis of colon cancer paschke identified that zfp91 is a noncanonical nf‘κb signaling pathway regulator with oncogenic properties in prostate cancer in the present study we also confirmed that a decrease in zfp91 could significantly inhibit nf‘κbp65 and relb expression in bc cells therefore mir1885p overexpression reduced zfp91 via the noncanonical nf‘κb signaling pathway to inhibit the progression of bcin conclusion our data showed that mir1885p is downregulated in bc cell lines and tissues and the downregulated expression of mir1885p is associated with the poor prognosis of patients with bc we further investigated that overexpression of mir1885p could inhibit proliferation and induce the apoptosis of mda‘mb‘ cells furthermore zfp91 was predicted and confirmed as a target gene of mirna‘‘5p and the effects of mir1885p on bc cells were dependent on the inhibition of zfp91 additionally a decrease in zfp91 significantly inhibited the nfκbp65 and relb expression in bc cells moreover the expression levels of mir‘‘5p and zfp91 were highly correlated with bc progression therefore we suggest that mir1885p can inhibit breast cancer progression via the zfp91nf‘κbp65 axis and may be a potential diagnostic indicator for bcacknowledgementsnot applicablefundingthis study was supported by the national natural science foundation of china grant nos and and talent projectavailability of data and materialsthe datasets used and analyzed during the current study are available from the corresponding author upon reasonable requestauthors' contributionszy and zl conceived and designed the study zy zc gy performed the experiments zy wrote the paper zy zl zc and gy reviewed the results and data and edited the manuscript all authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolvedethics approval and consent to participatetissues used in this study were obtained from shengjing hospital of china medical university shenyang liaoning china with the informed consent of patients and all experiments were approved by the ethics committee of shengjing hospital of china medical university no 2016ps18jpatient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences davidson ne armstrong sa coussens lm cruzcorrea mr deberardinis rj doroshow jh foti m hwu p k

cancer7547

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " breast cancer bc is the most commonly diagnosed malignant cancer in women bc is the main cause of cancerrelated death in women and seriously threatens the life and health of women worldwide micrornas mirnasmirs have been reported to regulate the development and progression of different types of cancer however the regulatory functions of mir1885p in bc have not been thoroughly demonstrated in this present research we identified that mir‘‘5p was downregulated in bc tissues and several bc cell lines downregulation of mir‘‘5p was significantly associated with advanced tnm stage moreover we identified that mir‘‘5p mimics significantly inhibited proliferation using cck8 assay colony formation and xenograft animal model suppressed invasion and migration detected by transwell invasion assay and increased the cellular apoptosis of bc cells as determined by cell apoptosis assay moreover mir1885p mimics also reduced the expression of nfκb p65rel to further investigate its regulatory mechanism transcription factor zinc finger protein zfp91 was predicted as the targeted protein of mir1885p by bioinformatic method we confirmed their specific binding by dual luciferase dlr assay we demonstrated that the overexpression of mir1885p significantly inhibited the expression of zfp91 in bc cell lines and reduced the expression of nfκb p65rel an inverse correlation was found between the expression of mir1885p and zfp91 in bc tissues importantly we demonstrated that the restoration of zfp91 was able to block the effect of mir1885p on the progression of mdamb231 cells therefore our study showed that mir1885p may be one of the important indicators and could inhibit the progression of human bc via targeting the zfp91nf‘κb p65rel signaling pathway suggesting that mir1885p may be a promising future target for bc treatmentcorrespondence to dr zhaoyu liu department of radiology shengjing hospital of china medical university sanhao street shenyang liaoning pr chinaemail liuzy1226126comkey words breast cancer mir1885p zinc finger protein zfp91 proliferation apoptosisintroductionbreast cancer bc is one of the most commonly diagnosed malignancy in the world the mortality rate for bc ranks first among all female malignant tumors globally the number of newly diagnosed bc cases reached approximately million in accounting for almost of cancer cases among women bc exhibits a complex pathogenesis and is a clinically heterogeneous disease with a wide range of clinical behaviors and treatment responses although many dysregulated molecular pathways have been discovered in bc the development of effective therapeutic methods has been limited it is urgent to discover novel molecules to suppress bc proliferation induce apoptosis and inhibit invasion and provide potential therapeutic strategies to improve the survival and quality of life of bc patients micrornas mirnasmirs are a class of endogenous noncoding rnas of approximately nucleotides in length which are generally located in unstable regions of the human genome and are usually dysregulated in malignant tumors to regulate gene functions mirnas regulate target genes through binding to the 'untranslated regions 'utrs of the target mrnas subsequently inhibiting gene expression through regulation of the targeted proteins mirnas play an important role in many tumor cellular processes such as proliferation cell cycle apoptosis invasion and metastasis and participate in almost all signaling pathways mir1885p has been reported to be an inhibitor of tumor growth and metastasis in prostate cancer and hepatocellular carcinoma however to the best of our knowledge the functions of mir1885p in bc remain elusivein the present study we detected the expression of mir1885p in tumor tissues of bc patient tissues and several bc cell lines furthermore we investigated its regulatory role in bc proliferation apoptosis and invasion we also predicted and confirmed the targeted protein of mir‘‘5p transcription factor zinc finger protein zfp91 elucidating the regulatory mechanisms of mir1885p in bcmaterials and methodspatients and tissues one hundred paired bc tissue specimens including malignant and normal tissues used in this study were obtained from shengjing hospital of china medical university shenyang liaoning china during the period 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91from january to december with the informed consent of patients the age range of the patients was from to years with a mean age of years all experiments were approved by the ethics committee of shengjing hospital of china medical university no 2016ps18j the samples were snap‘frozen in liquid nitrogen and stored at ‘Ëšc tnm staging system was performed for tumor grading of bc and for evaluating and staging of patients respectively which was carried out according to the 7th edition of the american joint committee on cancer ajcc tnm classification system cell cultivation the bc cell lines mdamb231 atcc crmhtb26 bt549 atcc htb122 and mcf7 atcc crl3435 were cultured in rpmi1640 medium sigma‘aldrich merck kgaa containing heat‘inactivated fetal bovine serum fbs mp biomedicals penicillin‘streptomycin invitrogen thermo fisher scientific inc no the nonmalignant mammary epithelial cell line mcf 10a atcc crl10317 was cultivated in dmemf12 ham's mixture supplemented with equine serum hyclone ge healthcare egf ngml insulin µgml hydrocortisone mgml and cholera toxin ngml all from sigma‘aldrich merck kgaa all cells were incubated at ˚c in a humidified co2 atmosphererna isolation and quantitative qpcr total rnas from tissues or cells were isolated using rnx„¢plus reagent cinnagen and cdna was synthesized using the primescript„¢ rt reagent kit takara according to the manufacturer's instructions qpcr was performed using a sybr premix extaq„¢ kit takara with the following primer sets on the abi qpcr system applied biosystems actin was used to normalize the relative expression of the target genes mirnas were detected through a miscript ii rt kit qiagen in a fluorescence thermal cycler bio‘rad laboratories inc the primers for mir1885p and the reference gene u6 were purchased from novland biopharm the thermocycling condition were ˚c for min followed by cycles of ˚c for sec and ˚c for 1min followed by a hold at ˚c the relative expression ratio of mir‘‘5p was quantified using the 2δδcq method the relative expression of mir1885p was normalized to u6 the primer sequences are listed in table iplasmid preparation the coding region of human zfp91 was amplified from human breast cancer cell mda‘mb‘ cdna library by pcr then we cloned the prepared zfp91 fragment into pcmv‘tag2b stratagene to obtain pcmv‘tag2b‘zfp91 the primer sequences are listed in table icell transfection the mir1885p mimics and mirnc were purchased from thermo fisher scientific inc firstly lipofectamine transfection reagent thermo fisher scientific inc was used to transfect mir1885p mimics mirnc into mdamb231 cells in accordance with the manufacturer's instructionsafter the whole detection the pcmvtag2b vector pc was transfected into mdamb231 cells with mirnc ncpc or mir1885p mimics pcmir1885p pcmv‘tag2b‘zfp91 was transfected into mda‘mb‘ cells with mir‘‘5p mimics mir‘‘5pzfp91 as the cells were grown to mir mimics or vector was transfected into cells according to the manufacturer's instructions then cells were cultivated for up to h finally the total rna and protein were extracted and properly stored for further researchcell proliferation assay cell counting kit8 cck8 kit dojindo was performed to detect cell proliferation based on the reduction of wst‘ to wst‘ formazan briefly the mdamb231 cells were seeded in a 96well plate at a density of 5x103 cellswell on the day of the experiment the cells were transfected with empty vector and mir1885p cck8 reagent was added into the culture medium at the indicated time and incubated for min the absorbance at nm was measured by a microplate readercolony formation assay mdamb231 cells from the different treated groups were seeded in a 60mm dish containing cells followed by a 14day cultivation at ˚c with co2 the supernatant was discarded and cells were washed twice with pbs the colonies were fixed in paraformaldehyde for min and then stained with giemsa staining solution solarbio science technology co ltd beijing china for min colonies were counted and images were captured under an inverted microscope nikon tokyo japan this assay was repeated timescell apoptosis assay mdamb231 cells were stained by annexin valexa fluor488propidium iodide pi staining to identify the apoptotic mdamb231 cells after transfection with mir1885p for h mdamb231 cells were stained with annexin valexa fluor488 for min on ice followed by the addition of pi solution for the secondary staining process all experimental procedures were strictly protected from lights the data were calculated by flowjo software v87 tree star after facs calibur bd biosciences analysiscell migration and invasion assays after the counting mdamb231 cells in the different groups were inoculated equally at a density of 5x105 cellsml in the upper compartments of polycarbonate membrane filters cell migration and invasion assays were performed uncoated for the migration assay and coated with matrigel bd biosciences for the invasion assay after h the migrated and invaded cells in the membrane were fixed with methanol and then stained with crystal violet for min at room temperature cells were observed under a light microscope with magnification x100western blotting protein samples extracted from tissues or cultivated cells were lysed in ripa buffer containing protease and phosphatase inhibitor cocktail and incubated at ˚c followed by the quantified measurement of protein using bca kit fujifilm wako pure chemical corp after protein samples µgeach sample were loaded and separated on sdspage gels for electrophoresis the proteins were then transferred onto a polyvinylidene difluoride pvdf membranes millipore usa the membranes were blocked in wv skim milk for h at room temperature and incubated at ˚c overnight with primary antibodies anti‘zfp91 dilution 0concology reports bidirectional primer sequencetable i primer informationgene name f 'ccctctctcacatcccttgcat3'mir1885p r 'atcctgcaaaccctgcatgtg3' f '‘tgagacctacaaaccccactt‘'zfp91 r 'ccttttgggtaaacgtggacttt3'homoactin f 'ttcctccgcaaggatgacacgc3'r 'ccttttgggtaaacgtggacttt3' f 'cgggtttgttttgcatttct3'u6 snrna f 'agtcccag catgaacagctt3'zfp91 zinc finger protein homolog f forward r reverse abcam ab30970 and antivimentin dilution cell signaling technology inc antiecadherin dilution cell signaling technology inc ncadherin dilution cell signaling technology inc matrix metalloproteinase mmp2 dilution cell signaling technology inc mmp9 dilution cell signaling technology inc nfκb p65 dilution cell signaling technology inc relb dilution cell signaling technology inc and gapdh dilution cell signaling technology inc as internal control on the following day all membranes were incubated with antirabbit igg hrplabeled secondary antibodies dilution cell signaling technology inc finally the signals were detected and analyzed with the application of luminata forte western hrp substrate millipore in the biorad chemidox xrs imaging system biorad laboratoriesluciferase reporter assay to further investigate the specific correlation between mir‘‘5p and zfp91 targetscan wwwtargetscanorgmamm_31 and miranda wwwmicrornaorgmicrornahomedo were performed the zfp91 was selected to be the predicted targeting of mir1885p the fragments of the 'utr of zfp91 containing mir‘‘5p binding sites and its mutants were amplified by pcr and then the pcr products were inserted into pmirglo dualluciferase mirna target expression vector promega corp the reporter and control vector were transfected into 293t cells using lipofectamine thermo fisher scientific inc after cultivation for h the relative luciferase activity was examined by the dualluciferase reporter assay kit thermo fisher scientific inc according to the manufacturer's instructionspreparation of tumor xenograft animal model and treat‘ment with mir‘‘5p mimics thirtysix nude mice female weighing ± g were purchased from huafukang biotech beijing china the experiments were performed in the animal facility at the department of laboratory animal science of china medical university and approved by the animal ethics committee of shengjing hospital approval no nude mice were randomly divided into a control group n12 mir1885p group n12 and nc group n12 a density of 5x106 cells in logarithmic phase were transfected with 1x pbs control group nc or mir1885p then the different groups of cells were resuspended in 1x pbs and injected into the nude mice respectively then tumor size was measured every days using a slide caliper and the tumor volume v was calculated using the formula vlength x width22 after days the mice were euthanasia by cervical dislocation and the tumors were excised imaged weighed and stored properly for further investigationsstatistical analysis graphpad prism graphpad software inc was used to perform statistical analysis the results are represented as mean ± sd of at least independent experiments the comparisons between groups were evaluated by student' t‘test one‘way anova followed by tukey test was used to evaluate the differences for multiple comparisons the statistical significance of correlations between mir‘‘5p and zfp91 expression in bc tissue were analyzed by pearson's correlation coefficient p005 was considered to indicate a statistically significant differenceresultsmir‘‘5p is signif\ufeff\ufefficantly decreased in bc tissue and cell lines firstly we analyzed the expression level of mir1885p in cases of bc tissues and adjacent counterparts by rtqpcr the results showed that the level of mir1885p in bc tissues was significantly lower than that in the normal adjacent counterparts fig 1a p005 we also found that mir1885p was correlated with bc tnm stage fig 1b p005 the expression level of mir‘‘5p in advanced bc tumors was lower than that in early stage tumors suggesting that mir1885p is inversely correlated with the malignancy of bcwe also compared the expression level of mir1885p in the nonmalignant mammary epithelial cell line mcf10a and bc cell lines mdamb231 bt549 and mcf7 our data showed that the levels of mir1885p in the mdamb231 bt549 and mcf7 cells were lower than that in the mcf‘10a cells fig 1c p005 meanwhile the lowest mir1885p expression was detected in mdamb231 therefore mdamb231 cells were selected for further experimentsmir‘‘5p inhibits proliferation induces cell apoptosis and suppresses migration and invasion of bc cells as the expression of mir1885p in both bc cell lines and tumor tissues of bc patients were clearly downregulated we sought to investigate the effects of mir1885p on bc development by using both in vitro bc cell line cultivation and in vivo mouse tumor xenografts as shown in fig 2a transfection of mda‘mb‘ cells with mir‘‘5p mimics significantly elevated the expression level of mir1885p when compared to the control and mir‘nc groups p005 importantly the increased level of mir‘‘5p in mda‘mb‘ cells significantly inhibited the cell proliferation when compared to the control and mir‘nc groups fig 2b and c p005 it was also observed that the apoptotic mdamb231 cell numbers were significantly increased by the upregulation of mir‘‘5p when compared to the control and mirnc groups fig 2d 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure mir1885p is downregulated in bc tissue and bc cell lines a rtqpcr results showed that the level of mir1885p in bc tissues was downregulated compared with counterpart bc tissues the comparisons between groups were evaluated by student's ttest b relative expression level of mir1885p in patients at different clinical stages c relative expression level of mir1885p in bc cell lines mdamb231 bt549 and mcf7 relative to the normal human breast epithelial cell line mcf‘10a one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons in b and c all data are presented as means ± sd n3 bc breast cancerp005 importantly mir‘‘5p mimics significantly inhibited the invasion and migration abilities of the mdamb23 cells under transwell assay detection when compared to the control and mir‘nc groups fig 2e p005 moreover mir‘‘5p mimics significantly enhanced the expression of vimentin and ncadherin and reduced the level of ecadherin when compared to the control and mirnc groups fig 2f p005 the matrix metalloproteinases mmp2 and mmp9 mmp29 possess the ability to hydrolyze components of the basement membrane and stimulate tumor growth metastasis and epithelialmesenchymal transition emt mir‘‘5p mimics were demonstrated to significantly inhibit the expression of mmp2 and mmp9 fig 2f p005 these data provide robust evidence that mir1885p inhibits the tumor proliferation induces apoptosis reduces tumor invasion and migration and inhibits emt of bc which may be through the regulation of mmp29 expressionzfp91 is the downstream target of mir‘‘5p to further investigate the specific correlation between mir1885p and zfp91 targetscan wwwtargetscanorgmamm_31 and miranda wwwmicrornaorgmicrornahomedo were performed the results predicted that mir1885p possesses the binding sites of zfp91 fig 3a hence we sought to discover the regulatory mechanisms of mir1885p on bc development through targeting on zfp91 as hypothesized the upregulation of mir‘‘5p in mda‘mb‘ cells decreased zfp91 mrna and protein levels when compared to the mirnc and control groups fig 3b p005 moreover the luciferase assay confirmed that mir1885p specifically binds to the 'utr of zfp91 fig 3c p005 it was also discovered that the injection of mdamb231 transfected with mir1885p mimics in tumor xenograft mice inhibited zfp91 expression fig 6b p005 these results suggested that zfp91 is the downstream target gene of mir1885p in bcmir‘‘5p regulates bc cell progression through targeting zfp91 to further investigate the biological functions of mir1885p in bc development we established a zfp91‘overexpressing mda‘mb‘ cell line the expression of zfp91 was confirmed by rt‘qpcr fig s1 p005 with this system inhibition of zfp91 by mir‘‘5p mimics was reversed fig 4a p005 then it was found that the co‘transfection of mda‘mb‘ cells mir‘‘5pzfp91 group significantly enhanced the cell proliferation compared to that in pcmir‘‘5p group fig 4b and c p005 significantly suppressed cell apoptosis fig 4d p005 and significantly promoted invasion migration fig 4e p005 and emt fig 4f p005 in contrast with the mono‘transfection of mir1885p mimics in mdamb231 cells moreover the regulatory role of mir1885p on mmp2 and mmp9 was also reversed by overexpression of zfp91mir‘‘5p and zfp91 are correlated in tumor tissues of bc patients furthermore we examined the levels of zfp91 in the tumor tissues and adjacent normal tissues of bc patients the aberrantly high level of zfp91 was observed in the tumor tissues of the bc patients fig 5a p005 spearman's correlation analysis showed a significantly inverse correlation between mir‘‘5p and zfp91 in the bc patient tissues fig 5b p005 taken together these results further confirmed that the proliferation and apoptosis of bc is regulated by mir‘‘5pzfp91mir‘‘5p inhibits the proliferation of mda‘mb‘ cells and reduces the expression of zpf91 in a bc xenograft mouse model moreover to evaluate the regulatory role of mir1885p in a bc xenograft mouse model we injected the mdamb231 cells transfected with mir1885p mimics or mirnc into nude mice the results showed that mir1885p mimics inhibited the tumor volume and weight compared to the mir‘nc group fig 6a p005 protein expression and the mrna level of zpf91 were also suppressed by mir1885p mimics in the tumor tissues of the xenograft mouse model when compared with the mirnc and control groups fig 6b p005mir‘‘5pzfp91 axis regulates nf‘kbp65 and relb expression numerous studies have reported that zinc finger protein zfp91 promotes proliferation and tumorigenesis 0concology reports figure effect of mir1885p on bc cell line mdamb231 mdamb231 cells were transfected with mir1885p mimics and mirnc a the mrna levels of mir1885p in mdamb231 cells were determined by rtqpcr p005 the proliferation of mda‘mb‘ cells was examined by b cck‘ kit post transfection and c colony formation p005 compared to control group p005 compared to mir‘nc group d the apoptotic mda‘mb‘ cells were analyzed using annexin vpi staining and facs p005 e the invasion and migration capability of mda‘mb‘ cells were detected by transwell assay p005 f expression of e‘cadherin n‘cadherin vimentin mmp2 mmp9 and nf‘κb p65rel were detected by western blotting the data are represented as the mean ± sd n3 p005 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer mmp matrix metalloproteinase nc negative controlof different cancer types via regulation of the nfκb p65 pathway therefore to further investigate the regulatory mechanism of the mir‘‘5pzfp91 axis we detected the expression of nfκbp65 and relb in bc cells the results showed that mir‘‘5p mimics significantly reduced the expression of nfκbp65 and relb together fig s2a p005 moreover the co‘transfection of mir‘‘5p mimics and zfp91 also upregulated the expression levels of 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure zfp91 is a target of mir‘‘5p a zfp91 provides binding sites with mir‘‘5p b rt‘qpcr and western blotting were used for analysis of transcription and translation of zfp91 in vitro and in vivo p005 c the binding between mir‘‘5p and zfp91 was confirmed by luciferase assay p005 relative gene expression was normalized by gapdh expression the data are represented as the mean ± sd n3 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons in b the comparisons between two groups were evaluated by t‘test in c zfp91 zinc finger protein nfκbp65 and relb compared to the monotransfection of mir1885p mimics fig s2b p005 in summary these results illustrated that the mir‘‘5pzfp91 axis regulates the progression of bc via the noncanonical nfκb signaling pathwaydiscussionbreast cancer bc is one of the most common types of tumors diagnosed in women worldwide bc is the second leading cause of cancerrelated mortality worldwide in more than women were diagnosed with bc in china and almost percent of all newly diagnosed cancer cases were in women however the molecular mechanisms of bc still await elucidation and effective molecular targets for the diagnosis and treatment of bc are urgently required recently research has reported that mirnas are small noncoding rna molecules which regulate target protein expression to play critical roles as tumorpromotors or suppressors several studies have demonstrated that mir1885p promotes cell proliferation migration and metastasis in gastric cancer and hepatocellular carcinoma moreover iwakawa detected higher expression of mir1885p in stage iii breast cancer and tnbc wang reported that circulating mir1885p was upregulated in bc patients and associated with tnm of bc interestingly mir1885p was downregulated in bc mdamb231 and mcf7 cells moreover using gainof and lossoffunction analyses of mir1885p in breast cancer cells the authors demonstrated that mir1885p inhibited the proliferation and invasion of bc mdamb231 cells via targeting il6st however we demonstrated that the expression of mir1885p was drastically downregulated in bc tissue specimens which was also decreased in bc cell lines mdamb231 bt549 and mcf7 compared to normal breast epithelial cell line mcf10a moreover the downregulation of mir‘‘5p was significantly 0concology reports figure mda‘mb‘ cell proliferation and apoptosis are regulated by mir‘‘5pzfp91 pcmv‘tag2b vector pc was transfected into bc mda‘mb‘ cells with mir‘nc ncpc or mir‘‘5p mimics pcmir‘‘5p pcmv‘tag2b‘zfp91 was transfected into mda‘mb‘ cells with mir‘‘5p mimics mir‘‘5pzfp91 a the mrna levels of mir‘‘5p in the co‘transfected overexpressing zfp91mda‘mb‘ cells were quantified by rtqpcr p005 cell proliferation was measured by b the cck‘ kit and c colony formation assay p005 compared to control group p005 compared to mirnc group d cell apoptosis was detected by annexin vpi staining and facs p005 e the invasion and migration capability of mdamb231 cells were detected by transwell assay p005 f expression of e‘cadherin n‘cadherin vimentin mmp2 mmp9 and nf‘κb p65rel were detected by western blotting relative genes expression was normalized by gapdh expression the data are represented as the mean ± sd n3 p005 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer zfp91 zinc finger protein mmp matrix metalloproteinase nc negative control 0cyang mirna1885p inhibits proliferation and induces apoptosis in bc via zfp91figure mrna and protein levels of zpf91 in bc tissues a the mrna and protein levels of zpf91 in bc tissue and corresponding non‘tumor normal tissue were quantified using rt‘qpcr and western blot analysis the comparisons between two groups were evaluated by t‘test n non‘cancer tissue c cancer tissue p005 b correlation between zpf91 mrna and mir‘‘5p was analyzed using pearson's correlation coefficient the data are represented as the mean ± sd n100 bc breast cancer zfp91 zinc finger protein figure the regulatory role of mir1885p in a bc xenograft mouse model bc mdamb231 cells were transfected with mir1885p mimics and mirnc and then injected into nude mice a the tumor volume and weight in the tumor xenograft mouse were compared among the mir1885p mimics group mirnc group and control group p005 compared to control group p005 compared to mir‘nc group b the protein expression and mrna level of zpf91 were detected by c western blotting and rt‘qpcr the data are represented as the mean ± sd n3 p005 one‘way anova followed by tukey's test was used to evaluate the difference for multiple comparisons bc breast cancer zfp91 zinc finger protein nc negative controlassociated with advanced tnm stage however to investigate the relationship of mir1885p and bc patient prognosis we found that kaplanmeier analysis of mir1885p was limited due to the small sample size in tcga these results illustrated that the downregulation of mir1885p may be related with bc progression in the clinic suggesting that mir1885p may be a valuable bc diagnostic indicator moreover we confirmed that mir1885p mimics considerably inhibited the proliferation induced the apoptosis and inhibited the invasion of bc cells suggesting that mir1885p plays an inhibitory role in bc cellstranscription factor zinc finger protein zfp91 was firstly identified in the mouse in which was found to be overexpressed in colon liver prostate stomach and breast cancer zfp91 has a molecular mass of kda with amino acids containing five zinc‘finger motives a leucine zipper a coiledcoil structure and nuclear localization sequences zfp91 was confirmed to be a transcription factor located in the cellular nucleus ma reported that zfp91 functions as an oncogene in cancer development by activating hif1α transcription the overexpression of zfp91 0concology reports was also found to result in the promotion of nkκb signaling pathway activation through increasing nkκb inducing kinase nik whose activity and overexpression are related to cancer progression in melanoma pancreatic breast and lung cancer the inhibition of zfp91 was demonstrated to promote apoptosis in bc stomach cancer cells colon cancer and endometrial cancer in addition the overexpression of zfp91 was found to increase the cancer cell growth rate and metastatic capability zfp91 was also reported to interact with cyclin‘dependent kinase inhibitor 2a cdkn2a which is an alternative reading frame arf tumor suppressor inhibiting the induction of p53dependent cell death to illuminate the molecular mechanisms of mir188 we predicted that il6st foxn2 zfp91 may be the targets of mir‘‘5p using targetscan and miranda furthermore peng reported that zfp91 is the target protein of mir‘‘5p in gastric cancer in addition overexpression of mir1885p was confirmed to inhibit the progression of breast cancer thus we chose the reported oncogene zfp91 for further investigation in the present study we confirmed that the 'untranslated region 'utr of zfp91 was bound by mir‘‘5p through dual luciferase assay moreover transfection of mir1885p mimics in mda‘mb‘ cells reduced the zfp91 mrna and protein levels together mirnas usually bind to the 'utrs of target mrnas and do not reduce the level of mrnas however mirnas also were reported to decay the target mrnas and decease mrna level restoration of zfp91 largely reversed the decreased proliferation and induced apoptosis which were both regulated by mir1885p overexpression moreover in the tumor xenograft mouse model we observed that the expression of zfp91 was downregulated by an increased level of mir1885p furthermore the expression of mir‘‘5p and zfp91 were negatively correlated in bc patient tissues therefore our studies confirmed that mir‘‘5p can inhibit the progression of human bc via targeting zfp91zfp91 has been reported to promote proliferation in colon cancer prostate cancer and gastric cancer ma reported that zfp91 activates nf‘kappabp65 to promote proliferation and tumorigenesis of colon cancer paschke identified that zfp91 is a noncanonical nf‘κb signaling pathway regulator with oncogenic properties in prostate cancer in the present study we also confirmed that a decrease in zfp91 could significantly inhibit nf‘κbp65 and relb expression in bc cells therefore mir1885p overexpression reduced zfp91 via the noncanonical nf‘κb signaling pathway to inhibit the progression of bcin conclusion our data showed that mir1885p is downregulated in bc cell lines and tissues and the downregulated expression of mir1885p is associated with the poor prognosis of patients with bc we further investigated that overexpression of mir1885p could inhibit proliferation and induce the apoptosis of mda‘mb‘ cells furthermore zfp91 was predicted and confirmed as a target gene of mirna‘‘5p and the effects of mir1885p on bc cells were dependent on the inhibition of zfp91 additionally a decrease in zfp91 significantly inhibited the nfκbp65 and relb expression in bc cells moreover the expression levels of mir‘‘5p and zfp91 were highly correlated with bc progression therefore we suggest that mir1885p can inhibit breast cancer progression via the zfp91nf‘κbp65 axis and may be a potential diagnostic indicator for bcacknowledgementsnot applicablefundingthis study was supported by the national natural science foundation of china grant nos and and talent projectavailability of data and materialsthe datasets used and analyzed during the current study are available from the corresponding author upon reasonable requestauthors' contributionszy and zl conceived and designed the study zy zc gy performed the experiments zy wrote the paper zy zl zc and gy reviewed the results and data and edited the manuscript all authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolvedethics approval and consent to participatetissues used in this study were obtained from shengjing hospital of china medical university shenyang liaoning china with the informed consent of patients and all experiments were approved by the ethics committee of shengjing hospital of china medical university no 2016ps18jpatient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences davidson ne armstrong sa coussens lm cruzcorrea mr deberardinis rj doroshow jh foti m hwu p k Answer:

Colon_Cancer

" lung carcinoma is a prominent cause of mortality among patients with cancer previous studies have reported the vital role of long noncoding rnas lncrnas in the malignant progression of lung cancer lncrna rp11284f219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown therefore the present study aimed to elucidate the role of lncrna rp11284f219 in lung carcinoma progression the expression of rp11‘284f219 in lung cell lines and tissues was measured using reverse transcription‘quantitative pcr the endogenous expression of rp11284f219 was silenced using rna interference and cell viabilities were measured with a cell counting kit‘ assay the invasion and apoptosis of cells were determined via transwell assays and flow cytometry respectively the protein expression levels were measured by western blotting an increased expression of rp11‘284f219 was identified in both lung carcinoma tissues and cells knockdown of rp11‘284f219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis the present study identified the existence of a direct interaction between rp11‘284f219 and microrna mirnamir6273p mechanistically it was demonstrated that rp11284f219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of mir6273p furthermore cell division cycle and apoptosis regulator ccar1 was identified as a target gene of mir6273p the in vivo tumor growth assay also demonstrated that the knockdown of rp11‘284f219 suppressed tumor growth upregulated mir6273p and downregulated correspondence to dr yuan wang department of medical imaging the first affiliated hospital of xi'an jiaotong university west yanta road xi'an shaanxi pr chinaemail wangyuan8003126comabbreviations ccar1 cell division cycle and apoptosis regulator nsclc non‘small cell lung cancer sclc small cell lung cancerkey words rp11284f219 lung carcinoma proliferation invasion microrna6273p ccarccar1 in the xenograft model of nude mice thus the present findings indicated the tumor promoting functions of rp11284f219 in the progression of lung carcinoma and provided a novel lncrnamirna axis as a target for the management of lung cancerintroductionpulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women furthermore of lung cancer cases are categorized as nonsmall cell lung cancer nsclc while the remaining are classified as sclc although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentiallong noncoding rnas lncrnas are nucleotides in length and have little or no protein coding capacity the mechanisms via which lncrnas regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small rnas generating different splice variants via regulating mrna splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes in recent years previous studies have reported that various human cancer types exhibit lncrnas dysfunction and these lncrnas are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells in lung cancer lncrna metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncrna hox transcript antisense rna is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with nsclc furthermore studies have shown that the expression of lncrna urothelial carcinoma‘associated 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionis significantly upregulated in nsclc and may induce resistance to treatment of egfr‘tyrosine kinase inhibitors by activating the aktmtor pathway lncrna rp11284f219 was primarily discovered in a pancancer transcriptomic analysis lncrna rp11‘284f219 exists as a cluster of three annotated lncrnas rp11284f219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas however the specific function and the underlying mechanism of rp11284f219 in lung carcinoma remain unknownto the best of our knowledge the present study demonstrated for the first time that lncrna rp11284f219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer together with microrna mirnamir6273p and cell division cycle and apoptosis regulator ccar1 the regulatory axis of rp11‘284f219mir‘‘3pccar1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo the present study aimed to investigate rp11284f219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the rp11‘284f219mir‘‘3pccar1 axismaterials and methodstissue samples and cell lines between may and jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range ‘ years nine male patients four female patients who were diagnosed and treated in first affiliated hospital of xi'an jiaotong university the samples were dissected during the surgery and immediately flash‘frozen in liquid nitrogen and transferred to ‘Ëšc storage for further extraction of both rna and protein all the tissue samples were obtained with written informed consent from the patients the protocol was approved by the first affiliated hospital of xi'an jiaotong university approval no a normal lung epithelial cell line beas2b and lung carcinoma cell lines ncih460 ncih1299 and a549 were purchased from american type culture collection atcc and cultured according to the atcc guidelines 293t cells were purchased from procell life sciencetechnology co ltd and cultured in dmem supplemented with fbs cat no ‘ atcc and 1x penicillin‘streptomycin thermo fisher scientific inc beas2b cells were cultured in bronchial epithelial growth medium begm cat no cc‘ clonetics corporation according to the manufacturer's instructions nci‘h460 and nci‘h1299 cells were cultured in rpmi‘ medium cat no ‘ atcc and a549 cells in f12k medium cat no ‘ atcc supplemented with fbs cat no atcc and 1x penicillin‘streptomycin thermo fisher scientific inc all cells were culture at ˚c with co2rna extraction and reverse transcription‘quantitative pcr rt‘qpcr total rna from both tissue samples and cell lines were extracted using trizol® reagent invitrogen thermo fisher scientific inc for each sample ng total rna was reverse transcribed to synthesize the first‘strand cdna using the primescript rt reagent kit takara bio inccdna samples were diluted times to perform the rt‘qpcr using sybr premix ex taq takara bio inc on a cfx96 realtime pcr detection system biorad laboratories inc expression levels of mrnas lncrnas and mirnas were normalized to gapdh the primers used for rtqpcr analyses were as follows gapdh forward '‘aac gac ccc ttc att gac c‘' and reverse '‘tcc acg aca tac tca gca cc‘' rp11‘284f219 forward '‘agg att ggc act cac ttc gg‘' and reverse '‘tct ctc acc acg tct ggt ct‘' and ccar1 forward '‘ctg atg gct agc cct agt atg ga‘' and reverse '‘tgc ctt tca tgc cca cta aaa ‘' the temperature protocol used to perform rt was ˚c for h followed by ˚c for min thermal conditions of pcr reactions were initial denaturation at ˚c for min followed by cycles for sec at ˚c and sec at ˚c the mrna expression levels were determined using the 2δδcq method oligonucleotides and cell transfection the small interfering rna sirna synthetic negative control sinc rp11284f219 sirnas sirp11284f219 mirnc mir‘‘3p mimics and mir‘‘3p inhibitor were purchased from shanghai genepharma co ltdall primer sequence information is presented in table i at a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency all of the oligonucleotides were transfected at a final concentration of nm using lipofectamine® reagent invitrogen thermo fisher scientific inc according to the manufacturer's instruction cells were collected at h posttransfection for subsequent experimentscell counting kit cck‘ assay and edu labeling of prolif‘erating cells a cck‘ was used for cell proliferation assay the cells were seeded into ‘well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions dojindo molecular technologies inc the optical density was measured at nm using a spectrophotometer thermo fisher scientific incfor the edu assay cells were incubated with µm edu cat no ab219801 abcam for h at ˚c and fixed with formaldehyde at room temperature for min after a brief washing with pbs click reagent was added into each well and incubated in the dark for min at room temperature followed by pbs washing the cells were stained with µgml dapi at room temperature for min images were captured using a fluorescence microscope nikon corporation and measured using adobe photoshop software adobe systems inc the edu labeled cells were analyzed with moflo astrios beckman‘coulter inc magnification x200transwell assay and flow cytometry measurement of cell apoptosis transwell assays were performed with a coating of matrigel bd biosciences mixed with culture medium mixed at ratio at ˚c for h a total of 1x105 cells in µl serum‘free medium were added to the upper layer of the transwell chambers µm pore size corning inc and cultured for h the lower chamber contained the culture medium with fbs the migrated cells were fixed with 0concology reports table i sequence of sirnas and mirna mimics and inhibitorsoligonucleotides si‘nc si‘rp11‘284f219 mir‘nc mir‘‘3p mimics mir‘‘3p inhibitor mir microrna sirna small interfering rna nc negative controlsequence '†’'uucuccgaacgugucacguttuauuggcaccaaggauagcucguuaaucggcuauaauacgcucuuuucuuugagacucacuucuuuucuuugagacucacu paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope magnification x200cellular apoptosis was detected using the apoptosis detection kit cat no kgf001 nanjing keygen biotech co ltd according to the manufacturer's instructions cells were stained with fluorescein isothiocyanateconjugated annexin v and pi after incubated for min at ˚c in the dark µl 1x binding buffer was added to each tube and stained cells were analyzed using bd facs canto ii flow cytometry facs calibur bd biosciences data were analyzed using flowjo software version tree star incluciferase reporter assay the rp11284f219 wildtype wt or mutant mut '‘untranslated region '‘utr and ccar1 wt or mut '‘utr sequences were cloned into the pmirglo plasmid youbio httpwwwyoubiocn cat no vt1439 the vectors µgml were co‘transfected with mir‘nc or mir6273p mimic nm and renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using lipofectamine® reagent invitrogen thermo fisher scientific inc cell lysates were collected at h after transfection and the luciferase activities were detected with the dualluciferase reporter assay system promega corporation according to the manufacturer's instructionswestern blotting cell were lysed using ripa lysis buffer sigma‘aldrich merck kgaa and protein concentrations were assessed with the bca protein assay kit according to the manufacturer's instructions beyotime institute of biotechnology shanghai china equal amounts µg of cell protein lysates were loaded and separated by sds‘page transferred to a pvdf membrane and blocked with non‘fat milk at room temperature for h the membranes were then incubated with ccar1 primary antibody cat no ab70243 abcam overnight at ˚c followed by incubation with goat anti‘mouse or goat anti‘rabbit igg‘horseradish peroxidase conjugate secondary antibodies cat no ab205718 abcam at room temperature for h gapdh cat no ab181602 abcam was used as loading control the signals were detected using the ecl system protein simple according to the manufacturer's instructionsin vivo tumorigenicity analysis in mice male balbc nude mice age weeks weight ‘ g were obtained from beijing vital river laboratory animal technology co ltd and housed at a room temperature of ˚c with a h lightdark cycle the mice were maintained in an individually ventilated cage system under specific pathogen‘free conditions temperature ˚c humidity and fed with sterile food and water free access to evaluate the effect of rp11‘284f219 knockdown on the growth of lung carcinoma in vivo 5x106 sinc or sirp11284f219 treated nci‘h1299 cells in µl serum‘free medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin the animals were monitored daily and the following criteria for humane endpoint was used severe tumor burden mm in diameter difficulty breathing significant body‘weight loss and clinical signs such as prostration hypothermia and significant abdominal distension tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] then weeks after inoculation the mice were euthanized by co2 inhalation co2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat the xenografts were imaged and weighedthe total rna was then extracted from the xenografts as aforementioned animal care and study were approved by the institutional animal care and use committee of the first affiliated hospital of xi'an jiaotong university approval no target prediction potential target mirnas of rp11284f219 were predicted using lncbase v2 httpcarolinaimisathena‘ innovationgrdiana_toolswebindexphprlncbasev2index the target genes of mir‘‘3p were predicted using three bioinformatics algorithms targetscanv72 httpwwwtargetscanorgvert_72 and mirdb httpwwwmirdborgmininghtmlstatistics analysis data were analyzed using the graphpad prism software graphpad software inc and presented as the mean ± sd from ‰¥ independent experiments a two‘tailed unpaired student's t‘test or one‘way anova with tukey's post‘hoc analysis were performed to evaluate the statistical significance p005 was considered to indicate a statistically significant difference 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure rp11‘284f219 expression is upregulated in lc tissues and cell lines a expression of rp11‘284f219 in lc tissues in comparison with adjacent healthy tissues was analyzed using rtqpcr p0001 vs adjacent tissues n13 b expression of rp11‘284f219 in human lung carcinoma cell lines ncih460 ncih1299 and a549 compared with normal human lung epithelial cell line beas2b was analyzed using rtqpcr p005 p0001 vs beas‘2b n3 lc lung carcinoma rt‘qpcr reverse transcription‘quantitative pcrresultsexpression of rp11‘284f219 is upregulated in lung carci‘noma to investigate the potential role of rp11284f219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma the results demonstrated that the expression of rp11‘284f219 was significantly upregulated in tumor tissues compared with healthy tissues fig 1a the expression of rp11‘284f219 was also analyzed in human lung carcinoma cell lines ncih460 ncih1299 and a549 and normal human lung epithelial cell line beas2b consistent with the findings in the tissue samples the expression of rp11‘284f219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line fig 1b these results indicated that rp11284f219 may serve an oncogenic role in lung carcinomaknockdown of rp11‘284f219 exerts anti‘oncogenic effects in lung carcinoma cells to study the specific role of rp11284f219 in lung carcinoma cells rp11284f219 sirna was transfected into ncih1299 and ncih460 cells fig 2a after transfection the proliferation of these cells was measured using cck‘ and edu assays fig 2b‘d the results suggested that knocking down rp11‘284f219 significantly reduced the proliferation of lung carcinoma cells compared with the nc group fig 2bd the invasiveness of si‘rp11‘284f219 transfected cells also significantly decreased as indicated by the data from the transwell assay fig 2f to further validate the invasive capability a rt‘qpcr assay was performed to detect the expression levels of invasion‘related genes and the results identified that both mmp2 and mmp9 were significantly decreased when rp11‘284f219 was downregulated fig s1the results of flow cytometry measurement based apoptosis assay suggested that cells transfected with sirp11284f219 had a higher apoptotic rate compared with the sinc transfected group fig 2e these data demonstrated the antitumor effects of rp11‘284f219 knockdown in lung carcinoma cells indicating an oncogenic role of rp11284f219rp11‘284f219 directly interacts with mir‘‘3p based on the prediction of the online tool lncbase v2 from diana prediction module httpcarolinaimisathena‘innovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream mirnas of rp11284f219 the first five mirnas in the output list were tested among the predicted potential targets it was found that mir6273p had the most significant upregulation in ncih1299 cells transfected with sirp11284f219 fig s2using sequence alignment it was identified that mir‘‘3p was partially complementary with the '‘utr of rp11‘284f219 fig 3a subsequently 293t cells were transfected with the pmirglorp11284f219wt or mut vector containing the wt or mut sequence of rp11284f219 '‘utr with or without mir‘‘3p mimics results from the luciferase reporter assay suggested that mir6273p mimics significantly decrease the signal of rp11‘284f219‘wt transfected cells but not the rp11‘284f219‘mut transfected cells indicating a direct interaction between the two non‘coding rnas fig 3a furthermore transfection of sirp11284f219 into ncih1299 and ncih460 cells resulted in the suppression of endogenous rp11‘284f219 leading to a significant increase in mir‘‘3p expression fig 3b thus these findings suggested an inhibitory effect of rp11‘284f219 on the expression of mir‘‘3p in lung carcinoma cellsthe expression of mir‘‘3p was detected in both lung carcinoma tissues and cell lines it was demonstrated that mir‘‘3p was significantly downregulated in carcinoma tissues fig 3c and ncih460 ncih1299 and a549 cells fig 3d compared with healthy tissues and cells collectively these data suggested a direct interaction between rp11284f219 and mir6273p in which rp11284f219 suppresses the expression of mir‘‘3prp11‘284f219 regulates the proliferation and invasiveness of lung carcinoma cells via mir‘‘3p to rescue the antitumor effects of sirp11284f219 in lung carcinoma cells the mir‘‘3p inhibitor which specifically downregulates the expression of mir‘‘3p was transfected into nci‘h1299 and nci‘h460 cells fig 4a the results from the cck‘ and edu assays demonstrated that treatment with si‘rp11‘284f219 0concology reports figure rp11‘284f219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis a rp11‘284f219 knockdown was achieved via rp11‘284f219 sirna and the knockdown efficiency was verified using reverse transcription‘quantitative pcr n3 cell counting kit‘ assay was performed to measure the proliferation of b ncih1299 and c ncih460 cells after transfection with sirp11284f219 compared with the si‘nc group n5 d an edu assay was performed to measure the proliferation of nci‘h1299 and nci‘h460 cells after transfection with si‘nc and si‘rp11‘284f219 magnification x200 e flow cytometry analysis was performed to determine the effects of rp11‘284f219 knockdown on apoptotic rates in nci‘h1299 and nci‘h460 cells n3 f transwell assay was performed to determine the effects of rp11‘284f219 knockdown on nci‘h1299 and nci‘h460 cell invasion n3 magnification x200 p005 p001 vs control group nc negative control sirna small interfering rna od optical density and mir‘nc significantly decrease the proliferation of both ncih1299 and ncih460 cells fig 4bd however the administration of mir‘‘3p inhibitor partially reversed the antiproliferative effect of sirp11284f219 indicating that rp11284f219 regulates the proliferation of lung carcinoma cells partially via mir6273p fig 4bd in addition the 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure rp11‘284f219 directly interacts with mir‘‘3p a binding site between rp11‘284f219 and mir‘‘3p that was identified using the diana tools and a luciferase reporter assay was conducted in pmirglorp11284f219wt or mut treated cells in the presence of mir6273p mimics or mir‘nc n3 p005 vs mir‘nc b expression of mir‘‘3p in nci‘h1299 and nci‘h460 cells transfected with si‘rp11‘284f219 was analyzed using rtqpcr p001 vs si‘nc n3 mir‘‘3p expression in c lc tissues and d nci‘h460 nci‘h1299 and a549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using rt‘qpcr n3 p005 p001 vs adjacent tissue or beas‘2b cells nc negative control sirna small interfering rna wt wild‘type mut mutant mir microrna lc lung carcinoma mir‘‘3p inhibitor restored the reduction in the number of ncih1299 and ncih460 cells that migrated through the transwell membrane induced by si‘rp11‘284f219 treatment fig 4f these data indicated the participation of mir6273p in the rp11284f219mediated invasive effectthe qpcr assay results identified that both mmp2 and mmp9 expression levels were restored in rp11284f219downregulated cells when mir6273p was inhibited compared with the mir‘nc group fig s3 in addition transfection with mir‘‘3p inhibitor also diminished the pro‘apoptosis effect of si‘rp11‘284f219 in both ncih1299 and ncih460 cells fig 4e therefore it was suggested that rp11284f219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of mir‘‘3prp11‘284f219 regulates ccar1 via targeting mir‘‘3p to further evaluate how rp11‘284f219 exerts an oncogenic role via mir‘‘3p the publicly available algorithms of targetscan httpwwwtargetscanorg and mirdb were used which identified ccar1 as a potential target for mir6273p fig 5a in order to validate this prediction mir6273p mimic was transfected into cells and the transfection efficiency was assessed the results demonstrated that transfection of mir6273p mimic increased the expression of mir‘‘3p by times compared with cells transfected with mirnc fig s4after validating the upregulation of mir6273p mimic a ccar1wt vector was constructed which contained the wt binding site between mir‘‘3p and the ccar1 '‘utr and ccar1mut vector containing the mut sequence fig 5a the results from luciferase reporter assays indicated that compared with the mirnc group the mir6273p mimic significantly decreased the luciferase activity of ccar1‘wt treated cells but not the ccar1‘mut treated cells suggesting a direct binding of mir‘‘3p to the '‘utr of ccar1 fig 5b increased expression levels of ccar1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues fig 5c moreover a significant decrease in both mrna and protein expression levels of ccar1 was detected upon transfecting ncih1299 and ncih460 cells with mir6273p mimics fig 5d and e thus ccar1 may be a direct target of mir‘‘3p in lung carcinoma cells and tissuesrp11‘284f219 knockdown inhibits tumor growth and the expression of ccar1 in vivo in order to investigate the effect of rp11284f219 on in vivo tumorigenicity ncih1299 cells were transfected with sinc or sirp11284f219 and injected into the nude mice after weeks a significantly 0concology reports figure rp11‘284f219 regulates proliferation and invasiveness in lung carcinoma cells via mir‘‘3p a expression of mir‘‘3p in nci‘h1299 and nci‘h460 cells transfected with mir‘nc or mir‘‘3p inhibitor was detected using rt‘qpcr analysis n3 p005 vs mir‘nc cell counting kit‘ assay was performed in b nci‘h1299 and c nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor n5 d edu assay was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor magnification x200 e flow cytometry analysis was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor n3 f transwell assay was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor magnification x200 n3 p005 vs si‘nc nc negative control sirna small interfering rna od optical density mir microrna 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure mir‘‘3p directly targets ccar1 a bioinformatic analysis of the predicted binding sites between the ccar1 '‘untranslated region and mir‘‘3p b luciferase reporter assay in ccar1‘wt or ccar1‘mut treated cells in the presence of mir‘nc or mir‘‘3p mimic n3 p005 vs mir‘nc c ccar1 expression in lc tissues compared with adjacent healthy tissues was analyzed using rt‘qpcr n13 p001 vs adjacent tissue expression of ccar1 in nci‘h1299 and nci‘h460 cells transfected with mir‘nc or mir‘‘3p mimics was detected using d rt‘qpcr and e western blotting n3 p005 vs mir‘nc mir microrna nc negative control wt wild‘type mut mutant rt‘qpcr reverse transcription‘quantitative pcr ccar1 cell division cycle and apoptosis regulator lc lung carcinoma slower proliferative rate of the tumors was observed in the sirp11284f219 group compared with the sinc group fig 6a and b furthermore the tumor volume and weight were significantly decreased in the si‘rp11‘284f219 group compared with the control group fig 6a and b rtqpcr analysis also demonstrated that compared with the sinc group the tumors in the si‘rp11‘284f219 group expressed higher levels of mir6273p fig 6c and lower levels of ccar1 fig 6d providing further evidence to the existence of the rp11‘284f219mir‘‘3pccar1 regulatory axis in lung carcinoma tumor tissuesdiscussionthe present study investigated the function of rp11284f219 in lung carcinoma it was initially found that rp11284f219 was significantly upregulated in both lung cancer tissues and cell lines following the deduction of a potential oncogenic role of this lncrna sirp11284f219 was transfected into ncih460 and ncih1299 cells and it was demonstrated that knockdown of rp11‘284f219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells in the mechanistic studies using online prediction tools and in vitro assays the results indicated that mir‘‘3p directly interacts with rp11‘284f219 by binding to its '‘utrthe function of mir627 was initially reported in colorectal cancer crc padi found that when upregulated by calcitriol mir627 targets the histone demethylase jumonji domain containing 1a to increase methylation of histone h3k9 and suppresses the proliferative factors of crc cells thus inhibiting the proliferation of crc both in vitro and in vivo moreover in crc sun discovered the role of mir‘ in vitamin d‘enhanced efficacy of irinotecan via inhibition of the cytochrome p450 enzyme‘mediated intratumoral drug metabolism mir‘ is also reported to be a potential non‘invasive diagnostic marker in gastric and breast cancer types in pulmonary diseases mir627 is downregulated in patients with chronic obstructive pulmonary disease and targets the high‘mobility group box protein to inhibit its expression thus improving transforming growth factorβ1‘induced pulmonary fibrosis the present results demonstrated the inhibitory effect of rp11‘284f219 on the expression of mir‘‘3p in addition it was identified that the mir‘‘3p inhibitor can neutralize the anti‘tumor effects of rp11‘284f219 knockdown indicating that rp11‘284f219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating mir‘‘3p this anti‘tumor role of mir6273p under the regulation of rp11284f219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human crc gastric and breast cancer types 0concology reports figure rp11‘284f219 knockdown inhibits tumor growth in vivo a macroscopic image of xenografted tumors b tumor volume in nude mice injected with nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 measured over weeks n5 c weight of tumors in nude mice at weeks after injection of nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 n5 expression levels of d mir‘‘3p and e ccar1 in the tumor tissues from nude mice injected with nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 for weeks were detected using reverse transcription‘quantitative pcr n5 p005 p001 p0001 vs si‘nc mir microrna nc negative control sh short hairpin rna ccar1 cell division cycle and apoptosis regulator using the publicly available rna interaction prediction algorithms the current study identified that ccar1 which was initially shown as the target gene of mir6273p is also regulated by rp11‘284f219 furthermore the regulatory axis of rp11‘284f219mir‘‘3pccar1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model the interaction between rp11‘284f219 and mir‘‘3p and the interaction between mir‘‘3p and ccar1 were demonstrated by the dual‘luciferase assay although this method has been used to validate rna‘rna interactions in previous studies other assays such as rna pulldown and rna binding protein immunoprecipitation that would provide more direct evidence for the rnarna and rna‘protein interactions should be performedccar1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as adriamycin and etoposide subsequently kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors in human breast cancer cells as ccar1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells in crc cells ou reported that ccar1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor ccar1 is essential for the expression of wnt target genes as well as the neoplastic transformation of crc cells in gastric cancer cells researchers have revealed the cooperation between ccar1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells in lung cancer ccar1 was reported to be an effector of doxorubicin‘induced apoptosis moreover muthu demonstrated that certain chemical compounds that bind with ccar1 can increase the expression of ccar1 and induce apoptosis however a contradictory conclusion was reported in a recent study which observed that ccar1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate corroborating this finding in the current study via the targeting of mir‘‘3p the expr

cancer7548

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: " lung carcinoma is a prominent cause of mortality among patients with cancer previous studies have reported the vital role of long noncoding rnas lncrnas in the malignant progression of lung cancer lncrna rp11284f219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown therefore the present study aimed to elucidate the role of lncrna rp11284f219 in lung carcinoma progression the expression of rp11‘284f219 in lung cell lines and tissues was measured using reverse transcription‘quantitative pcr the endogenous expression of rp11284f219 was silenced using rna interference and cell viabilities were measured with a cell counting kit‘ assay the invasion and apoptosis of cells were determined via transwell assays and flow cytometry respectively the protein expression levels were measured by western blotting an increased expression of rp11‘284f219 was identified in both lung carcinoma tissues and cells knockdown of rp11‘284f219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis the present study identified the existence of a direct interaction between rp11‘284f219 and microrna mirnamir6273p mechanistically it was demonstrated that rp11284f219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of mir6273p furthermore cell division cycle and apoptosis regulator ccar1 was identified as a target gene of mir6273p the in vivo tumor growth assay also demonstrated that the knockdown of rp11‘284f219 suppressed tumor growth upregulated mir6273p and downregulated correspondence to dr yuan wang department of medical imaging the first affiliated hospital of xi'an jiaotong university west yanta road xi'an shaanxi pr chinaemail wangyuan8003126comabbreviations ccar1 cell division cycle and apoptosis regulator nsclc non‘small cell lung cancer sclc small cell lung cancerkey words rp11284f219 lung carcinoma proliferation invasion microrna6273p ccarccar1 in the xenograft model of nude mice thus the present findings indicated the tumor promoting functions of rp11284f219 in the progression of lung carcinoma and provided a novel lncrnamirna axis as a target for the management of lung cancerintroductionpulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women furthermore of lung cancer cases are categorized as nonsmall cell lung cancer nsclc while the remaining are classified as sclc although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentiallong noncoding rnas lncrnas are nucleotides in length and have little or no protein coding capacity the mechanisms via which lncrnas regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small rnas generating different splice variants via regulating mrna splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes in recent years previous studies have reported that various human cancer types exhibit lncrnas dysfunction and these lncrnas are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells in lung cancer lncrna metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncrna hox transcript antisense rna is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with nsclc furthermore studies have shown that the expression of lncrna urothelial carcinoma‘associated 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionis significantly upregulated in nsclc and may induce resistance to treatment of egfr‘tyrosine kinase inhibitors by activating the aktmtor pathway lncrna rp11284f219 was primarily discovered in a pancancer transcriptomic analysis lncrna rp11‘284f219 exists as a cluster of three annotated lncrnas rp11284f219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas however the specific function and the underlying mechanism of rp11284f219 in lung carcinoma remain unknownto the best of our knowledge the present study demonstrated for the first time that lncrna rp11284f219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer together with microrna mirnamir6273p and cell division cycle and apoptosis regulator ccar1 the regulatory axis of rp11‘284f219mir‘‘3pccar1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo the present study aimed to investigate rp11284f219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the rp11‘284f219mir‘‘3pccar1 axismaterials and methodstissue samples and cell lines between may and jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range ‘ years nine male patients four female patients who were diagnosed and treated in first affiliated hospital of xi'an jiaotong university the samples were dissected during the surgery and immediately flash‘frozen in liquid nitrogen and transferred to ‘Ëšc storage for further extraction of both rna and protein all the tissue samples were obtained with written informed consent from the patients the protocol was approved by the first affiliated hospital of xi'an jiaotong university approval no a normal lung epithelial cell line beas2b and lung carcinoma cell lines ncih460 ncih1299 and a549 were purchased from american type culture collection atcc and cultured according to the atcc guidelines 293t cells were purchased from procell life sciencetechnology co ltd and cultured in dmem supplemented with fbs cat no ‘ atcc and 1x penicillin‘streptomycin thermo fisher scientific inc beas2b cells were cultured in bronchial epithelial growth medium begm cat no cc‘ clonetics corporation according to the manufacturer's instructions nci‘h460 and nci‘h1299 cells were cultured in rpmi‘ medium cat no ‘ atcc and a549 cells in f12k medium cat no ‘ atcc supplemented with fbs cat no atcc and 1x penicillin‘streptomycin thermo fisher scientific inc all cells were culture at ˚c with co2rna extraction and reverse transcription‘quantitative pcr rt‘qpcr total rna from both tissue samples and cell lines were extracted using trizol® reagent invitrogen thermo fisher scientific inc for each sample ng total rna was reverse transcribed to synthesize the first‘strand cdna using the primescript rt reagent kit takara bio inccdna samples were diluted times to perform the rt‘qpcr using sybr premix ex taq takara bio inc on a cfx96 realtime pcr detection system biorad laboratories inc expression levels of mrnas lncrnas and mirnas were normalized to gapdh the primers used for rtqpcr analyses were as follows gapdh forward '‘aac gac ccc ttc att gac c‘' and reverse '‘tcc acg aca tac tca gca cc‘' rp11‘284f219 forward '‘agg att ggc act cac ttc gg‘' and reverse '‘tct ctc acc acg tct ggt ct‘' and ccar1 forward '‘ctg atg gct agc cct agt atg ga‘' and reverse '‘tgc ctt tca tgc cca cta aaa ‘' the temperature protocol used to perform rt was ˚c for h followed by ˚c for min thermal conditions of pcr reactions were initial denaturation at ˚c for min followed by cycles for sec at ˚c and sec at ˚c the mrna expression levels were determined using the 2δδcq method oligonucleotides and cell transfection the small interfering rna sirna synthetic negative control sinc rp11284f219 sirnas sirp11284f219 mirnc mir‘‘3p mimics and mir‘‘3p inhibitor were purchased from shanghai genepharma co ltdall primer sequence information is presented in table i at a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency all of the oligonucleotides were transfected at a final concentration of nm using lipofectamine® reagent invitrogen thermo fisher scientific inc according to the manufacturer's instruction cells were collected at h posttransfection for subsequent experimentscell counting kit cck‘ assay and edu labeling of prolif‘erating cells a cck‘ was used for cell proliferation assay the cells were seeded into ‘well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions dojindo molecular technologies inc the optical density was measured at nm using a spectrophotometer thermo fisher scientific incfor the edu assay cells were incubated with µm edu cat no ab219801 abcam for h at ˚c and fixed with formaldehyde at room temperature for min after a brief washing with pbs click reagent was added into each well and incubated in the dark for min at room temperature followed by pbs washing the cells were stained with µgml dapi at room temperature for min images were captured using a fluorescence microscope nikon corporation and measured using adobe photoshop software adobe systems inc the edu labeled cells were analyzed with moflo astrios beckman‘coulter inc magnification x200transwell assay and flow cytometry measurement of cell apoptosis transwell assays were performed with a coating of matrigel bd biosciences mixed with culture medium mixed at ratio at ˚c for h a total of 1x105 cells in µl serum‘free medium were added to the upper layer of the transwell chambers µm pore size corning inc and cultured for h the lower chamber contained the culture medium with fbs the migrated cells were fixed with 0concology reports table i sequence of sirnas and mirna mimics and inhibitorsoligonucleotides si‘nc si‘rp11‘284f219 mir‘nc mir‘‘3p mimics mir‘‘3p inhibitor mir microrna sirna small interfering rna nc negative controlsequence '†’'uucuccgaacgugucacguttuauuggcaccaaggauagcucguuaaucggcuauaauacgcucuuuucuuugagacucacuucuuuucuuugagacucacu paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope magnification x200cellular apoptosis was detected using the apoptosis detection kit cat no kgf001 nanjing keygen biotech co ltd according to the manufacturer's instructions cells were stained with fluorescein isothiocyanateconjugated annexin v and pi after incubated for min at ˚c in the dark µl 1x binding buffer was added to each tube and stained cells were analyzed using bd facs canto ii flow cytometry facs calibur bd biosciences data were analyzed using flowjo software version tree star incluciferase reporter assay the rp11284f219 wildtype wt or mutant mut '‘untranslated region '‘utr and ccar1 wt or mut '‘utr sequences were cloned into the pmirglo plasmid youbio httpwwwyoubiocn cat no vt1439 the vectors µgml were co‘transfected with mir‘nc or mir6273p mimic nm and renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using lipofectamine® reagent invitrogen thermo fisher scientific inc cell lysates were collected at h after transfection and the luciferase activities were detected with the dualluciferase reporter assay system promega corporation according to the manufacturer's instructionswestern blotting cell were lysed using ripa lysis buffer sigma‘aldrich merck kgaa and protein concentrations were assessed with the bca protein assay kit according to the manufacturer's instructions beyotime institute of biotechnology shanghai china equal amounts µg of cell protein lysates were loaded and separated by sds‘page transferred to a pvdf membrane and blocked with non‘fat milk at room temperature for h the membranes were then incubated with ccar1 primary antibody cat no ab70243 abcam overnight at ˚c followed by incubation with goat anti‘mouse or goat anti‘rabbit igg‘horseradish peroxidase conjugate secondary antibodies cat no ab205718 abcam at room temperature for h gapdh cat no ab181602 abcam was used as loading control the signals were detected using the ecl system protein simple according to the manufacturer's instructionsin vivo tumorigenicity analysis in mice male balbc nude mice age weeks weight ‘ g were obtained from beijing vital river laboratory animal technology co ltd and housed at a room temperature of ˚c with a h lightdark cycle the mice were maintained in an individually ventilated cage system under specific pathogen‘free conditions temperature ˚c humidity and fed with sterile food and water free access to evaluate the effect of rp11‘284f219 knockdown on the growth of lung carcinoma in vivo 5x106 sinc or sirp11284f219 treated nci‘h1299 cells in µl serum‘free medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin the animals were monitored daily and the following criteria for humane endpoint was used severe tumor burden mm in diameter difficulty breathing significant body‘weight loss and clinical signs such as prostration hypothermia and significant abdominal distension tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] then weeks after inoculation the mice were euthanized by co2 inhalation co2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat the xenografts were imaged and weighedthe total rna was then extracted from the xenografts as aforementioned animal care and study were approved by the institutional animal care and use committee of the first affiliated hospital of xi'an jiaotong university approval no target prediction potential target mirnas of rp11284f219 were predicted using lncbase v2 httpcarolinaimisathena‘ innovationgrdiana_toolswebindexphprlncbasev2index the target genes of mir‘‘3p were predicted using three bioinformatics algorithms targetscanv72 httpwwwtargetscanorgvert_72 and mirdb httpwwwmirdborgmininghtmlstatistics analysis data were analyzed using the graphpad prism software graphpad software inc and presented as the mean ± sd from ‰¥ independent experiments a two‘tailed unpaired student's t‘test or one‘way anova with tukey's post‘hoc analysis were performed to evaluate the statistical significance p005 was considered to indicate a statistically significant difference 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure rp11‘284f219 expression is upregulated in lc tissues and cell lines a expression of rp11‘284f219 in lc tissues in comparison with adjacent healthy tissues was analyzed using rtqpcr p0001 vs adjacent tissues n13 b expression of rp11‘284f219 in human lung carcinoma cell lines ncih460 ncih1299 and a549 compared with normal human lung epithelial cell line beas2b was analyzed using rtqpcr p005 p0001 vs beas‘2b n3 lc lung carcinoma rt‘qpcr reverse transcription‘quantitative pcrresultsexpression of rp11‘284f219 is upregulated in lung carci‘noma to investigate the potential role of rp11284f219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma the results demonstrated that the expression of rp11‘284f219 was significantly upregulated in tumor tissues compared with healthy tissues fig 1a the expression of rp11‘284f219 was also analyzed in human lung carcinoma cell lines ncih460 ncih1299 and a549 and normal human lung epithelial cell line beas2b consistent with the findings in the tissue samples the expression of rp11‘284f219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line fig 1b these results indicated that rp11284f219 may serve an oncogenic role in lung carcinomaknockdown of rp11‘284f219 exerts anti‘oncogenic effects in lung carcinoma cells to study the specific role of rp11284f219 in lung carcinoma cells rp11284f219 sirna was transfected into ncih1299 and ncih460 cells fig 2a after transfection the proliferation of these cells was measured using cck‘ and edu assays fig 2b‘d the results suggested that knocking down rp11‘284f219 significantly reduced the proliferation of lung carcinoma cells compared with the nc group fig 2bd the invasiveness of si‘rp11‘284f219 transfected cells also significantly decreased as indicated by the data from the transwell assay fig 2f to further validate the invasive capability a rt‘qpcr assay was performed to detect the expression levels of invasion‘related genes and the results identified that both mmp2 and mmp9 were significantly decreased when rp11‘284f219 was downregulated fig s1the results of flow cytometry measurement based apoptosis assay suggested that cells transfected with sirp11284f219 had a higher apoptotic rate compared with the sinc transfected group fig 2e these data demonstrated the antitumor effects of rp11‘284f219 knockdown in lung carcinoma cells indicating an oncogenic role of rp11284f219rp11‘284f219 directly interacts with mir‘‘3p based on the prediction of the online tool lncbase v2 from diana prediction module httpcarolinaimisathena‘innovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream mirnas of rp11284f219 the first five mirnas in the output list were tested among the predicted potential targets it was found that mir6273p had the most significant upregulation in ncih1299 cells transfected with sirp11284f219 fig s2using sequence alignment it was identified that mir‘‘3p was partially complementary with the '‘utr of rp11‘284f219 fig 3a subsequently 293t cells were transfected with the pmirglorp11284f219wt or mut vector containing the wt or mut sequence of rp11284f219 '‘utr with or without mir‘‘3p mimics results from the luciferase reporter assay suggested that mir6273p mimics significantly decrease the signal of rp11‘284f219‘wt transfected cells but not the rp11‘284f219‘mut transfected cells indicating a direct interaction between the two non‘coding rnas fig 3a furthermore transfection of sirp11284f219 into ncih1299 and ncih460 cells resulted in the suppression of endogenous rp11‘284f219 leading to a significant increase in mir‘‘3p expression fig 3b thus these findings suggested an inhibitory effect of rp11‘284f219 on the expression of mir‘‘3p in lung carcinoma cellsthe expression of mir‘‘3p was detected in both lung carcinoma tissues and cell lines it was demonstrated that mir‘‘3p was significantly downregulated in carcinoma tissues fig 3c and ncih460 ncih1299 and a549 cells fig 3d compared with healthy tissues and cells collectively these data suggested a direct interaction between rp11284f219 and mir6273p in which rp11284f219 suppresses the expression of mir‘‘3prp11‘284f219 regulates the proliferation and invasiveness of lung carcinoma cells via mir‘‘3p to rescue the antitumor effects of sirp11284f219 in lung carcinoma cells the mir‘‘3p inhibitor which specifically downregulates the expression of mir‘‘3p was transfected into nci‘h1299 and nci‘h460 cells fig 4a the results from the cck‘ and edu assays demonstrated that treatment with si‘rp11‘284f219 0concology reports figure rp11‘284f219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis a rp11‘284f219 knockdown was achieved via rp11‘284f219 sirna and the knockdown efficiency was verified using reverse transcription‘quantitative pcr n3 cell counting kit‘ assay was performed to measure the proliferation of b ncih1299 and c ncih460 cells after transfection with sirp11284f219 compared with the si‘nc group n5 d an edu assay was performed to measure the proliferation of nci‘h1299 and nci‘h460 cells after transfection with si‘nc and si‘rp11‘284f219 magnification x200 e flow cytometry analysis was performed to determine the effects of rp11‘284f219 knockdown on apoptotic rates in nci‘h1299 and nci‘h460 cells n3 f transwell assay was performed to determine the effects of rp11‘284f219 knockdown on nci‘h1299 and nci‘h460 cell invasion n3 magnification x200 p005 p001 vs control group nc negative control sirna small interfering rna od optical density and mir‘nc significantly decrease the proliferation of both ncih1299 and ncih460 cells fig 4bd however the administration of mir‘‘3p inhibitor partially reversed the antiproliferative effect of sirp11284f219 indicating that rp11284f219 regulates the proliferation of lung carcinoma cells partially via mir6273p fig 4bd in addition the 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure rp11‘284f219 directly interacts with mir‘‘3p a binding site between rp11‘284f219 and mir‘‘3p that was identified using the diana tools and a luciferase reporter assay was conducted in pmirglorp11284f219wt or mut treated cells in the presence of mir6273p mimics or mir‘nc n3 p005 vs mir‘nc b expression of mir‘‘3p in nci‘h1299 and nci‘h460 cells transfected with si‘rp11‘284f219 was analyzed using rtqpcr p001 vs si‘nc n3 mir‘‘3p expression in c lc tissues and d nci‘h460 nci‘h1299 and a549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using rt‘qpcr n3 p005 p001 vs adjacent tissue or beas‘2b cells nc negative control sirna small interfering rna wt wild‘type mut mutant mir microrna lc lung carcinoma mir‘‘3p inhibitor restored the reduction in the number of ncih1299 and ncih460 cells that migrated through the transwell membrane induced by si‘rp11‘284f219 treatment fig 4f these data indicated the participation of mir6273p in the rp11284f219mediated invasive effectthe qpcr assay results identified that both mmp2 and mmp9 expression levels were restored in rp11284f219downregulated cells when mir6273p was inhibited compared with the mir‘nc group fig s3 in addition transfection with mir‘‘3p inhibitor also diminished the pro‘apoptosis effect of si‘rp11‘284f219 in both ncih1299 and ncih460 cells fig 4e therefore it was suggested that rp11284f219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of mir‘‘3prp11‘284f219 regulates ccar1 via targeting mir‘‘3p to further evaluate how rp11‘284f219 exerts an oncogenic role via mir‘‘3p the publicly available algorithms of targetscan httpwwwtargetscanorg and mirdb were used which identified ccar1 as a potential target for mir6273p fig 5a in order to validate this prediction mir6273p mimic was transfected into cells and the transfection efficiency was assessed the results demonstrated that transfection of mir6273p mimic increased the expression of mir‘‘3p by times compared with cells transfected with mirnc fig s4after validating the upregulation of mir6273p mimic a ccar1wt vector was constructed which contained the wt binding site between mir‘‘3p and the ccar1 '‘utr and ccar1mut vector containing the mut sequence fig 5a the results from luciferase reporter assays indicated that compared with the mirnc group the mir6273p mimic significantly decreased the luciferase activity of ccar1‘wt treated cells but not the ccar1‘mut treated cells suggesting a direct binding of mir‘‘3p to the '‘utr of ccar1 fig 5b increased expression levels of ccar1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues fig 5c moreover a significant decrease in both mrna and protein expression levels of ccar1 was detected upon transfecting ncih1299 and ncih460 cells with mir6273p mimics fig 5d and e thus ccar1 may be a direct target of mir‘‘3p in lung carcinoma cells and tissuesrp11‘284f219 knockdown inhibits tumor growth and the expression of ccar1 in vivo in order to investigate the effect of rp11284f219 on in vivo tumorigenicity ncih1299 cells were transfected with sinc or sirp11284f219 and injected into the nude mice after weeks a significantly 0concology reports figure rp11‘284f219 regulates proliferation and invasiveness in lung carcinoma cells via mir‘‘3p a expression of mir‘‘3p in nci‘h1299 and nci‘h460 cells transfected with mir‘nc or mir‘‘3p inhibitor was detected using rt‘qpcr analysis n3 p005 vs mir‘nc cell counting kit‘ assay was performed in b nci‘h1299 and c nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor n5 d edu assay was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor magnification x200 e flow cytometry analysis was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor n3 f transwell assay was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor magnification x200 n3 p005 vs si‘nc nc negative control sirna small interfering rna od optical density mir microrna 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure mir‘‘3p directly targets ccar1 a bioinformatic analysis of the predicted binding sites between the ccar1 '‘untranslated region and mir‘‘3p b luciferase reporter assay in ccar1‘wt or ccar1‘mut treated cells in the presence of mir‘nc or mir‘‘3p mimic n3 p005 vs mir‘nc c ccar1 expression in lc tissues compared with adjacent healthy tissues was analyzed using rt‘qpcr n13 p001 vs adjacent tissue expression of ccar1 in nci‘h1299 and nci‘h460 cells transfected with mir‘nc or mir‘‘3p mimics was detected using d rt‘qpcr and e western blotting n3 p005 vs mir‘nc mir microrna nc negative control wt wild‘type mut mutant rt‘qpcr reverse transcription‘quantitative pcr ccar1 cell division cycle and apoptosis regulator lc lung carcinoma slower proliferative rate of the tumors was observed in the sirp11284f219 group compared with the sinc group fig 6a and b furthermore the tumor volume and weight were significantly decreased in the si‘rp11‘284f219 group compared with the control group fig 6a and b rtqpcr analysis also demonstrated that compared with the sinc group the tumors in the si‘rp11‘284f219 group expressed higher levels of mir6273p fig 6c and lower levels of ccar1 fig 6d providing further evidence to the existence of the rp11‘284f219mir‘‘3pccar1 regulatory axis in lung carcinoma tumor tissuesdiscussionthe present study investigated the function of rp11284f219 in lung carcinoma it was initially found that rp11284f219 was significantly upregulated in both lung cancer tissues and cell lines following the deduction of a potential oncogenic role of this lncrna sirp11284f219 was transfected into ncih460 and ncih1299 cells and it was demonstrated that knockdown of rp11‘284f219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells in the mechanistic studies using online prediction tools and in vitro assays the results indicated that mir‘‘3p directly interacts with rp11‘284f219 by binding to its '‘utrthe function of mir627 was initially reported in colorectal cancer crc padi found that when upregulated by calcitriol mir627 targets the histone demethylase jumonji domain containing 1a to increase methylation of histone h3k9 and suppresses the proliferative factors of crc cells thus inhibiting the proliferation of crc both in vitro and in vivo moreover in crc sun discovered the role of mir‘ in vitamin d‘enhanced efficacy of irinotecan via inhibition of the cytochrome p450 enzyme‘mediated intratumoral drug metabolism mir‘ is also reported to be a potential non‘invasive diagnostic marker in gastric and breast cancer types in pulmonary diseases mir627 is downregulated in patients with chronic obstructive pulmonary disease and targets the high‘mobility group box protein to inhibit its expression thus improving transforming growth factorβ1‘induced pulmonary fibrosis the present results demonstrated the inhibitory effect of rp11‘284f219 on the expression of mir‘‘3p in addition it was identified that the mir‘‘3p inhibitor can neutralize the anti‘tumor effects of rp11‘284f219 knockdown indicating that rp11‘284f219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating mir‘‘3p this anti‘tumor role of mir6273p under the regulation of rp11284f219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human crc gastric and breast cancer types 0concology reports figure rp11‘284f219 knockdown inhibits tumor growth in vivo a macroscopic image of xenografted tumors b tumor volume in nude mice injected with nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 measured over weeks n5 c weight of tumors in nude mice at weeks after injection of nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 n5 expression levels of d mir‘‘3p and e ccar1 in the tumor tissues from nude mice injected with nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 for weeks were detected using reverse transcription‘quantitative pcr n5 p005 p001 p0001 vs si‘nc mir microrna nc negative control sh short hairpin rna ccar1 cell division cycle and apoptosis regulator using the publicly available rna interaction prediction algorithms the current study identified that ccar1 which was initially shown as the target gene of mir6273p is also regulated by rp11‘284f219 furthermore the regulatory axis of rp11‘284f219mir‘‘3pccar1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model the interaction between rp11‘284f219 and mir‘‘3p and the interaction between mir‘‘3p and ccar1 were demonstrated by the dual‘luciferase assay although this method has been used to validate rna‘rna interactions in previous studies other assays such as rna pulldown and rna binding protein immunoprecipitation that would provide more direct evidence for the rnarna and rna‘protein interactions should be performedccar1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as adriamycin and etoposide subsequently kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors in human breast cancer cells as ccar1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells in crc cells ou reported that ccar1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor ccar1 is essential for the expression of wnt target genes as well as the neoplastic transformation of crc cells in gastric cancer cells researchers have revealed the cooperation between ccar1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells in lung cancer ccar1 was reported to be an effector of doxorubicin‘induced apoptosis moreover muthu demonstrated that certain chemical compounds that bind with ccar1 can increase the expression of ccar1 and induce apoptosis however a contradictory conclusion was reported in a recent study which observed that ccar1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate corroborating this finding in the current study via the targeting of mir‘‘3p the expr Answer:

Colon_Cancer

dysregulation of ribosome production can lead to a number of developmental disorderscalled ribosomopathies despite the ubiquitous requirement for these cellular machinesused in protein synthesis ribosomopathies manifest in a tissuespecific manner with manyaffecting the development of the face here we reveal yet another connection between craniofacial development and making ribosomes through the protein paired box pax9pax9 functions as an rna polymerase ii transcription factor to regulate the expression ofproteins required for craniofacial and tooth development in humans we now expand thisfunction of pax9 by demonstrating that pax9 acts outside of the cell nucleolus to regulatethe levels of proteins critical for building the small subunit of the ribosome this function ofpax9 is conserved to the anism xenopus tropicalis an established model for humanribosomopathies depletion of pax9 leads to craniofacial defects due to abnormalities inneural crest development a result consistent with that found for depletion of other ribosomebiogenesis factors this work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic developmentauthor summarywe are only beginning to understand the complex process of making human ribosomesthe cellular machines critical for all protein synthesis in humans making a ribosomerequires hundreds of regulatory factors to ensure proper cellular growth and development dysregulation of this process can lead to a number of tissue specific disorderstermed ribosomopathies here we have discovered a new role for the protein pairedbox pax9 in making human ribosomes while pax9 has traditionally been known toa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation farleybarnes ki deniz e overton mmkhokha mk baserga sj paired box pax9 the rna polymerase ii transcription factorregulates human ribosome biogenesis andcraniofacial development genet e1008967 101371 pgen1008967editor paul a trainor stowers institute formedical research united statesreceived february accepted june published august copyright farleybarnes this is anopen access distributed under the terms ofthe creative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all rnaseq andrnapii chipseq files are available from the geneexpression omnibus geo database and areaccessible through the geo series accessionnumber gse154764 wwwncbinlmnihgovgeoqueryacccgiaccgse154764 allnumerical data that underlies graphs or summarystatistics is available in the supporting informations4 table genetics 101371 pgen1008967 august genetics 0cfunding this work was supported by the nationalinstitutes of health r01gm115710r01gm122926 and r35gm131687 to sjbr01hd081379 to mkk t32gm007223 to sjb andkif f31de026946 to kif and a pilot grant from theyale cancer center to sjb the funders had no rolein study design data collection and analysisdecision to publish or preparation of themanuscriptcompeting interests the authors have declaredthat no competing interests existpaired box pax9 regulates human ribosome biogenesisplay a role in regulating the levels of proteins required for craniofacial and tooth development in humans we expand this function of pax9 by showing that pax9 acts outside ofthe cell nucleolus to regulate the levels of proteins critical for building the small subunit ofthe ribosome in addition we show that this function is conserved to the model anismxenopus tropicalis this link between pax9™s role in craniofacial development and inribosome biogenesis may lead to new insights into the pathogenesis of these pax9 mutations in humansintroductionwhen ribosome biogenesis is genetically disrupted in humans a number of surprisingly tissuespecific disorders called ribosomopathies arise for example genetic disruption of thetcof1 polr1c or polr1d genes in the ribosomopathy treacher collins syndrome tcsomim results in reduced preribosomal rna prerrna transcription [“] interestingly while these mutations each affect the global process of prerrna transcriptionpatients have specific defects in craniofacial development tcs patients present with hypoplasia of the facial bones micrognathia with or without cleft palate narrowing of the ear canaland bilateral conductive hearing loss [“] modeling the disease in mice has shown that thistissue specificity arises from differential tissue susceptibility to p53 levels p53 levels are stabilized upon disruptions in ribosome biogenesis when free ribosomal proteins bind to mdm2the e3 ligase for p53 [ ] this stabilization of p53 leads to apoptosis of the developing neural crest cells ultimately resulting in the mandibulofacial dysostosis of tcstcs is not the only ribosomopathy to affect craniofacial development for example diamond blackfan anemia dba omim is characterized by anemia low reticulocytecount and elevated erythrocyte adenosine deaminase activity [ ] however dba patientsoften also have craniofacial anomalies and cleft palate reviewed in additionally theribosomopathy acrofacial dysostosis cincinnati type omim is caused by mutationsin polr1a that inhibit prerrna transcription resulting in craniofacial defects amore precise understanding of the role of the factors involved in human ribosome biogenesiscan therefore shed light on the molecular mechanisms underlying aberrant craniofacialdevelopmentthe process of making the cellular machines required for protein synthesis called ribosomebiogenesis includes a large number of factors that work together to control cell growth anddevelopment in humans these proteins are still being defined previous studies have shownthat ribosome biogenesis begins with the transcription of the tandemly repeated ribosomaldna rdna into the 47s polycistronic prerrna fig 1a the prerrna is further modified and processed to create the mature 18s 58s and 28s rrnas these rrnas are incorporated along with the 5s rrna and ribosomal proteins into the small ssu and large lsusubunits of the ribosome the 47s prerrna is transcribed by rna polymerase i rnapiwhile the 5s rrna is transcribed by rna polymerase iii rnapiii various assembly factorsand the ribosomal proteins are transcribed by rna polymerase ii rnapii in addition torequiring all rna polymerases synthesis and assembly of functional ribosomes integrates anumber of cellular signaling pathways to ensure proper regulation of this essential process inresponse to stimulithe complex development of the face is controlled by a number of proteins including several rnapii transcription factors such as paired box pax9 pax9 belongs to a family oftranscription factors that play key roles in anogenesis and neural crest cell development by genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisfig pax9 is required for human ribosome biogenesis a ribosome biogenesis at a glance the tandemly repeated ribosomal dna rdna istranscribed into the 47s polycistronic preribosomal rna prerrna by rna polymerase i rnapi this 47s prerrna is processed throughmultiple steps to form the mature 18s 58s and 28s rrnas which are incorporated into the small and large subunits of the ribosome along with the5s rrna and ribosomal proteins ribosomes perform cytoplasmic cellular protein synthesis through the translation of mrnas b pax9 depletionreduces nucleolar number from “ to only in mcf10a cells left panel nuclei stained in hoechst are shown in blue nucleoli are shown in pinkand stained with antifibrillarin antibody as in sigfp top was used as a negative control “ nucleolinucleus and siutp4 middle was usedas a positive control nucleolusnucleus sipax9 is shown at the bottom right panel quantitation of the number of nucleoli per nucleus for sigfptop siutp4 middle or sipax9 bottom c pax9 is not required for rnapi transcription in mcf10a cells a dualluciferase reporter assay wasused to quantify luminescence after sirna depletion of pax9 the plasmids are phrdiresluc firefly to report rnapi transcription and arenilla transfection control as in the ratio of firefly to renilla luciferase was normalized to the sint control n sinol11 was used as apositive control data were analyzed by student™s t test using graphpad prism ���� p � d pax9 is required for pre18s rrnaprocessing in mcf10a cells left schematic of prerrna processing steps in human cells intermediates detected by probe p3 are indicated with ablack box below center northern blot with probe p3 a probe for the 7sl rna was used as a loading control intermediates detected by probe p3 areshown to the right of the northern blot negative controls were mock no sirna and sint nontargeting siutp4 was used as a positive control right quantitation by ramp of probe p3 upper and 7sl lower northern blots graph is mean ± sem n data were analyzed by2way anova using graphpad prism ���� p � ��� p � �� p � and � p � ptp indicates the 47s 45s and 43s processingintermediates e pax9 sirna depletion in mcf10a cells results in an increased ratio of 28s18s by agilent bioanalyzer significance was calculatedby student™s t test in graphpad prism where �� p � f pax9 sirna depletion in mcf10a cells results in decreased global protein synthesis asassessed by the puromycin incorporation assay a representative western blot using an antipuromycin antibody with a β actin loading control isshown to the left protein was harvested after knockdown for hours using the indicated sirnas mock indicates no sirna and mock μmindicates no sirna and half the concentration of puromycin sint nontargeting was used a positive control quantitation of replicates using cellsof different passage numbers is shown to the right significance was calculated by oneway anova in graphpad prism where ���� p � and��� p � g pax9 depletion in mcf10a cells results in decreased 40s 60s and 80s ribosome subunit levels representative polysome profile ofmcf10a cells depleted using sirnas targeting either pax9 red or a nontargeting sint blue control equal amounts of protein were loaded oneach gradient this experiment was performed times using cells of different passage numbers101371 pgen1008967g001controlling gene expression [ ] in humans mutations in pax9 cause tooth agenesis aswell as hair loss [“] and reviewed in indeed pax9 mutations are the most prevalent mutation in patients with nonsyndromic tooth agenesis including oligodontia additionally mice homozygous for a partial deletion of pax9 likely null have craniofacialmalformations including cleft palate skeletal abnormalities and arrested tooth developmentand die a few hours after birth pax9 mice also exhibit a range of cardiac malformations another commonly impacted tissue in ribosomopathies while some research hasbeen done to identify the signaling pathways regulated by pax9 attempts to correct the developmental defects have been only partially successful [“] therefore further studies areneeded to identify all factors regulated by pax9 in order to understand pax9™s role in craniofacial developmentour work ties together pax9 and ribosome biogenesis filling in some gaps in our knowledge of the many cell growth and signaling pathways influenced by pax9 depletion we originally identified pax9 as a potential regulator of ribosome biogenesis in an sirna screen forproteins required to maintain nucleolar number probing pax9™s specific role in makingribosomes in human tissue culture cells we discovered that pax9 is required both for the prerrna processing that produces the small subunit 18s rrna and for global protein synthesiswe employed the genomewide transcriptomics analysis rnaseq to define pax9 dependentmrnas necessary for making ribosomes several of the differentially expressed mrnasincluding several ribosomal proteins were further examined to pinpoint roles for these proteins in prerrna processing and global protein synthesis finally given an established rolefor pax9 in human craniofacial development we sought to model this disease in xenopus tropicalis x tropicalis embryos an established model of human ribosomopathies depletion ofpax9 in x tropicalis did indeed alter craniofacial patterning as well as neural crest development a migratory cell population that plays a major role in establishing craniofacial structurex tropicalis embryos depleted of pax9 also show defective prerrna processing these resultsshed light on the plethora of factors whose expression is regulated by pax9 and open the doorto likely connections between pax9™s role in craniofacial development and human ribosomebiogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisresultspax9 depletion disrupts small subunit ribosome biogenesispreviously we performed an sirna screen for new regulators of nucleolar number in humanmcf10a cells we identified pax9 by this screening approach as a protein that whendepleted reduced the number of nucleoli per nucleus from “ to only fig 1b as in cells were fixed after hours of knockdown using pools of sirnas targeting sigfp as a negative control siutp4 as a positive control or sipax9 this transient knockdown approach successfully depleted pax9 s1a and s1b fig the mcf10a cells were then stained with anantibody to fibrillarin fbl a nucleolar protein to detect nucleoli and with hoechst todetect nuclei a cellprofiler pipeline was used to quantify the number of nucleoli per cellnucleus which shifted from “ in sigfp control cells to only in sipax9 and siutp4treatedcells we had previously used oligonucleotide deconvolution to additionally confirm thatpax9 depletion leads to reductions in nucleolar number using this approach individualdepletion of pax9 using of the sirnas from the original pax9 pool reduced the numberof nucleoli from “ to only per cell nucleus [ ] as the sirna screen served as a phenotypic readout of nucleolar function we hypothesized that pax9 plays a role in humanribosome biogenesis through its function as an rnapii transcription factorwe sought to investigate the extent to which pax9 depletion affects human ribosome biogenesis using a panel of assays the first assay probes pax9™s role in rnapi transcriptionusing a dualluciferase reporter system previously published by our laboratory and others [ ] as pax9 is a known rnapii transcription factor it was relevant to test it for a possibleadditional role in rnapi transcription in this reporter assay the ratio of firefly luciferasewhich is under the control of the rdna promoter and measures rnapi transcription wasquantified relative to a renilla luciferase transfection control relative to a nontargeting control sirna sint sirnas targeting pax9 had no significant effect on rnapi transcriptionlevels after hours of knockdown in mcf10a cells fig 1c mock no sirna and sinol11were used as negative and positive controls respectively pax9 is therefore not required forrnapi transcription in mcf10a cellsnorthern blotting was used to define pax9™s role in prerrna processing in humans prerrna processing occurs via a number of different pathways fig 1d left and requires a number of transacting factors we therefore employed different probes to pinpoint any prerrna processing defects occurring after pax9 depletion in mcf10a cells s2a fig after hours of sirna knockdown pax9 depletion resulted in a significant increase in the 30s prerrna intermediate as well as a decrease in the levels of its 21s processing product relative tothe nontargeting sirna sint fig 1d middle and right and s2 fig additionally 41s levelswere decreased relative to the primary processing transcript 47s plus the 45s and 43s processing intermediates herein termed the primary transcript plus or ptp fig 1d middle andright and s2 fig quantitation of the ratios of each intermediate relative to its precursor in theprocessing pathway by ratio analysis of multiple precursors ramp confirmed the statistical significance of these results fig 1d right and s2 fig these effects were also significant relative to a 7sl loading control fig 1d right and s2 fig because the 30s and 21sintermediates are both precursors to the 18s rrna pax9 is required for ssu biogenesis thesame prerrna processing defect was also detected in human embryonic kidney hek293ftand colon carcinoma rko cells depleted of pax9 indicating that pax9™s role in ribosomebiogenesis is conserved among diverse human cell lines s1 figbecause the prerrna processing defects indicate aberrant ssu biogenesis we sought todetermine the extent to which pax9 depletion affects the production of the mature 18s rrnaagilent bioanalyzer quantitation shows an increase in the ratio of 28s to 18s fig 1e genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesiscombined with the northern blot results indicating defects in the processing of the precursorsto the 18s rrna this result is consistent with a predicted reduction in 18s rrna levels takentogether these results argue that pax9 is required likely indirectly for the biogenesis of thesmall subunit of the ribosome which contains the 18s rrnawe also utilized a puromycin incorporation assay to test the extent to which pax9 depletion alters the final product of ribosome biogenesis global cellular translation fig 1f cellswith or without pax9 depletion are treated with a low dose μm of puromycin which isincorporated into all nascent peptides produced during a hour pulse western blottingfor incorporated puromycin shows decreased protein synthesis after hours of pax9 depletion relative to a nontargeting sirna control sint fig 1f mock μm puromycin withno sirna and mock at a halfdose of puromycin μm were used as negative controls fig1f these results confirm that pax9 depletion leads to reduced protein synthesis consistentwith a role for pax9 in ssu biogenesisbecause the above assays indicated a role for pax9 in small subunit prerrna processingand global protein synthesis we also tested the extent to which pax9 depletion is required forribosomal subunit biogenesis and assembly using polysome profiling we determined thatpax9 depletion does result in significantly decreased ssu 40s levels in mcf10a cells fig1g consistent with the reduction in 18s rrna levels seen in fig 1e additionally 60s and80s polysome fractions also showed significant decreases after pax9 knockdown fig 1ginterestingly both here and in previous experiments mcf10a cells do not demonstraterobust polysome fractions using this technique regardless we conclude that pax9 depletion results in decreased ssu biogenesis that impacts the assembly and function of theribosomeas disruptions in ribosome biogenesis result in interruptions in the cell cycle [“] wealso examined the extent to which pax9 depletion changed the distribution of mcf10a cellswithin the cell cycle using flow cytometry s3 fig relative to a sint control depletion ofpax9 for hours resulted in a minor increase in the proportion of cells in g1 phase of thecell cycle but this was not statistically significant s3 fig sirnas targeting the ribosome biogenesis factor nol11 were used as a positive control and depletion of this protein resulted inan increase in the proportion of cells in g2 s3 fig consistent with previous findings inall these assays allowed us to conclude that pax9 regulates human ribosome biogenesisrnaseq analysis upon pax9 depletion reveals decreased levels ofnucleolar mrnas responsible for small subunit maturationas pax9 is a known rnapii transcription factor we hypothesized that pax9 works indirectlyto modulate ssu biogenesis fig 2a this is consistent with a nuclear but not nucleolar localization of pax9 in existing databases [“] pax9 may act directly as a transcription factorfor nucleolar proteins or indirectly for proteins that affect the expression or function of nucleolar proteins to test the hypothesis that pax9 affects nucleolar protein expression through itsfunction as a rnapii transcription factor we used rnaseq in mcf10a cells to define the setof mrnas that were differentially expressed after pax9 depletion relative to a nontargetingcontrol sirna sint pax9 depletion resulted in the differential expression of over mrnas fold change � or � q � fig 2b and s1 table approximately half of these were reduced in their levels consistent with the hypothesis that pax9 acts as a transcription factor to drive their expressionwhen considered as a whole the rnaseq dataset is enriched for several pathways knownto be regulated by pax9 s4a fig ingenuity pathway analysis of the differentiallyexpressed mrnas reveals enrichment of both wntca2 signaling differential expression of genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisfig rnaseq transcriptomics analysis in human tissue culture cells reveals changes in the expression levels of over nucleolar mrnas afterpax9 knockdown a schematic of how pax9 would act as a rnapii transcription factor to drive the levels of mrnas required for making the smallsubunit ssu of the ribosome in the cell nucleus pax9 binds to dna to affect the transcription of mrnas that either encode nucleolar proteinsdirect solid arrow or to transcribe mrnas that affect the levels of mrnas encoding nucleolar proteins indirect dotted arrows the resulting mrnasare translated in the cytoplasm into proteins that function in ssu prerrna processing in the nucleolus b rnaseq analysis after pax9 sirnadepletion in mcf10a cells reveals decreased levels of mrnas encoding nucleolar proteins relative to a nontargeting sirna control sint pax9depletion resulted in differential expression of mrnas fold change � or and fdr � of these mrnas had a decreased foldchange � and of those mrnas code for proteins designated as nucleolar in at least one of three databases [“] of the mrnas whoselevels were decreased and that also code for nucleolar proteins were chosen as candidates for followup studies c qrtpcr confirms reduced mrnalevels of the rnaseq candidates after pax9 sirna knockdown in mcf10a cells after depletion using sirnas targeting either pax9 or a nontargeting control sirna sint the levels of the indicated mrnas were quantified by qrtpcr using primers to each target gene relative to a 7slcontrol and sint data are shown as mean ± sem three replicates using cells of different passage numbers with technical replicates each wereperformed significance was calculated by oneway anova using graphpad prism where ���� p � d depletion of of the candidatemrnas rps6es6 rps9us4 rps28es28 and fbl individually results in the same prerrna processing defect as pax9 sirna depletion in mcf10acells representative northern blot after knockdown of the indicated sirnas using probe p3 a probe for the 7sl rna was used as a loading control prerrna processing intermediates detected by probe p3 are shown to the right of the northern blot ptp indicates the 47s 45s and 43s prerrnaprocessing intermediates e quantitation of northern blots using probe p3 as shown in fig 2d using ramp graph is mean ± sem n datawere analyzed using 2way anova in graphpad prism where ���� p � ��� p � and �� p � quantitation relative to the 7sl loadingcontrol is shown in s5 fig f sirna depletion of rnaseq candidates in mcf10a cells results in decreased global protein synthesis after hoursof knockdown with the indicated sirnas mcf10a cells were pulsed with puromycin for hour and protein was harvested western blotting with anantipuromycin antibody as well as a β actin loading control was carried out representative western blots shown to the left mock mock at half theconcentration of puromycin μm and sint nontargeting sirnas were used as negative controls g quantitation of three replicates usingmcf10a cells of different passage numbers of the puromycin incorporation assays following depletion with the indicated sirnas relative to the sint andβ actin loading controls is shown as mean ± sem n significance was calculated by student™s ttest using graphpad prism where ���� p � ���p � and � p � 101371 pgen1008967g002 pathway members p x ˆ’ s4b fig and wntβcatenin signaling differentialexpression of pathway members p x ˆ’ s4c fig in cells depleted of pax9expression levels are increased for many of the mrnas in the wnt signaling pathway this isconsistent with previous results suggesting a role for pax9 in the negative regulation of wntsignaling [ ]as an additional validation of the rnaseq dataset we determined that the genomesequences kb upstream of the start sites of the differentially expressed mrnas containmultiple potential pax9 binding sites making these mrnas candidates for direct transcriptional regulation by pax9 scanning for known pax9 binding sequences [™sgtcacgcwtgantgma3™ ™cgcgtgaccg3™ cd192ains ™gcgtgacca3™ and e5 ™gcggaacgg3™] in the kb upstream of the mrnas using centrimo analysis reveals and potential pax9 binding sites respectively in the kbupstream of the mrnas this number of potential binding sites upstream of the mrnas sites per gene is similar to that observed in pax9 chip experiments in the vertebral column of e125 mice sites per gene additionally centrimo enrichmentanalysis of the sequence kb upstream of each of the differentially expressedmrnas reveals significant enrichment of different dna binding sequences including thepax3 pax5 pax6 and pax7 dna binding domains as multiple pax proteins can bindthe same dna sequence this provides further evidence for pax9 regulation of these mrnas these analyses therefore support the hypothesis that pax9 regulates the levels of themrnas identified in our rnaseq datasetin the rnaseq dataset many of the differentially expressed mrnas have knownroles in nucleolar function for example have appeared in other genomewidesirna screens for nucleolar function s1 table [ ] additionally of the differentially expressed mrnas code for proteins designated as nucleolar in at least of nucleolar databases s1 table [“] this is a significant enrichment in the expected number of nucleolar proteins assuming that nucleolar proteins make up only of the proteins inhuman cells surprisingly expression of most of the mrnas encoding nucleolar proteins genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis was downregulated upon pax9 depletion indicating that pax9 is requiredto maintain normal levels of many mrnas whose protein products are destined for functionin the nucleolus s1 tableto determine the mechanism of pax9™s function in ssu biogenesis we chose candidatesfrom the rnaseq dataset to follow up on in greater detail the mrna levels for the candidates were all downregulated after pax9 depletion and all code for nucleolar proteins fig 2band s1 table [“] four of the candidates rps6es6 rps9us4 rps28es28 and fblwere chosen on the basis of literature suggesting a role for these proteins in ssu prerrnaprocessing in hela cells [ ] additionally it was pertinent to analyze rpl5ul18 as it hasa known role in the p53dependent nucleolar stress response qrtpcr confirmed thernaseq results with reduced levels of each mrna when pax9 is depleted in mcf10a cellsfig 2c interestingly depletion of either rps9us4 or rps28es28 resulted in a decrease innucleolar number from “ to only in our original sirna screen therefore it is possible that the mechanism through which pax9 depletion results in decreased nucleolar numberrelies upon reduced expression of rps9us4 andor rps28es28 fig 2awe were able to confirm that depletion of of the tested candidates rps6es6 rps9us4 rps28es28 and fbl in mcf10a cells resulted in prerrna processing defects similarto that of pax9 depletion fig 2d and 2e s5 fig only rpl5ul18 did not give the 30sincrease characteristic of pax9 depletion although this was expected given its known role inlsu prerrna processing additionally depletion of several of the candidates individually resulted in significantly decreased global protein synthesis by the puromycin incorporation assay similar to the effect seen after pax9 depletion fig 2f and 2g puromycinincorporation was also reduced after rps6es6 depletion although it was not statistically significant fig 2f and 2g therefore pax9 may function as a transcription factor to directly orindirectly increase the expression of rps6es6 rps9us4 rps28es28 andor fbl fig 2aas the proteins encoded by these mrnas are required for ssu ribosome biogenesis fig their depletion after pax9 knockdown is a plausible mechanism through which pax9 regulates prerrna processing and global protein synthesisrnapii chipseq analysis reveals decreased transcription of mrnasencoding nucleolar proteins after pax9 depletionas the rnaseq analysis confirmed that levels of nucleolar mrnas were decreased afterpax9 depletion fig we sought to map how rnapii distributes on genes using rnapiichipseq as a readout of transcription through this approach we aimed to untangle the effectsof pax9 depletion on rnapii transcription from its effects on mrna stability since rnapiichipseq is able to detect genomewide changes in rnapii occupancy as pax proteins areable to both activate and repress target protein expression we have included genes withboth increased and decreased rnapii occupancy in this analysis approximately mrnaswere differentially occupied by rnapii upon pax9 knockdown compared to the nontargeting control sirna sint in mcf10a cells fold change cutoff � or � and maxtags � s2 table of these had decreased rnapii occupancy consistent with pax9 acting as a transcriptional driver of these mrnasto assess the validity of the rnapii chipseq dataset we again used centrimo to identifypotential pax9 dnabinding sites in the kb upstream of the genes with differentialrnapii occupancy searching for the ™ cgcgtgaccg ™ pax9 binding motif definedin revealed possible sites in the bp upstream of the differentially occupiedgenes also the known pax9 dna binding motifs cd192ains ™gcgtgacca3™ ande5 ™gcggaacgg3™ had and binding sites in these sequences respectively genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisadditionally analysis of motif enrichment ame identified the pax5 and pax6 dnabinding motifs as being significantly enriched in the kb of sequence upstream of the genes p � since multiple pax proteins can bind the same motif this suggests thatthis dataset does contain mrnas that are regulated by pax9 of the differentially occupied genes have also been shown to be differentially regulated by pax9 directly in pax9chipseq experiments on e125 wt vertebral column murine tissue fig 3a and s2 table these analyses confirm the ability of rnapii chipseq to detect changes in rnapiimediated transcription after pax9 knockdownbased on the hypothesis that pax9 acts as an rnapii transcription factor for regulators ofnucleolar function fig we expected to detect changes in the rnapiimediated transcription of a number of mrnas encoding nucleolar proteins after pax9 depletion indeedmrnas coding for nucleolar proteins were enriched with of the genes with differentialrnapii occupancy coding for nucleolar proteins in at least one of three databases s2table [“] this is again higher than would be expected assuming that nucleolar proteins account for approximately of all cellular proteins additionally depletion of onegene with differential rnapii occupancy anln resulted in decreased nucleolar number inour sirna screen similar to the phenotype seen after pax9 depletion fig 1b notably of the genes with differential rnapii occupancy appeared in other genomewide screens for ribosome biogenesis factors s2 table [ ] another targeted screeninvestigated the effects of of the genes top2a and cdca8 on prerrna processingwhen depleted by sirna in hela cells however depletion of neither gave the 30s prerrna increase on northern blots characteristic of pax9 depletion in al

cancer7549

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: dysregulation of ribosome production can lead to a number of developmental disorderscalled ribosomopathies despite the ubiquitous requirement for these cellular machinesused in protein synthesis ribosomopathies manifest in a tissuespecific manner with manyaffecting the development of the face here we reveal yet another connection between craniofacial development and making ribosomes through the protein paired box pax9pax9 functions as an rna polymerase ii transcription factor to regulate the expression ofproteins required for craniofacial and tooth development in humans we now expand thisfunction of pax9 by demonstrating that pax9 acts outside of the cell nucleolus to regulatethe levels of proteins critical for building the small subunit of the ribosome this function ofpax9 is conserved to the anism xenopus tropicalis an established model for humanribosomopathies depletion of pax9 leads to craniofacial defects due to abnormalities inneural crest development a result consistent with that found for depletion of other ribosomebiogenesis factors this work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic developmentauthor summarywe are only beginning to understand the complex process of making human ribosomesthe cellular machines critical for all protein synthesis in humans making a ribosomerequires hundreds of regulatory factors to ensure proper cellular growth and development dysregulation of this process can lead to a number of tissue specific disorderstermed ribosomopathies here we have discovered a new role for the protein pairedbox pax9 in making human ribosomes while pax9 has traditionally been known toa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation farleybarnes ki deniz e overton mmkhokha mk baserga sj paired box pax9 the rna polymerase ii transcription factorregulates human ribosome biogenesis andcraniofacial development genet e1008967 101371 pgen1008967editor paul a trainor stowers institute formedical research united statesreceived february accepted june published august copyright farleybarnes this is anopen access distributed under the terms ofthe creative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all rnaseq andrnapii chipseq files are available from the geneexpression omnibus geo database and areaccessible through the geo series accessionnumber gse154764 wwwncbinlmnihgovgeoqueryacccgiaccgse154764 allnumerical data that underlies graphs or summarystatistics is available in the supporting informations4 table genetics 101371 pgen1008967 august genetics 0cfunding this work was supported by the nationalinstitutes of health r01gm115710r01gm122926 and r35gm131687 to sjbr01hd081379 to mkk t32gm007223 to sjb andkif f31de026946 to kif and a pilot grant from theyale cancer center to sjb the funders had no rolein study design data collection and analysisdecision to publish or preparation of themanuscriptcompeting interests the authors have declaredthat no competing interests existpaired box pax9 regulates human ribosome biogenesisplay a role in regulating the levels of proteins required for craniofacial and tooth development in humans we expand this function of pax9 by showing that pax9 acts outside ofthe cell nucleolus to regulate the levels of proteins critical for building the small subunit ofthe ribosome in addition we show that this function is conserved to the model anismxenopus tropicalis this link between pax9™s role in craniofacial development and inribosome biogenesis may lead to new insights into the pathogenesis of these pax9 mutations in humansintroductionwhen ribosome biogenesis is genetically disrupted in humans a number of surprisingly tissuespecific disorders called ribosomopathies arise for example genetic disruption of thetcof1 polr1c or polr1d genes in the ribosomopathy treacher collins syndrome tcsomim results in reduced preribosomal rna prerrna transcription [“] interestingly while these mutations each affect the global process of prerrna transcriptionpatients have specific defects in craniofacial development tcs patients present with hypoplasia of the facial bones micrognathia with or without cleft palate narrowing of the ear canaland bilateral conductive hearing loss [“] modeling the disease in mice has shown that thistissue specificity arises from differential tissue susceptibility to p53 levels p53 levels are stabilized upon disruptions in ribosome biogenesis when free ribosomal proteins bind to mdm2the e3 ligase for p53 [ ] this stabilization of p53 leads to apoptosis of the developing neural crest cells ultimately resulting in the mandibulofacial dysostosis of tcstcs is not the only ribosomopathy to affect craniofacial development for example diamond blackfan anemia dba omim is characterized by anemia low reticulocytecount and elevated erythrocyte adenosine deaminase activity [ ] however dba patientsoften also have craniofacial anomalies and cleft palate reviewed in additionally theribosomopathy acrofacial dysostosis cincinnati type omim is caused by mutationsin polr1a that inhibit prerrna transcription resulting in craniofacial defects amore precise understanding of the role of the factors involved in human ribosome biogenesiscan therefore shed light on the molecular mechanisms underlying aberrant craniofacialdevelopmentthe process of making the cellular machines required for protein synthesis called ribosomebiogenesis includes a large number of factors that work together to control cell growth anddevelopment in humans these proteins are still being defined previous studies have shownthat ribosome biogenesis begins with the transcription of the tandemly repeated ribosomaldna rdna into the 47s polycistronic prerrna fig 1a the prerrna is further modified and processed to create the mature 18s 58s and 28s rrnas these rrnas are incorporated along with the 5s rrna and ribosomal proteins into the small ssu and large lsusubunits of the ribosome the 47s prerrna is transcribed by rna polymerase i rnapiwhile the 5s rrna is transcribed by rna polymerase iii rnapiii various assembly factorsand the ribosomal proteins are transcribed by rna polymerase ii rnapii in addition torequiring all rna polymerases synthesis and assembly of functional ribosomes integrates anumber of cellular signaling pathways to ensure proper regulation of this essential process inresponse to stimulithe complex development of the face is controlled by a number of proteins including several rnapii transcription factors such as paired box pax9 pax9 belongs to a family oftranscription factors that play key roles in anogenesis and neural crest cell development by genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisfig pax9 is required for human ribosome biogenesis a ribosome biogenesis at a glance the tandemly repeated ribosomal dna rdna istranscribed into the 47s polycistronic preribosomal rna prerrna by rna polymerase i rnapi this 47s prerrna is processed throughmultiple steps to form the mature 18s 58s and 28s rrnas which are incorporated into the small and large subunits of the ribosome along with the5s rrna and ribosomal proteins ribosomes perform cytoplasmic cellular protein synthesis through the translation of mrnas b pax9 depletionreduces nucleolar number from “ to only in mcf10a cells left panel nuclei stained in hoechst are shown in blue nucleoli are shown in pinkand stained with antifibrillarin antibody as in sigfp top was used as a negative control “ nucleolinucleus and siutp4 middle was usedas a positive control nucleolusnucleus sipax9 is shown at the bottom right panel quantitation of the number of nucleoli per nucleus for sigfptop siutp4 middle or sipax9 bottom c pax9 is not required for rnapi transcription in mcf10a cells a dualluciferase reporter assay wasused to quantify luminescence after sirna depletion of pax9 the plasmids are phrdiresluc firefly to report rnapi transcription and arenilla transfection control as in the ratio of firefly to renilla luciferase was normalized to the sint control n sinol11 was used as apositive control data were analyzed by student™s t test using graphpad prism ���� p � d pax9 is required for pre18s rrnaprocessing in mcf10a cells left schematic of prerrna processing steps in human cells intermediates detected by probe p3 are indicated with ablack box below center northern blot with probe p3 a probe for the 7sl rna was used as a loading control intermediates detected by probe p3 areshown to the right of the northern blot negative controls were mock no sirna and sint nontargeting siutp4 was used as a positive control right quantitation by ramp of probe p3 upper and 7sl lower northern blots graph is mean ± sem n data were analyzed by2way anova using graphpad prism ���� p � ��� p � �� p � and � p � ptp indicates the 47s 45s and 43s processingintermediates e pax9 sirna depletion in mcf10a cells results in an increased ratio of 28s18s by agilent bioanalyzer significance was calculatedby student™s t test in graphpad prism where �� p � f pax9 sirna depletion in mcf10a cells results in decreased global protein synthesis asassessed by the puromycin incorporation assay a representative western blot using an antipuromycin antibody with a β actin loading control isshown to the left protein was harvested after knockdown for hours using the indicated sirnas mock indicates no sirna and mock μmindicates no sirna and half the concentration of puromycin sint nontargeting was used a positive control quantitation of replicates using cellsof different passage numbers is shown to the right significance was calculated by oneway anova in graphpad prism where ���� p � and��� p � g pax9 depletion in mcf10a cells results in decreased 40s 60s and 80s ribosome subunit levels representative polysome profile ofmcf10a cells depleted using sirnas targeting either pax9 red or a nontargeting sint blue control equal amounts of protein were loaded oneach gradient this experiment was performed times using cells of different passage numbers101371 pgen1008967g001controlling gene expression [ ] in humans mutations in pax9 cause tooth agenesis aswell as hair loss [“] and reviewed in indeed pax9 mutations are the most prevalent mutation in patients with nonsyndromic tooth agenesis including oligodontia additionally mice homozygous for a partial deletion of pax9 likely null have craniofacialmalformations including cleft palate skeletal abnormalities and arrested tooth developmentand die a few hours after birth pax9 mice also exhibit a range of cardiac malformations another commonly impacted tissue in ribosomopathies while some research hasbeen done to identify the signaling pathways regulated by pax9 attempts to correct the developmental defects have been only partially successful [“] therefore further studies areneeded to identify all factors regulated by pax9 in order to understand pax9™s role in craniofacial developmentour work ties together pax9 and ribosome biogenesis filling in some gaps in our knowledge of the many cell growth and signaling pathways influenced by pax9 depletion we originally identified pax9 as a potential regulator of ribosome biogenesis in an sirna screen forproteins required to maintain nucleolar number probing pax9™s specific role in makingribosomes in human tissue culture cells we discovered that pax9 is required both for the prerrna processing that produces the small subunit 18s rrna and for global protein synthesiswe employed the genomewide transcriptomics analysis rnaseq to define pax9 dependentmrnas necessary for making ribosomes several of the differentially expressed mrnasincluding several ribosomal proteins were further examined to pinpoint roles for these proteins in prerrna processing and global protein synthesis finally given an established rolefor pax9 in human craniofacial development we sought to model this disease in xenopus tropicalis x tropicalis embryos an established model of human ribosomopathies depletion ofpax9 in x tropicalis did indeed alter craniofacial patterning as well as neural crest development a migratory cell population that plays a major role in establishing craniofacial structurex tropicalis embryos depleted of pax9 also show defective prerrna processing these resultsshed light on the plethora of factors whose expression is regulated by pax9 and open the doorto likely connections between pax9™s role in craniofacial development and human ribosomebiogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisresultspax9 depletion disrupts small subunit ribosome biogenesispreviously we performed an sirna screen for new regulators of nucleolar number in humanmcf10a cells we identified pax9 by this screening approach as a protein that whendepleted reduced the number of nucleoli per nucleus from “ to only fig 1b as in cells were fixed after hours of knockdown using pools of sirnas targeting sigfp as a negative control siutp4 as a positive control or sipax9 this transient knockdown approach successfully depleted pax9 s1a and s1b fig the mcf10a cells were then stained with anantibody to fibrillarin fbl a nucleolar protein to detect nucleoli and with hoechst todetect nuclei a cellprofiler pipeline was used to quantify the number of nucleoli per cellnucleus which shifted from “ in sigfp control cells to only in sipax9 and siutp4treatedcells we had previously used oligonucleotide deconvolution to additionally confirm thatpax9 depletion leads to reductions in nucleolar number using this approach individualdepletion of pax9 using of the sirnas from the original pax9 pool reduced the numberof nucleoli from “ to only per cell nucleus [ ] as the sirna screen served as a phenotypic readout of nucleolar function we hypothesized that pax9 plays a role in humanribosome biogenesis through its function as an rnapii transcription factorwe sought to investigate the extent to which pax9 depletion affects human ribosome biogenesis using a panel of assays the first assay probes pax9™s role in rnapi transcriptionusing a dualluciferase reporter system previously published by our laboratory and others [ ] as pax9 is a known rnapii transcription factor it was relevant to test it for a possibleadditional role in rnapi transcription in this reporter assay the ratio of firefly luciferasewhich is under the control of the rdna promoter and measures rnapi transcription wasquantified relative to a renilla luciferase transfection control relative to a nontargeting control sirna sint sirnas targeting pax9 had no significant effect on rnapi transcriptionlevels after hours of knockdown in mcf10a cells fig 1c mock no sirna and sinol11were used as negative and positive controls respectively pax9 is therefore not required forrnapi transcription in mcf10a cellsnorthern blotting was used to define pax9™s role in prerrna processing in humans prerrna processing occurs via a number of different pathways fig 1d left and requires a number of transacting factors we therefore employed different probes to pinpoint any prerrna processing defects occurring after pax9 depletion in mcf10a cells s2a fig after hours of sirna knockdown pax9 depletion resulted in a significant increase in the 30s prerrna intermediate as well as a decrease in the levels of its 21s processing product relative tothe nontargeting sirna sint fig 1d middle and right and s2 fig additionally 41s levelswere decreased relative to the primary processing transcript 47s plus the 45s and 43s processing intermediates herein termed the primary transcript plus or ptp fig 1d middle andright and s2 fig quantitation of the ratios of each intermediate relative to its precursor in theprocessing pathway by ratio analysis of multiple precursors ramp confirmed the statistical significance of these results fig 1d right and s2 fig these effects were also significant relative to a 7sl loading control fig 1d right and s2 fig because the 30s and 21sintermediates are both precursors to the 18s rrna pax9 is required for ssu biogenesis thesame prerrna processing defect was also detected in human embryonic kidney hek293ftand colon carcinoma rko cells depleted of pax9 indicating that pax9™s role in ribosomebiogenesis is conserved among diverse human cell lines s1 figbecause the prerrna processing defects indicate aberrant ssu biogenesis we sought todetermine the extent to which pax9 depletion affects the production of the mature 18s rrnaagilent bioanalyzer quantitation shows an increase in the ratio of 28s to 18s fig 1e genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesiscombined with the northern blot results indicating defects in the processing of the precursorsto the 18s rrna this result is consistent with a predicted reduction in 18s rrna levels takentogether these results argue that pax9 is required likely indirectly for the biogenesis of thesmall subunit of the ribosome which contains the 18s rrnawe also utilized a puromycin incorporation assay to test the extent to which pax9 depletion alters the final product of ribosome biogenesis global cellular translation fig 1f cellswith or without pax9 depletion are treated with a low dose μm of puromycin which isincorporated into all nascent peptides produced during a hour pulse western blottingfor incorporated puromycin shows decreased protein synthesis after hours of pax9 depletion relative to a nontargeting sirna control sint fig 1f mock μm puromycin withno sirna and mock at a halfdose of puromycin μm were used as negative controls fig1f these results confirm that pax9 depletion leads to reduced protein synthesis consistentwith a role for pax9 in ssu biogenesisbecause the above assays indicated a role for pax9 in small subunit prerrna processingand global protein synthesis we also tested the extent to which pax9 depletion is required forribosomal subunit biogenesis and assembly using polysome profiling we determined thatpax9 depletion does result in significantly decreased ssu 40s levels in mcf10a cells fig1g consistent with the reduction in 18s rrna levels seen in fig 1e additionally 60s and80s polysome fractions also showed significant decreases after pax9 knockdown fig 1ginterestingly both here and in previous experiments mcf10a cells do not demonstraterobust polysome fractions using this technique regardless we conclude that pax9 depletion results in decreased ssu biogenesis that impacts the assembly and function of theribosomeas disruptions in ribosome biogenesis result in interruptions in the cell cycle [“] wealso examined the extent to which pax9 depletion changed the distribution of mcf10a cellswithin the cell cycle using flow cytometry s3 fig relative to a sint control depletion ofpax9 for hours resulted in a minor increase in the proportion of cells in g1 phase of thecell cycle but this was not statistically significant s3 fig sirnas targeting the ribosome biogenesis factor nol11 were used as a positive control and depletion of this protein resulted inan increase in the proportion of cells in g2 s3 fig consistent with previous findings inall these assays allowed us to conclude that pax9 regulates human ribosome biogenesisrnaseq analysis upon pax9 depletion reveals decreased levels ofnucleolar mrnas responsible for small subunit maturationas pax9 is a known rnapii transcription factor we hypothesized that pax9 works indirectlyto modulate ssu biogenesis fig 2a this is consistent with a nuclear but not nucleolar localization of pax9 in existing databases [“] pax9 may act directly as a transcription factorfor nucleolar proteins or indirectly for proteins that affect the expression or function of nucleolar proteins to test the hypothesis that pax9 affects nucleolar protein expression through itsfunction as a rnapii transcription factor we used rnaseq in mcf10a cells to define the setof mrnas that were differentially expressed after pax9 depletion relative to a nontargetingcontrol sirna sint pax9 depletion resulted in the differential expression of over mrnas fold change � or � q � fig 2b and s1 table approximately half of these were reduced in their levels consistent with the hypothesis that pax9 acts as a transcription factor to drive their expressionwhen considered as a whole the rnaseq dataset is enriched for several pathways knownto be regulated by pax9 s4a fig ingenuity pathway analysis of the differentiallyexpressed mrnas reveals enrichment of both wntca2 signaling differential expression of genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisfig rnaseq transcriptomics analysis in human tissue culture cells reveals changes in the expression levels of over nucleolar mrnas afterpax9 knockdown a schematic of how pax9 would act as a rnapii transcription factor to drive the levels of mrnas required for making the smallsubunit ssu of the ribosome in the cell nucleus pax9 binds to dna to affect the transcription of mrnas that either encode nucleolar proteinsdirect solid arrow or to transcribe mrnas that affect the levels of mrnas encoding nucleolar proteins indirect dotted arrows the resulting mrnasare translated in the cytoplasm into proteins that function in ssu prerrna processing in the nucleolus b rnaseq analysis after pax9 sirnadepletion in mcf10a cells reveals decreased levels of mrnas encoding nucleolar proteins relative to a nontargeting sirna control sint pax9depletion resulted in differential expression of mrnas fold change � or and fdr � of these mrnas had a decreased foldchange � and of those mrnas code for proteins designated as nucleolar in at least one of three databases [“] of the mrnas whoselevels were decreased and that also code for nucleolar proteins were chosen as candidates for followup studies c qrtpcr confirms reduced mrnalevels of the rnaseq candidates after pax9 sirna knockdown in mcf10a cells after depletion using sirnas targeting either pax9 or a nontargeting control sirna sint the levels of the indicated mrnas were quantified by qrtpcr using primers to each target gene relative to a 7slcontrol and sint data are shown as mean ± sem three replicates using cells of different passage numbers with technical replicates each wereperformed significance was calculated by oneway anova using graphpad prism where ���� p � d depletion of of the candidatemrnas rps6es6 rps9us4 rps28es28 and fbl individually results in the same prerrna processing defect as pax9 sirna depletion in mcf10acells representative northern blot after knockdown of the indicated sirnas using probe p3 a probe for the 7sl rna was used as a loading control prerrna processing intermediates detected by probe p3 are shown to the right of the northern blot ptp indicates the 47s 45s and 43s prerrnaprocessing intermediates e quantitation of northern blots using probe p3 as shown in fig 2d using ramp graph is mean ± sem n datawere analyzed using 2way anova in graphpad prism where ���� p � ��� p � and �� p � quantitation relative to the 7sl loadingcontrol is shown in s5 fig f sirna depletion of rnaseq candidates in mcf10a cells results in decreased global protein synthesis after hoursof knockdown with the indicated sirnas mcf10a cells were pulsed with puromycin for hour and protein was harvested western blotting with anantipuromycin antibody as well as a β actin loading control was carried out representative western blots shown to the left mock mock at half theconcentration of puromycin μm and sint nontargeting sirnas were used as negative controls g quantitation of three replicates usingmcf10a cells of different passage numbers of the puromycin incorporation assays following depletion with the indicated sirnas relative to the sint andβ actin loading controls is shown as mean ± sem n significance was calculated by student™s ttest using graphpad prism where ���� p � ���p � and � p � 101371 pgen1008967g002 pathway members p x ˆ’ s4b fig and wntβcatenin signaling differentialexpression of pathway members p x ˆ’ s4c fig in cells depleted of pax9expression levels are increased for many of the mrnas in the wnt signaling pathway this isconsistent with previous results suggesting a role for pax9 in the negative regulation of wntsignaling [ ]as an additional validation of the rnaseq dataset we determined that the genomesequences kb upstream of the start sites of the differentially expressed mrnas containmultiple potential pax9 binding sites making these mrnas candidates for direct transcriptional regulation by pax9 scanning for known pax9 binding sequences [™sgtcacgcwtgantgma3™ ™cgcgtgaccg3™ cd192ains ™gcgtgacca3™ and e5 ™gcggaacgg3™] in the kb upstream of the mrnas using centrimo analysis reveals and potential pax9 binding sites respectively in the kbupstream of the mrnas this number of potential binding sites upstream of the mrnas sites per gene is similar to that observed in pax9 chip experiments in the vertebral column of e125 mice sites per gene additionally centrimo enrichmentanalysis of the sequence kb upstream of each of the differentially expressedmrnas reveals significant enrichment of different dna binding sequences including thepax3 pax5 pax6 and pax7 dna binding domains as multiple pax proteins can bindthe same dna sequence this provides further evidence for pax9 regulation of these mrnas these analyses therefore support the hypothesis that pax9 regulates the levels of themrnas identified in our rnaseq datasetin the rnaseq dataset many of the differentially expressed mrnas have knownroles in nucleolar function for example have appeared in other genomewidesirna screens for nucleolar function s1 table [ ] additionally of the differentially expressed mrnas code for proteins designated as nucleolar in at least of nucleolar databases s1 table [“] this is a significant enrichment in the expected number of nucleolar proteins assuming that nucleolar proteins make up only of the proteins inhuman cells surprisingly expression of most of the mrnas encoding nucleolar proteins genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis was downregulated upon pax9 depletion indicating that pax9 is requiredto maintain normal levels of many mrnas whose protein products are destined for functionin the nucleolus s1 tableto determine the mechanism of pax9™s function in ssu biogenesis we chose candidatesfrom the rnaseq dataset to follow up on in greater detail the mrna levels for the candidates were all downregulated after pax9 depletion and all code for nucleolar proteins fig 2band s1 table [“] four of the candidates rps6es6 rps9us4 rps28es28 and fblwere chosen on the basis of literature suggesting a role for these proteins in ssu prerrnaprocessing in hela cells [ ] additionally it was pertinent to analyze rpl5ul18 as it hasa known role in the p53dependent nucleolar stress response qrtpcr confirmed thernaseq results with reduced levels of each mrna when pax9 is depleted in mcf10a cellsfig 2c interestingly depletion of either rps9us4 or rps28es28 resulted in a decrease innucleolar number from “ to only in our original sirna screen therefore it is possible that the mechanism through which pax9 depletion results in decreased nucleolar numberrelies upon reduced expression of rps9us4 andor rps28es28 fig 2awe were able to confirm that depletion of of the tested candidates rps6es6 rps9us4 rps28es28 and fbl in mcf10a cells resulted in prerrna processing defects similarto that of pax9 depletion fig 2d and 2e s5 fig only rpl5ul18 did not give the 30sincrease characteristic of pax9 depletion although this was expected given its known role inlsu prerrna processing additionally depletion of several of the candidates individually resulted in significantly decreased global protein synthesis by the puromycin incorporation assay similar to the effect seen after pax9 depletion fig 2f and 2g puromycinincorporation was also reduced after rps6es6 depletion although it was not statistically significant fig 2f and 2g therefore pax9 may function as a transcription factor to directly orindirectly increase the expression of rps6es6 rps9us4 rps28es28 andor fbl fig 2aas the proteins encoded by these mrnas are required for ssu ribosome biogenesis fig their depletion after pax9 knockdown is a plausible mechanism through which pax9 regulates prerrna processing and global protein synthesisrnapii chipseq analysis reveals decreased transcription of mrnasencoding nucleolar proteins after pax9 depletionas the rnaseq analysis confirmed that levels of nucleolar mrnas were decreased afterpax9 depletion fig we sought to map how rnapii distributes on genes using rnapiichipseq as a readout of transcription through this approach we aimed to untangle the effectsof pax9 depletion on rnapii transcription from its effects on mrna stability since rnapiichipseq is able to detect genomewide changes in rnapii occupancy as pax proteins areable to both activate and repress target protein expression we have included genes withboth increased and decreased rnapii occupancy in this analysis approximately mrnaswere differentially occupied by rnapii upon pax9 knockdown compared to the nontargeting control sirna sint in mcf10a cells fold change cutoff � or � and maxtags � s2 table of these had decreased rnapii occupancy consistent with pax9 acting as a transcriptional driver of these mrnasto assess the validity of the rnapii chipseq dataset we again used centrimo to identifypotential pax9 dnabinding sites in the kb upstream of the genes with differentialrnapii occupancy searching for the ™ cgcgtgaccg ™ pax9 binding motif definedin revealed possible sites in the bp upstream of the differentially occupiedgenes also the known pax9 dna binding motifs cd192ains ™gcgtgacca3™ ande5 ™gcggaacgg3™ had and binding sites in these sequences respectively genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisadditionally analysis of motif enrichment ame identified the pax5 and pax6 dnabinding motifs as being significantly enriched in the kb of sequence upstream of the genes p � since multiple pax proteins can bind the same motif this suggests thatthis dataset does contain mrnas that are regulated by pax9 of the differentially occupied genes have also been shown to be differentially regulated by pax9 directly in pax9chipseq experiments on e125 wt vertebral column murine tissue fig 3a and s2 table these analyses confirm the ability of rnapii chipseq to detect changes in rnapiimediated transcription after pax9 knockdownbased on the hypothesis that pax9 acts as an rnapii transcription factor for regulators ofnucleolar function fig we expected to detect changes in the rnapiimediated transcription of a number of mrnas encoding nucleolar proteins after pax9 depletion indeedmrnas coding for nucleolar proteins were enriched with of the genes with differentialrnapii occupancy coding for nucleolar proteins in at least one of three databases s2table [“] this is again higher than would be expected assuming that nucleolar proteins account for approximately of all cellular proteins additionally depletion of onegene with differential rnapii occupancy anln resulted in decreased nucleolar number inour sirna screen similar to the phenotype seen after pax9 depletion fig 1b notably of the genes with differential rnapii occupancy appeared in other genomewide screens for ribosome biogenesis factors s2 table [ ] another targeted screeninvestigated the effects of of the genes top2a and cdca8 on prerrna processingwhen depleted by sirna in hela cells however depletion of neither gave the 30s prerrna increase on northern blots characteristic of pax9 depletion in al Answer:

Colon_Cancer

"it is well understood that the level of molecular oxygen o2 in tissue is a very important factor impacting both physiology and pathological processes as well as responsiveness to some treatments data on o2 in tissue could be effectively utilized to enhance precision medicine however the nature of the data that can be obtained using existing clinically applicable techniques is often misunderstood and this can confound the effective use of the information attempts to make clinical measurements of o2 in tissues will inevitably provide data that are aggregated over time and space and therefore will not fully represent the inherent heterogeneity of o2 in tissues additionally the nature of existing techniques to measure o2 may result in uneven sampling of the volume of interest and therefore may not provide accurate information on the œaverage o2 in the measured volume by recognizing the potential limitations of the o2 measurements one can focus on the important and useful information that can be obtained from these techniques the most valuable clinical characterizations of oxygen are likely to be derived from a series of measurements that provide data about factors that can change levels of o2 which then can be exploited both diagnostically and therapeutically the clinical utility of such data ultimately needs to be verified by careful studies of outcomes related to the measured changes in levels of o2k e y w o r d sclinical measures of oxygen oxygen in tissues partial pressure1department of radiology dartmouth medical school hanover nh usa2department of medicine section of radiation oncology dartmouthhitchcock medical center lebanon nh usa3thayer school of engineering dartmouth college hanover nh usa4department radiation and cellular oncology university of chicago chicago il usa5department of surgery dartmouthhitchcock medical center lebanon nh usa6louvain drug research institute universit catholique de louvain brussels belgium7department radiation oncology university medical center university of freiburg freiburg germany8german cancer center consortium dktk partner site freiburg german cancer research center dkfz heidelberg germanycorrespondenceann barry flood clinepr llc river road lyme nh usaemail annbarryflooddartmouthedufunding informationmajor funding for this work was from the national institutes of health national cancer institute ppg grant p01ca190193 r01 ca p30 ca023108 and national institute of biomedical imaging and bioengineering p41 this is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided the original work is properly cited the authors physiological reports published by wiley periodicals llc on behalf of the physiological society and the american physiological societyphysiological reports 20208e14541 1014814phy214541 wileyonlinelibrarycom phy2 of 0c of introduction the overall goal of this review is to facilitate clinically effective use of measurements of molecular oxygen o2 in tissues with the explicit intent of improving clinical care that is improving the accuracy and effectiveness of diagnoses treatments and prognoses for individual patients this review focusses especially on improving personalized medicine and outcomes of care by carefully considering the basis and validity of clinical measurements of o2 in tissues and how those measurements can be used to advance diagnosis and therapy while measurements of o2 in tissues have been recognized as an important factor in the clinical evaluation and treatment of many diseases especially cancer busk overgaard horsman a0 and pathologies involving ischemia such as in peripheral vascular disease and wound healing insufficient attention often has been paid to the meaning of the values that have been obtained note this review is derived in part from a series of recent papers on this topic flood et a0al a0 swartz flood swartz vaupel instead all too often when a measurement technique has indicated that the level of o2 in a given tissue is œx that is is some specific quantitative number for the o2 in the tissue researchers and clinicians alike assume that œx is a reliable accurate representation of the œtrue oxygenation status of the tissue this approach ignores the complexity and dynamics of o2 in living biological systems the reality is that any o2 measurement has been taken at only one point in time of a distribution in the subvolume that was interrogated by the method while the o2 is in fact varying with time and across space in the tissue and is unlikely to be uniform in the volume that is being interrogatedin this review we focus on the biologicalclinical meaningfulness of o2 measurements made in living anisms while recognizing that tissue o2 is in constant flux we emphasize that to obtain maximum clinical utility of the measurements it is necessary to consider the goal of the measurements and the limitations of the data that are obtained we particularly focus on the clinical value of making repeated measurements of o2 especially in association with strategiesevents that potentially change o2 levels what are oxygen levels physical concepts and terminology for in tissues reporting on oxygen levels in tissuesthe level of molecular oxygen that is o2 is usually reported as partial pressure of oxygen po2 or concentration of oxygen [o2] or co2 these terms have physically rigorous meanings that can usefully be extended to describe gases such as o2 that are dissolved in liquids or solids including tissues partial pressure is the pressure exerted by oxygen in a mixture of gases while concentration is the content of oxygen in the gas mixture or solid partial pressure is commonly expressed in mmhg and these units are sometimes referred to as torr or kpa si unit used in the eu while concentration of o2 is commonly expressed in ml of o2 per a0ml for example in bloodhowever the solubility of oxygen varies greatly in different media bennett swartz brown koenig a0 jordan et a0al a0 and this affects the relationship between po2 and [o2] the transport of o2 across lipid membranes is known to depend on both diffusion and solubility in the bilayer and to be affected by changes in the physical state and by the lipid composition especially the content of cholesterol and unsaturated fatty acids for example because o2 partitions preferentially into lipophilic media such as membranes the solubility of o2 in membranes is about four times greater than in aqueous solutions m¶ller et a0 al a0 this difference has significant consequences for physical and chemical interactions involving molecular oxygen in biological systems because these interactions depend on the number of oxygen molecules that are present and their rate of diffusiondoes it matter clinically to know whether the technique is reporting [o2] or po2 while these measures are not identical there is a known relationship between them according to the ideal gas law po2 is directly proportional to concentration assuming the volume and temperature are constant that ispv nrtwhere p a0 a0pressure po2 v a0 a0volume n a0 a0amount of substance [o2] r a0 a0ideal gas constant t a0 a0temperatureit is less straightforward in biological systems if the solubility of oxygen in each component of tissue is known and this is not always fairly readily derived experimentally and po2 can be measured then it is relatively straightforward to calculate [o2] conversely if the component in which [o2] is measured and the solubility of o2 in that compartment is sufficiently known then it should be feasible to determine po2 however it often is not feasible to measure these parameters readilybecause of the biological complexities in assessing o2 in tissues each reported measure of o2 level in a tissue can be better considered as an average value average is used here in its more colloquial usage rather than as a statistically defined term because different techniques output their measurement of o2 using differing methods pertinent to that technique each technique gathers information from a particular volume of tissue irrespective of whether that volume is well characterized which we refer to hereafter as the œinterrogated swartz 0cvolume the sampling of data within the interrogated volume is then used to produce a measure based on a sort of average o2 within that volume characterizing that œaverage measure is made difficult both by the imprecision of knowing the exact volume queried but also because the detection of o2 in that volume may be affected by factors such as its distance from the detector or for optical techniques different rates of scattering that also may not be well characterized hence we conclude that it is important to bear in mind that the measurements of o2 in vivo are fundamentally based on a sort of œaverage within the interrogated volumebecause of all of these challenges in obtaining precise measurements of relevant parameters necessary to assess whether the data obtained by any technique is truly measuring either po2 or [o2] and because of the imprecisions of knowing the volume being assessed and the tissues within that volume it is more realistic to acknowledge the complexity of these issues for in vivo measurements in tissues by using a less precise term for measures of molecular oxygen in tissues such as œo2 levels which is the convention we follow in this review we also argue that while it is important to recognize the biological imprecisions in these measures there are still many clinically viable uses of this information such as assessing change in o2 levelsnote too within this paper while focusing on the uncertainties due to sampling issues of each technique and variations due to biological factors we are not taking into consideration further uncertainties due to inevitable instrumental noise variations in the placement of the detector etc expected levels of o2 in tissues table a0 presents some illustrative data on the o2 levels in various tissues both in normal states and as altered by some diseases these measures are presented here as reported in the literature the first column presents the median po2 obtained using the eppendorf electrode or comparable polarographic techniques to measure o2 levels in patients also presented are two other indications of o2 levels the hypoxic fraction the percentage of measurements in a given type of tissue that is below a defined œhypoxic level in this case a0mmhg and the range of po2 values found experimentally these data illustrate both the variation in median o2 levels between types of tissues and how they may vary with physiology or disease for example intertissue in general the median o2 levels are lower in skeletal muscle and heart compared to the spleen intratissue the median o2 in skeletal muscle at rest is higher than with exercise while in contrast there is almost no variation between the normal spleen and with hodgkin's disease the data in column illustrate the wide range that any given measurement can have in the œsame type of tissue that is almost all tissues range from of to a0mmhg even when their median value is quite different see again spleen vs bone however these very high values in the upper range may include experimental artifacts due to the measurement being taken within or very close to an arteriole for example a vessel feeding the microcirculatory bedtable a0 presents the same types of information for a more detailed analysis of changes in an important pathology cancer where o2 levels have been an especially important focus for informing clinical treatment and prognosis to give the reader a sense of how well supported the numbers are the data in table a0 have been ordered by the number of patients included in each rowof interest here all seven cancer types with at least patients studied have a fairly consistent and fairly hypoxic median o2 level a0mmhg similarly all but soft tissue sarcoma have a similar hypoxic fraction sarcoma appears to be about half that glioma appears to be an outlier on the range glioma had no patient whose o2 level was above while all others as was true for the tissues in table a0 have at least one measurement in the upper 80s or 90s these occasional high readings are not surprising since it is plausible that randomly some readings will have been obtained in or very near to arteriolesfeeding microvessels in contrast to the first seven cancers the types of cancers with fewer than patients appear to be more varied in their o2 levels but this is possibly due to being based on few patientsnevertheless even though as noted elsewhere in this review the data presented are not unconditionally œabsolute values of o2 levels as they are sometimes referred to eg koch a0 macnab gagnon gagnon minchinton fry a0 nevertheless as argued in this review they can provide very useful data as long as their limitations are recognized by researchers and clinicians heterogeneity of distributions of levels of oxygen in tissues examples and causesheterogeneity of distributions of oxygen values in the tissues of interest exists over many dimensions including time and space and over a wide range of scales harrison vaupel a0 tables a0 and focus on intertissue and intratissue variation in overall levels of o2 figures a0 and illustrate heterogeneity using more refined data points to illustrate the skewed nature of the data particularly for malignancies the data in figure a0 are based on multiple measurements made in a series of patients using a computerized polarographic microsensor technique which enables direct assessment of the o2 levels with an o2sensitive needle electrode subject to the limitations of providing true absolute values as discussed swartz 0c of table oxygenation status of anstissuesantissuekidneycortexouter medullainner medullaliverpancreasspleennormalin hypersplenismhodgkin's diseasemyocardiumsubepicardialsubendocardialmucosaoralrectallarge bowelbreastnormalfibrocystic diseaseprostateuterine cervixsubcutisbonecorticalhematopoietic marrowadipose marrowskeletal musclerestingexercisehypovolemic shockpaodskinthermoneutral conditionscritical limb ischemialimbs venous diseasebraingray matterwhite mattermedian po2 mmhghf po2 range mmhgreferences““““““““““““““nanana““nanana“““““““““““““““““““““““““““““nag¼nther aum¼ller kunke vaupel and thews samesamekallinowski and buhr 1995akoong vaupel wendling thom and fischer wendling vaupel fischer and br¼nner samewinbury howe and weiss moss for bothkallinowski and buhr 1995asamesamevaupel schlenger knoop and h¶ckel vaupel and harrison samevaupel and kelleher h¶ckel schlenger knoop and vaupel samespencer et a0al a0samesamelandgraf and ehrly jung kessler pindur sternitzky and franke harrison and vaupel landgraf schultehuermann vallbracht and ehrly carreau et a0al a0harrison and vaupel clyne ramsden chant and webster vaupel samecontinuesswartz 0c of table continuedantissueretinamedian po2 mmhghf napo2 range mmhg“referenceshogeboom van buggenum van der heijde tangelder and reichertthoen linsenmeier and zhang white adipose tissuenonobeseobesenana““pasarica et a0al a0 hodson lempesis van meijel manolopoulos and goossens abbreviations arterial hf25 hypoxic fraction ‰¡ fraction of po2 values ‰¤ a0mmhg na information not available paod peripheral arterial occlusive diseaseelsewhere in the paper oxygen was measured along several electrode tracks in each individual during a given measurement session from near the tumor surface up to tissue depths of a0 a0 a0mm in breast cancers and in cancers of the uterine cervix each row in figure a0 presents a type of tissue breast and uterine cervix with comparisons of o2 levels made in normal tissue green versus malignancies prior to treatment of these ans red the summary measures median po2 values parallel to data reported in tables a0 and are included in the text boxesnote that the data in figure a0 are not normally distributed the distribution of o2 levels made in normal breast tissue is the closest approximation to a normal distribution comparing the two distributions for malignancies we see that breast cancer is more highly skewed than cervical cancer although they have the same median thus using a single measure such as the median could overlook potentially important clinical informationsimilarly comparing the distributions for the normal tissues the normal cervix had a substantial number of o2 measurements within a hypoxic range defined as ‰¤ a0 mmhg while there were no hypoxic measurements in the normal breast while figure a0 does not differentiate the measurements made per patient it illustrates why several authors report the hypoxic fraction when trying to capture a meaningful overall number to characterize a tissue finally these data underscore why it is important to understand what is well captured by a given measure of o2 level”and what is missed or obscuredanother example of heterogeneity is presented in figure a0 these o2 measurements were taken in a breast cancer patient using epr oximetry with carlo erba ink as the o2 sensor flood et a0al a0 jeong et a0al a0 the data are presented as line widths because the epr oximetry technique using india ink as a sensor undoubtably gathers data from volumes too large to have homogenous oxygen levels however because this sensor remains in the same place in the tissue it still can provide very useful indications of changes over time andor the impact of interventions such as breathing enriched oxygen the data were taken in 30min sessions during which epr spectra were collected continuously but the period was divided into three 10min periods differing by the gas mixture the patient was breathing room air red baseline o2 delivered by a nonrebreather mask green followed by again breathing room air blue recovery the sessions were repeated approximately weekly throughout the period of radiation therapy thus mimicking a clinical course of radiation therapy although there was no attempt to impact therapy in this studythe data for this particular patient illustrate that the o2 levels responded to the patient's breathing an hyperoxic gas mixture and then returned rapidly to the baseline level after the 10min period in this patient there appeared to be some variation across the weeks of treatment with the final levels for each period baseline o2 and recovery all being slightly higher than at the beginning of radiation therapy based on nonoverlapping standard deviations of the first and last measurements causes of heterogeneity in normal tissuesthere are spatial variances in oxygen levels in normal tissues due to the longitudinal gradient in oxygen as the blood passes through the microcirculatory bed decreasing from the arterial inlet to the outlet of the microvessels erickson after the oxygen leaves the microvascular networks the partial pressure of o2 decreases due to radial gradients that is o2 diffuses through the tissues as it gets further from the vessels due to o2 being consumed by the cells as a result there are variations in o2 levels from cell to cell according to their distance from the microvessel within the cells o2 decreases in a microspatially complex manner as it is intracellularly consumed with most of the consumption occurring in the mitochondria there is growing evidence that diffusion of o2 into the cell may be constrained that is that o2 does not freely and rapidly flow into cells across the membrane and therefore there are gradients from outside to inside of cells khan et a0al a0 kurokawa et a0al a0 pias a0these variations of o2 levels that is gradients between and within cells cannot currently be measured as detailed below in discussing temporal variations even if such measurements of spatial heterogeneity could be made they would still be inadequate to understand the full complexity of heterogeneity of o2 for example in some normal tissues there is additional significant macroscopic heterogeneity of o2 swartz 0c of table pretherapeutic oxygenation status of human tumorstumor type ordered by no of patientscervix cancerhead and neck cancerprostate cancersoft tissue sarcomabreast cancerglioblastomavulvar cancermedian po2 mmhg“no of patientsrectal cancerlung cancermalignant melanoma metastaticnonhodgkin's lymphomapancreas cancerbrain metastasesnahf po2 range mmhg“““““““““““““liver metastasesrenal cell carcinomagall bladder cancerbile duct cancerabbreviations hf25 hypoxic fraction ‰¡ fraction of po2 values ‰¤ a0mmhg na information not available““““nanananareferencesvaupel et a0al a0vaupel vaupel data synopsesthese ref apply to allabove the linevaupel thews mayer h¶ckel and h¶ckel vaupel mayer and h¶ckel stone et a0al a0kallinowski and buhr 1995a mattern kallinowski herfarth and volm falk ward and bleehen le et a0al a0lartigau et a0al a0powell et a0al a0koong graffman bjork ederoth and ihse rampling cruickshank lewis fitzsimmons and workman kallinowski and buhr 1995a 1995blawrentschuk et a0al a0graffman et a0al a0graffman et a0al a0over space because of their physiology as a consequence of substantial differences in vascularity blood flow and oxygen consumption eg macroscopic heterogeneity between gray and white matter of the brain between subepicardial and subendocardial layers of the myocardium and between renal cortex and renal inner medullathere also are temporal changes in o2 levels in normal tissues griffith a0 moreover the supply of o2 can vary periodically due to rhythmic changes in microcirculatory blood flow which is reflected at all levels from the inflow arteries to the microcirculation finally within the microcirculation there are variations in microvascular flow due to regional regulation in response to varying metabolic demands kimura et a0al a0 important differences in o2 solubility across tissues affecting the relationship between po2 and [o2] were discussed earlier impact of pathology on heterogeneity of o2 levelsthe presence of pathology often significantly increases the amount and extent of oxygen heterogeneity both spatially and temporally vaupel harrison vaupel mayer a0 the presence of pathology often impacts the structuremorphology of the vessels in tumors there often is a significant amount of neoangiogenesis which results in much less ordered and less functional blood vessels busk et a0al a0 the resulting vessels are much less efficient in delivering blood and also tend to be much more prone to leak leakage from these vessels can cause increases in the interstitial pressure which can reduce the effectiveness of the microcirculation due to reduction of the perfusion pressure within the tumor capillaries fukumura duda munn jain a0pathological changes also can result in altered consumption of o2 in malignant tumors o2 consumption is likely to decrease due to poor oxygen delivery andor because of a switch to glycolysis due to metabolic reprogramming ie the warburg effect as a consequence of hif1α overexpression upregulation of oncogenes downregulation of suppressor genes and activation of certain signaling pathways vaupel multhoff a0 vaupel schmidberger mayer a0 pathology can also impact the integrity of the blood vessels for example tumor growth may physically impinge on the integrity of the blood vessels and the swartz 0c of figure distributions of multiple o2 levels made in patients with normal versus malignant tissue breast and cervix figure adapted from vaupel mayer a02017b p figure repeated o2 level measurementsa during each measurement session and over a0days breast cancer patient measured in skin and superficial breast tissue within the radiation field during a course of radiation therapy figure adapted from flood et a0al a0 p afor carlo erba ink epr line width increases with increasing o2 level but the relationship is nonlinear and can be impacted by several factors therefore the data are given here as line widthmetabolic abnormalities in diabetes can impact the structure of blood vessels causing either microangiopathy andor macroangiopathy the results of these processes can produce very significant local variations in the availability of fully functional vascular structures resulting in locally hypoxic regionsswartz 0c of pathology also can impact temporal changes of oxygen and the response to treatment the presence of pathology especially cancer eg acute and cycling hypoxia in cancers and peripheral vascular disease can result in significantly greater variability in o2 levels braun lanzen dewhirst a0 these include shortterm changes especially associated with the structural abnormalities of the microcirculation resulting in increased local variability in flow and longterm changes that develop over time such as those due to disease progression and responses to therapy baudelet et a0al a0 kimura et a0al a0 konerding fait gaumann a0 matsumoto there also is a potential for pathologies to interrelate with each other for example anemic hypoxia can develop in tumors due to the underlying systemic anemia of the patient vaupel mayer a0in addition to these underlying effects of pathology on tissue oxygen levels any applied therapeutic interventions are very likely to induce changes for example cell killing due to radiation or chemotherapy will alter oxygen consumption patterns these same therapies will also affect the o2 supplying vasculature via both antiangiogenic effects and”possibly”normalization of vessel structure jain a0 the effects of therapies will generally vary both spatially and temporally reiterating the complexity of meaningfully characterizing tissue oxygen levels analysis of the ability of clinically available techniques to directly measure levels of o2 in tissues andor resolve the heterogeneity of o2 distributions in tissuesalthough many techniques are often thought to measure actual o2 in tissues only a few actually have the potential to make o2 measurements directly in the tissues of interest springett swartz a0 tatum techniques that can potentially assess o2 directly in tissues include epr epel et a0al a0 swartz et a0al a0 swartz the eppendorf electrode vaupel h¶ckel mayer a0 some optical methods based on direct measurements of target molecules in tissues for example phosphorescence quenching of optical sensors placed directly in tissues or as part of a physical probe such as the œoxylite wen et a0al a0 and nmr relaxation techniques colliez et a0al a0two other types of measurements assess o2 in the vascular system blood gases do this directly while optical methods that measure both hemoglobin saturation and total hemoglobin especially near infrared spectroscopy [nirs] scheeren schober schwarte a0 provide a plausible link to the po2 in the bloodhowever the techniques most often used clinically to characterize tissue oxygenation do not in fact measure o2 directly instead they measure œindirect parameters that can be plausibly linked to actual o2 levels but only under appropriatedefined circumstances this latter group of techniques includes positron emission tomography pet imaging of glucose derivatives neveu et a0al a0 pet imaging of drugs that localize in hypoxic tissues tran laser doppler flow measures of metabolites that may be affected by o2 levels for example lactate and redox intermediates and several magnetic resonance imagingnuclear magnetic resonance mrinmr blood oxygenation level dependent bold imaging baudelet gallez a0 and mri egeland et a0al a0 note if their basis is understood and the data considered accordingly these can all provide clinically and physiologically useful information even though they do not provide direct information on the amount of o2 in the tissues direct measures of o2 in targeted tissues that potentially can be used in human subjectsthese are techniques that while they have the capability of providing direct quantitative measurements of o2 in homogeneous media cannot provide such data in tissues in vivo because the volumes that they sense are larger than the volumes of homogeneity of o2 in actively metabolizing tissues consequently all in vivo measurements of o2 are inherently averages of the actual oxygen content in that volume even neglecting the need to include measures of heterogeneity inside cells based on the usual volume of cells and assuming that differences are sought for aggregates of ‰¤ cells for a measurement of heterogeneity sensed within a a0mm diameter volume the spatial resolution needed to appropriately characterize o2 levels in this volume becomes million voxels the measuring techniques may not even provide a welldefined averaged po2 value within the volume that they sense for example sensors for the signal that is being measuredin the next sections we review the characteristics of each technique that can directly measure o2 levels in tissues we also briefly remark on the volumes they measure and how the measures obtained can be useful clinically see also ortezprado dunn vasconez castillo visco epr oximetry using appropriate particulate paramagnetic materials epr oximetry can provide direct measurements of o2 that is the epr signal is directly proportional to the amount of o2 epel bowman mailer halpern a0 swartz vaupel swartz 0cthe because each multisite sensor senses a volume that is much larger than capillary networks these techniques provide a volume averaged sampling of all compartments within the tissues the time resolution of the techniques can be milliseconds or shorterthe measured parameter of an epr spectrum that indicates the amount of o2 present is the line width of the observed resonance peak there usually is a fixed relationship between the line width and the amount of o2 with the relationship being specific for each type of paramagnetic material for example carbon charcoal or phthalocyanine particulates using particulate oximetric materials measurements can be continuous over any span of time and can be repeated indefinitely see example in figure a0 the method requires that the sensing material be injected or implanted in one or more regions of interest but thereafter all measurements can be made entirely noninvasively importantly the measurements can be carried out in a clinical setting and can fit into the workflow needed for patient careinitial clinical epr measurements of oxygen in tissues have used india ink as the oxygen sensor swartz et a0al a0 the carbon ps are the components that respond to oxygen lan beghein charlier gallez a0 after injection of “ a0µl of the suspension through a small needle the carbon ps disperse nonuniformly through the local region as small extracellular aggregates they are often engulfed by macrophages the resulting epr spectra in the region probed by the resonator ie the surface coil used for signal detection are a composite of the oxygendependent line widths from each of the ps in reality because of the relatively broad lines from the india ink ps the range of œoxygen levels that are likely to be present in the tissue and the limited number of ps in each subregion it is a challenge to resolve directly even the major groups of similar line widths therefore using the observed line width to provide a quantitative measure of oxygen would seem to have modest utility in itselfthe other method of clinical epr oximetry is based on the use of microcrystalline probes eg lipc lincbuo encapsulated in biocompatible polymers swartz et a0al a0 clinical measurements currently are being performed using the œoxychip which consists of oxygen sensitive microcrystals of lithium octanbutoxynaphthalocyanine lincbuo embedded in polydimethylsiloxane pdms hou khan gohain kuppusamy kuppusamy a0 hou et a0al a0 jarvis et a0al a0 the dimensions currently used in humans are cylinders that are a0 mm long with a diameter of a0mm the epr signal from the sensor oxychip reflects the po2 within the pdms which itself reflects an average of the po2 in contact with the external surface of the cylinder the dimensions of the oxy

cancer7550

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "it is well understood that the level of molecular oxygen o2 in tissue is a very important factor impacting both physiology and pathological processes as well as responsiveness to some treatments data on o2 in tissue could be effectively utilized to enhance precision medicine however the nature of the data that can be obtained using existing clinically applicable techniques is often misunderstood and this can confound the effective use of the information attempts to make clinical measurements of o2 in tissues will inevitably provide data that are aggregated over time and space and therefore will not fully represent the inherent heterogeneity of o2 in tissues additionally the nature of existing techniques to measure o2 may result in uneven sampling of the volume of interest and therefore may not provide accurate information on the œaverage o2 in the measured volume by recognizing the potential limitations of the o2 measurements one can focus on the important and useful information that can be obtained from these techniques the most valuable clinical characterizations of oxygen are likely to be derived from a series of measurements that provide data about factors that can change levels of o2 which then can be exploited both diagnostically and therapeutically the clinical utility of such data ultimately needs to be verified by careful studies of outcomes related to the measured changes in levels of o2k e y w o r d sclinical measures of oxygen oxygen in tissues partial pressure1department of radiology dartmouth medical school hanover nh usa2department of medicine section of radiation oncology dartmouthhitchcock medical center lebanon nh usa3thayer school of engineering dartmouth college hanover nh usa4department radiation and cellular oncology university of chicago chicago il usa5department of surgery dartmouthhitchcock medical center lebanon nh usa6louvain drug research institute universit catholique de louvain brussels belgium7department radiation oncology university medical center university of freiburg freiburg germany8german cancer center consortium dktk partner site freiburg german cancer research center dkfz heidelberg germanycorrespondenceann barry flood clinepr llc river road lyme nh usaemail annbarryflooddartmouthedufunding informationmajor funding for this work was from the national institutes of health national cancer institute ppg grant p01ca190193 r01 ca p30 ca023108 and national institute of biomedical imaging and bioengineering p41 this is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided the original work is properly cited the authors physiological reports published by wiley periodicals llc on behalf of the physiological society and the american physiological societyphysiological reports 20208e14541 1014814phy214541 wileyonlinelibrarycom phy2 of 0c of introduction the overall goal of this review is to facilitate clinically effective use of measurements of molecular oxygen o2 in tissues with the explicit intent of improving clinical care that is improving the accuracy and effectiveness of diagnoses treatments and prognoses for individual patients this review focusses especially on improving personalized medicine and outcomes of care by carefully considering the basis and validity of clinical measurements of o2 in tissues and how those measurements can be used to advance diagnosis and therapy while measurements of o2 in tissues have been recognized as an important factor in the clinical evaluation and treatment of many diseases especially cancer busk overgaard horsman a0 and pathologies involving ischemia such as in peripheral vascular disease and wound healing insufficient attention often has been paid to the meaning of the values that have been obtained note this review is derived in part from a series of recent papers on this topic flood et a0al a0 swartz flood swartz vaupel instead all too often when a measurement technique has indicated that the level of o2 in a given tissue is œx that is is some specific quantitative number for the o2 in the tissue researchers and clinicians alike assume that œx is a reliable accurate representation of the œtrue oxygenation status of the tissue this approach ignores the complexity and dynamics of o2 in living biological systems the reality is that any o2 measurement has been taken at only one point in time of a distribution in the subvolume that was interrogated by the method while the o2 is in fact varying with time and across space in the tissue and is unlikely to be uniform in the volume that is being interrogatedin this review we focus on the biologicalclinical meaningfulness of o2 measurements made in living anisms while recognizing that tissue o2 is in constant flux we emphasize that to obtain maximum clinical utility of the measurements it is necessary to consider the goal of the measurements and the limitations of the data that are obtained we particularly focus on the clinical value of making repeated measurements of o2 especially in association with strategiesevents that potentially change o2 levels what are oxygen levels physical concepts and terminology for in tissues reporting on oxygen levels in tissuesthe level of molecular oxygen that is o2 is usually reported as partial pressure of oxygen po2 or concentration of oxygen [o2] or co2 these terms have physically rigorous meanings that can usefully be extended to describe gases such as o2 that are dissolved in liquids or solids including tissues partial pressure is the pressure exerted by oxygen in a mixture of gases while concentration is the content of oxygen in the gas mixture or solid partial pressure is commonly expressed in mmhg and these units are sometimes referred to as torr or kpa si unit used in the eu while concentration of o2 is commonly expressed in ml of o2 per a0ml for example in bloodhowever the solubility of oxygen varies greatly in different media bennett swartz brown koenig a0 jordan et a0al a0 and this affects the relationship between po2 and [o2] the transport of o2 across lipid membranes is known to depend on both diffusion and solubility in the bilayer and to be affected by changes in the physical state and by the lipid composition especially the content of cholesterol and unsaturated fatty acids for example because o2 partitions preferentially into lipophilic media such as membranes the solubility of o2 in membranes is about four times greater than in aqueous solutions m¶ller et a0 al a0 this difference has significant consequences for physical and chemical interactions involving molecular oxygen in biological systems because these interactions depend on the number of oxygen molecules that are present and their rate of diffusiondoes it matter clinically to know whether the technique is reporting [o2] or po2 while these measures are not identical there is a known relationship between them according to the ideal gas law po2 is directly proportional to concentration assuming the volume and temperature are constant that ispv nrtwhere p a0 a0pressure po2 v a0 a0volume n a0 a0amount of substance [o2] r a0 a0ideal gas constant t a0 a0temperatureit is less straightforward in biological systems if the solubility of oxygen in each component of tissue is known and this is not always fairly readily derived experimentally and po2 can be measured then it is relatively straightforward to calculate [o2] conversely if the component in which [o2] is measured and the solubility of o2 in that compartment is sufficiently known then it should be feasible to determine po2 however it often is not feasible to measure these parameters readilybecause of the biological complexities in assessing o2 in tissues each reported measure of o2 level in a tissue can be better considered as an average value average is used here in its more colloquial usage rather than as a statistically defined term because different techniques output their measurement of o2 using differing methods pertinent to that technique each technique gathers information from a particular volume of tissue irrespective of whether that volume is well characterized which we refer to hereafter as the œinterrogated swartz 0cvolume the sampling of data within the interrogated volume is then used to produce a measure based on a sort of average o2 within that volume characterizing that œaverage measure is made difficult both by the imprecision of knowing the exact volume queried but also because the detection of o2 in that volume may be affected by factors such as its distance from the detector or for optical techniques different rates of scattering that also may not be well characterized hence we conclude that it is important to bear in mind that the measurements of o2 in vivo are fundamentally based on a sort of œaverage within the interrogated volumebecause of all of these challenges in obtaining precise measurements of relevant parameters necessary to assess whether the data obtained by any technique is truly measuring either po2 or [o2] and because of the imprecisions of knowing the volume being assessed and the tissues within that volume it is more realistic to acknowledge the complexity of these issues for in vivo measurements in tissues by using a less precise term for measures of molecular oxygen in tissues such as œo2 levels which is the convention we follow in this review we also argue that while it is important to recognize the biological imprecisions in these measures there are still many clinically viable uses of this information such as assessing change in o2 levelsnote too within this paper while focusing on the uncertainties due to sampling issues of each technique and variations due to biological factors we are not taking into consideration further uncertainties due to inevitable instrumental noise variations in the placement of the detector etc expected levels of o2 in tissues table a0 presents some illustrative data on the o2 levels in various tissues both in normal states and as altered by some diseases these measures are presented here as reported in the literature the first column presents the median po2 obtained using the eppendorf electrode or comparable polarographic techniques to measure o2 levels in patients also presented are two other indications of o2 levels the hypoxic fraction the percentage of measurements in a given type of tissue that is below a defined œhypoxic level in this case a0mmhg and the range of po2 values found experimentally these data illustrate both the variation in median o2 levels between types of tissues and how they may vary with physiology or disease for example intertissue in general the median o2 levels are lower in skeletal muscle and heart compared to the spleen intratissue the median o2 in skeletal muscle at rest is higher than with exercise while in contrast there is almost no variation between the normal spleen and with hodgkin's disease the data in column illustrate the wide range that any given measurement can have in the œsame type of tissue that is almost all tissues range from of to a0mmhg even when their median value is quite different see again spleen vs bone however these very high values in the upper range may include experimental artifacts due to the measurement being taken within or very close to an arteriole for example a vessel feeding the microcirculatory bedtable a0 presents the same types of information for a more detailed analysis of changes in an important pathology cancer where o2 levels have been an especially important focus for informing clinical treatment and prognosis to give the reader a sense of how well supported the numbers are the data in table a0 have been ordered by the number of patients included in each rowof interest here all seven cancer types with at least patients studied have a fairly consistent and fairly hypoxic median o2 level a0mmhg similarly all but soft tissue sarcoma have a similar hypoxic fraction sarcoma appears to be about half that glioma appears to be an outlier on the range glioma had no patient whose o2 level was above while all others as was true for the tissues in table a0 have at least one measurement in the upper 80s or 90s these occasional high readings are not surprising since it is plausible that randomly some readings will have been obtained in or very near to arteriolesfeeding microvessels in contrast to the first seven cancers the types of cancers with fewer than patients appear to be more varied in their o2 levels but this is possibly due to being based on few patientsnevertheless even though as noted elsewhere in this review the data presented are not unconditionally œabsolute values of o2 levels as they are sometimes referred to eg koch a0 macnab gagnon gagnon minchinton fry a0 nevertheless as argued in this review they can provide very useful data as long as their limitations are recognized by researchers and clinicians heterogeneity of distributions of levels of oxygen in tissues examples and causesheterogeneity of distributions of oxygen values in the tissues of interest exists over many dimensions including time and space and over a wide range of scales harrison vaupel a0 tables a0 and focus on intertissue and intratissue variation in overall levels of o2 figures a0 and illustrate heterogeneity using more refined data points to illustrate the skewed nature of the data particularly for malignancies the data in figure a0 are based on multiple measurements made in a series of patients using a computerized polarographic microsensor technique which enables direct assessment of the o2 levels with an o2sensitive needle electrode subject to the limitations of providing true absolute values as discussed swartz 0c of table oxygenation status of anstissuesantissuekidneycortexouter medullainner medullaliverpancreasspleennormalin hypersplenismhodgkin's diseasemyocardiumsubepicardialsubendocardialmucosaoralrectallarge bowelbreastnormalfibrocystic diseaseprostateuterine cervixsubcutisbonecorticalhematopoietic marrowadipose marrowskeletal musclerestingexercisehypovolemic shockpaodskinthermoneutral conditionscritical limb ischemialimbs venous diseasebraingray matterwhite mattermedian po2 mmhghf po2 range mmhgreferences““““““““““““““nanana““nanana“““““““““““““““““““““““““““““nag¼nther aum¼ller kunke vaupel and thews samesamekallinowski and buhr 1995akoong vaupel wendling thom and fischer wendling vaupel fischer and br¼nner samewinbury howe and weiss moss for bothkallinowski and buhr 1995asamesamevaupel schlenger knoop and h¶ckel vaupel and harrison samevaupel and kelleher h¶ckel schlenger knoop and vaupel samespencer et a0al a0samesamelandgraf and ehrly jung kessler pindur sternitzky and franke harrison and vaupel landgraf schultehuermann vallbracht and ehrly carreau et a0al a0harrison and vaupel clyne ramsden chant and webster vaupel samecontinuesswartz 0c of table continuedantissueretinamedian po2 mmhghf napo2 range mmhg“referenceshogeboom van buggenum van der heijde tangelder and reichertthoen linsenmeier and zhang white adipose tissuenonobeseobesenana““pasarica et a0al a0 hodson lempesis van meijel manolopoulos and goossens abbreviations arterial hf25 hypoxic fraction ‰¡ fraction of po2 values ‰¤ a0mmhg na information not available paod peripheral arterial occlusive diseaseelsewhere in the paper oxygen was measured along several electrode tracks in each individual during a given measurement session from near the tumor surface up to tissue depths of a0 a0 a0mm in breast cancers and in cancers of the uterine cervix each row in figure a0 presents a type of tissue breast and uterine cervix with comparisons of o2 levels made in normal tissue green versus malignancies prior to treatment of these ans red the summary measures median po2 values parallel to data reported in tables a0 and are included in the text boxesnote that the data in figure a0 are not normally distributed the distribution of o2 levels made in normal breast tissue is the closest approximation to a normal distribution comparing the two distributions for malignancies we see that breast cancer is more highly skewed than cervical cancer although they have the same median thus using a single measure such as the median could overlook potentially important clinical informationsimilarly comparing the distributions for the normal tissues the normal cervix had a substantial number of o2 measurements within a hypoxic range defined as ‰¤ a0 mmhg while there were no hypoxic measurements in the normal breast while figure a0 does not differentiate the measurements made per patient it illustrates why several authors report the hypoxic fraction when trying to capture a meaningful overall number to characterize a tissue finally these data underscore why it is important to understand what is well captured by a given measure of o2 level”and what is missed or obscuredanother example of heterogeneity is presented in figure a0 these o2 measurements were taken in a breast cancer patient using epr oximetry with carlo erba ink as the o2 sensor flood et a0al a0 jeong et a0al a0 the data are presented as line widths because the epr oximetry technique using india ink as a sensor undoubtably gathers data from volumes too large to have homogenous oxygen levels however because this sensor remains in the same place in the tissue it still can provide very useful indications of changes over time andor the impact of interventions such as breathing enriched oxygen the data were taken in 30min sessions during which epr spectra were collected continuously but the period was divided into three 10min periods differing by the gas mixture the patient was breathing room air red baseline o2 delivered by a nonrebreather mask green followed by again breathing room air blue recovery the sessions were repeated approximately weekly throughout the period of radiation therapy thus mimicking a clinical course of radiation therapy although there was no attempt to impact therapy in this studythe data for this particular patient illustrate that the o2 levels responded to the patient's breathing an hyperoxic gas mixture and then returned rapidly to the baseline level after the 10min period in this patient there appeared to be some variation across the weeks of treatment with the final levels for each period baseline o2 and recovery all being slightly higher than at the beginning of radiation therapy based on nonoverlapping standard deviations of the first and last measurements causes of heterogeneity in normal tissuesthere are spatial variances in oxygen levels in normal tissues due to the longitudinal gradient in oxygen as the blood passes through the microcirculatory bed decreasing from the arterial inlet to the outlet of the microvessels erickson after the oxygen leaves the microvascular networks the partial pressure of o2 decreases due to radial gradients that is o2 diffuses through the tissues as it gets further from the vessels due to o2 being consumed by the cells as a result there are variations in o2 levels from cell to cell according to their distance from the microvessel within the cells o2 decreases in a microspatially complex manner as it is intracellularly consumed with most of the consumption occurring in the mitochondria there is growing evidence that diffusion of o2 into the cell may be constrained that is that o2 does not freely and rapidly flow into cells across the membrane and therefore there are gradients from outside to inside of cells khan et a0al a0 kurokawa et a0al a0 pias a0these variations of o2 levels that is gradients between and within cells cannot currently be measured as detailed below in discussing temporal variations even if such measurements of spatial heterogeneity could be made they would still be inadequate to understand the full complexity of heterogeneity of o2 for example in some normal tissues there is additional significant macroscopic heterogeneity of o2 swartz 0c of table pretherapeutic oxygenation status of human tumorstumor type ordered by no of patientscervix cancerhead and neck cancerprostate cancersoft tissue sarcomabreast cancerglioblastomavulvar cancermedian po2 mmhg“no of patientsrectal cancerlung cancermalignant melanoma metastaticnonhodgkin's lymphomapancreas cancerbrain metastasesnahf po2 range mmhg“““““““““““““liver metastasesrenal cell carcinomagall bladder cancerbile duct cancerabbreviations hf25 hypoxic fraction ‰¡ fraction of po2 values ‰¤ a0mmhg na information not available““““nanananareferencesvaupel et a0al a0vaupel vaupel data synopsesthese ref apply to allabove the linevaupel thews mayer h¶ckel and h¶ckel vaupel mayer and h¶ckel stone et a0al a0kallinowski and buhr 1995a mattern kallinowski herfarth and volm falk ward and bleehen le et a0al a0lartigau et a0al a0powell et a0al a0koong graffman bjork ederoth and ihse rampling cruickshank lewis fitzsimmons and workman kallinowski and buhr 1995a 1995blawrentschuk et a0al a0graffman et a0al a0graffman et a0al a0over space because of their physiology as a consequence of substantial differences in vascularity blood flow and oxygen consumption eg macroscopic heterogeneity between gray and white matter of the brain between subepicardial and subendocardial layers of the myocardium and between renal cortex and renal inner medullathere also are temporal changes in o2 levels in normal tissues griffith a0 moreover the supply of o2 can vary periodically due to rhythmic changes in microcirculatory blood flow which is reflected at all levels from the inflow arteries to the microcirculation finally within the microcirculation there are variations in microvascular flow due to regional regulation in response to varying metabolic demands kimura et a0al a0 important differences in o2 solubility across tissues affecting the relationship between po2 and [o2] were discussed earlier impact of pathology on heterogeneity of o2 levelsthe presence of pathology often significantly increases the amount and extent of oxygen heterogeneity both spatially and temporally vaupel harrison vaupel mayer a0 the presence of pathology often impacts the structuremorphology of the vessels in tumors there often is a significant amount of neoangiogenesis which results in much less ordered and less functional blood vessels busk et a0al a0 the resulting vessels are much less efficient in delivering blood and also tend to be much more prone to leak leakage from these vessels can cause increases in the interstitial pressure which can reduce the effectiveness of the microcirculation due to reduction of the perfusion pressure within the tumor capillaries fukumura duda munn jain a0pathological changes also can result in altered consumption of o2 in malignant tumors o2 consumption is likely to decrease due to poor oxygen delivery andor because of a switch to glycolysis due to metabolic reprogramming ie the warburg effect as a consequence of hif1α overexpression upregulation of oncogenes downregulation of suppressor genes and activation of certain signaling pathways vaupel multhoff a0 vaupel schmidberger mayer a0 pathology can also impact the integrity of the blood vessels for example tumor growth may physically impinge on the integrity of the blood vessels and the swartz 0c of figure distributions of multiple o2 levels made in patients with normal versus malignant tissue breast and cervix figure adapted from vaupel mayer a02017b p figure repeated o2 level measurementsa during each measurement session and over a0days breast cancer patient measured in skin and superficial breast tissue within the radiation field during a course of radiation therapy figure adapted from flood et a0al a0 p afor carlo erba ink epr line width increases with increasing o2 level but the relationship is nonlinear and can be impacted by several factors therefore the data are given here as line widthmetabolic abnormalities in diabetes can impact the structure of blood vessels causing either microangiopathy andor macroangiopathy the results of these processes can produce very significant local variations in the availability of fully functional vascular structures resulting in locally hypoxic regionsswartz 0c of pathology also can impact temporal changes of oxygen and the response to treatment the presence of pathology especially cancer eg acute and cycling hypoxia in cancers and peripheral vascular disease can result in significantly greater variability in o2 levels braun lanzen dewhirst a0 these include shortterm changes especially associated with the structural abnormalities of the microcirculation resulting in increased local variability in flow and longterm changes that develop over time such as those due to disease progression and responses to therapy baudelet et a0al a0 kimura et a0al a0 konerding fait gaumann a0 matsumoto there also is a potential for pathologies to interrelate with each other for example anemic hypoxia can develop in tumors due to the underlying systemic anemia of the patient vaupel mayer a0in addition to these underlying effects of pathology on tissue oxygen levels any applied therapeutic interventions are very likely to induce changes for example cell killing due to radiation or chemotherapy will alter oxygen consumption patterns these same therapies will also affect the o2 supplying vasculature via both antiangiogenic effects and”possibly”normalization of vessel structure jain a0 the effects of therapies will generally vary both spatially and temporally reiterating the complexity of meaningfully characterizing tissue oxygen levels analysis of the ability of clinically available techniques to directly measure levels of o2 in tissues andor resolve the heterogeneity of o2 distributions in tissuesalthough many techniques are often thought to measure actual o2 in tissues only a few actually have the potential to make o2 measurements directly in the tissues of interest springett swartz a0 tatum techniques that can potentially assess o2 directly in tissues include epr epel et a0al a0 swartz et a0al a0 swartz the eppendorf electrode vaupel h¶ckel mayer a0 some optical methods based on direct measurements of target molecules in tissues for example phosphorescence quenching of optical sensors placed directly in tissues or as part of a physical probe such as the œoxylite wen et a0al a0 and nmr relaxation techniques colliez et a0al a0two other types of measurements assess o2 in the vascular system blood gases do this directly while optical methods that measure both hemoglobin saturation and total hemoglobin especially near infrared spectroscopy [nirs] scheeren schober schwarte a0 provide a plausible link to the po2 in the bloodhowever the techniques most often used clinically to characterize tissue oxygenation do not in fact measure o2 directly instead they measure œindirect parameters that can be plausibly linked to actual o2 levels but only under appropriatedefined circumstances this latter group of techniques includes positron emission tomography pet imaging of glucose derivatives neveu et a0al a0 pet imaging of drugs that localize in hypoxic tissues tran laser doppler flow measures of metabolites that may be affected by o2 levels for example lactate and redox intermediates and several magnetic resonance imagingnuclear magnetic resonance mrinmr blood oxygenation level dependent bold imaging baudelet gallez a0 and mri egeland et a0al a0 note if their basis is understood and the data considered accordingly these can all provide clinically and physiologically useful information even though they do not provide direct information on the amount of o2 in the tissues direct measures of o2 in targeted tissues that potentially can be used in human subjectsthese are techniques that while they have the capability of providing direct quantitative measurements of o2 in homogeneous media cannot provide such data in tissues in vivo because the volumes that they sense are larger than the volumes of homogeneity of o2 in actively metabolizing tissues consequently all in vivo measurements of o2 are inherently averages of the actual oxygen content in that volume even neglecting the need to include measures of heterogeneity inside cells based on the usual volume of cells and assuming that differences are sought for aggregates of ‰¤ cells for a measurement of heterogeneity sensed within a a0mm diameter volume the spatial resolution needed to appropriately characterize o2 levels in this volume becomes million voxels the measuring techniques may not even provide a welldefined averaged po2 value within the volume that they sense for example sensors for the signal that is being measuredin the next sections we review the characteristics of each technique that can directly measure o2 levels in tissues we also briefly remark on the volumes they measure and how the measures obtained can be useful clinically see also ortezprado dunn vasconez castillo visco epr oximetry using appropriate particulate paramagnetic materials epr oximetry can provide direct measurements of o2 that is the epr signal is directly proportional to the amount of o2 epel bowman mailer halpern a0 swartz vaupel swartz 0cthe because each multisite sensor senses a volume that is much larger than capillary networks these techniques provide a volume averaged sampling of all compartments within the tissues the time resolution of the techniques can be milliseconds or shorterthe measured parameter of an epr spectrum that indicates the amount of o2 present is the line width of the observed resonance peak there usually is a fixed relationship between the line width and the amount of o2 with the relationship being specific for each type of paramagnetic material for example carbon charcoal or phthalocyanine particulates using particulate oximetric materials measurements can be continuous over any span of time and can be repeated indefinitely see example in figure a0 the method requires that the sensing material be injected or implanted in one or more regions of interest but thereafter all measurements can be made entirely noninvasively importantly the measurements can be carried out in a clinical setting and can fit into the workflow needed for patient careinitial clinical epr measurements of oxygen in tissues have used india ink as the oxygen sensor swartz et a0al a0 the carbon ps are the components that respond to oxygen lan beghein charlier gallez a0 after injection of “ a0µl of the suspension through a small needle the carbon ps disperse nonuniformly through the local region as small extracellular aggregates they are often engulfed by macrophages the resulting epr spectra in the region probed by the resonator ie the surface coil used for signal detection are a composite of the oxygendependent line widths from each of the ps in reality because of the relatively broad lines from the india ink ps the range of œoxygen levels that are likely to be present in the tissue and the limited number of ps in each subregion it is a challenge to resolve directly even the major groups of similar line widths therefore using the observed line width to provide a quantitative measure of oxygen would seem to have modest utility in itselfthe other method of clinical epr oximetry is based on the use of microcrystalline probes eg lipc lincbuo encapsulated in biocompatible polymers swartz et a0al a0 clinical measurements currently are being performed using the œoxychip which consists of oxygen sensitive microcrystals of lithium octanbutoxynaphthalocyanine lincbuo embedded in polydimethylsiloxane pdms hou khan gohain kuppusamy kuppusamy a0 hou et a0al a0 jarvis et a0al a0 the dimensions currently used in humans are cylinders that are a0 mm long with a diameter of a0mm the epr signal from the sensor oxychip reflects the po2 within the pdms which itself reflects an average of the po2 in contact with the external surface of the cylinder the dimensions of the oxy Answer:

Colon_Cancer

the recent focus is on the analysis of biological activities of extracts from thirteen folk medicinal plants from arid and semiarid zones of balochistan pakistan only a small proportionof them have been scientifically analyzed therefore the present investigation explores thebiochemical and bioactive potential of different plant parts superoxide dismutase wasdetected maximum in fagonia indica ± unitsg ascorbate peroxidase in tribulus pentandrus ± unitsg catalase and peroxidase were higher in peganum harmala ± and ± unitsg respectively maximum esterase and αamylaseactivity was found in zygophyllum fabago ± and ± mgg respectively flavonoid content was high in t pentandrus ± μgml the highest total phenolic content and tannin was revealed in f olivieri ± and ± μmg respectivelythe highest value of ascorbic acid was depicted in f bruguieri fbn ± μgg totalsoluble proteins and reducing sugars were detected higher in p harmala ± and ± mgg respectively the maximum total antioxidant capacity was depicted in tetraenasimplex ± μmg the highest value of lycopene and total carotenoids exhibited int terrestris ± and ± mgg respectively chlorophyll contents were foundmaximum in t pentandrus var pterophorus ± ± and ± uggrespectively all taxa exhibited antiinflammatory activity and antidiabetic potential z eurypterum seeds exhibited the highest antiinflammatory potential along with othertaxa indicated “ activity when compared with the standard drug diclofenac sodium seeds of t pentandrus exhibited the highest antidiabetic activity the othertaxa also exhibited inhibitory activity of αamylase ranging from “ compared withmetformin standard drug phytochemical screening revealed that selected taxaproved to be the potential source of natural antioxidants and could further be explored for invivo studies and utilized in pharmaceutical industries as potent therapeutic agents validatingtheir ethnopharmacological usesa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation ahmed a hameed a saeed s biochemical profile and bioactive potential ofthirteen wild folk medicinal plants frombalochistan pakistan one e0231612101371 pone0231612editor branislav t sˇiler institute for biologicalresearch s stanković university of belgradeserbiareceived march accepted july published august copyright ahmed this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation filesfunding the authors received no specificfunding for this workcompeting interests the authors have declaredthat no competing interests exist one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantsintroductionwild medicinal plants are being utilized as folk medicines globally since long before recordedhistory most of the world™s population depends mainly on these herbal medicines to cure various ailments and to reduce risks of chronic human illness such as inflammation [ ] currently most prevailing and powerful drugs used were derived from medicinal plants [ ]based on their antioxidants originates from their ethnopharmacological utilization manymedicinal plants extracts constituted various types of chemicals like alkaloids flavonoids terpenoids glycosides tannins etc each has a property to control a variety of biological andpharmacological activities such as antimicrobial antiparasitic antidiabetic antioxidantantiinflammatory and anticholinesterase [ ] diabetes mellitus is commonly related tometabolic disorders linked with numerous macro and microvascular problems that acceleratemorbidity and mortality [“] it has also been revealed that oxidative stress rise in free radicals and deterioration of antioxidant defense may mediate the prevalence of diabetesassociated complications in diabetic patients [ ] many degenerative disorders like rheumatoidarthritis joints and shoulder inflammation heart disease muscular inflammation asthmacancer and inflammation of the gastrointestinal tract are commonly related to inflammatoryprocesses [ ]it has been proved that herbal extracts containing phytoantioxidants particularly polyphenols flavonoids tannins and other associated compounds have progressive health effects anddecrease disease risk recently much attention has been paid to reveal the effects of bioactivecompounds and antioxidant potentials of medicinal plants to analyze their pharmacologicalactivities [ ] therefore this study explores and compares nineteen biochemical compounds including five pigments for the first time in thirteen species of zygophyllaceae moreover in vitro antioxidant antidiabetic and anti inflammation activities were testedthe zygophyllaceae consists of diverse habits of wild flora including succulents herbsundershrub shrubs and small trees the habitat of these plants is predominantly desert orsaline areas of temperate and tropical regions around the globe [ ] many taxa of the family are used ethnomedicinally [ ]fagonia l is a genus of wild flowering plants of the family zygophyllaceae having about species all over the world the distribution of the genus includes parts of africa the mediterranean basin asia and parts of the america in pakistan species are reported earlierseven species are found during the present study in southern zones of balochistan provincefagonia species were collected to determine the presence of phytochemical compounds earlier reported phytochemicals were alkaloids coumarins flavonols saponins triterpenoid saponins tannins cardiac glycosides and rosmarinic acid [ ]peganum harmala l earlier in zygophyllaceae now placed in nitraceae is a perennial herbaceous plant usually “ cm tall having yellowishwhite flowers blooming in april to mayand commonly dispersed in many regions of the province earlier commonly known phytochemicals from p harmala are alkaloids harmine harmaline flavonoids and anthraquinones [ ]genus tribulus l is considered as the most complex genus in zygophyllaceae because ofthe enormous number of invalid specific epithets and also of the variations present in variouspopulations in pakistan four species are reported tribulus species contain a number of steroidal saponins which may account for their use in muscle building conditioning and treatment of certain ailments t terristrus is earlier reported for rich phytochemicals steroidalsaponins flavonoids alkaloids and lignan amides two new furostanol glycosides namedtribufurosides i j were isolated from the fruits of t terrestris so far only few species of tribulus have been chemically analyzed one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantszygophyllum linn with about species grows mainly between northern africa and central asia particularly in arid and semiarid areas zygophyllum species have been phytochemically studied leading to the identification of various classes of compounds includingtriterpenes flavonoids saponins sterols simple phenolic compounds and esters themain chemical constituents described from zygophyllum species are zygophyllin quinovicacid and glycosides which have been shown to have antiinflammatory and antipyretic activity z simplex l is an annual succulent halophyte plant it was found that it belongs togenus tetraena a sister to genus zygophyllum and recently reassigned to such genus and couldbe referred as tetraena simplex l beier thulin syn z simplex l zygophyllaceae t simplex syn z simplex l was documented as a traditional remedy for the treatment ofdry scaly patches cleaning the skin and as an analgesic and antiinflammatory agent phytochemically it was reported to contain flavonoids steroids and saponins [ “]materials and methodsplant collectionthe selection of medicinal plants was based on ethnobotanical appraisal wild plants were collected from different areas of balochistan pakistan no specific permissions were required forthese locationsactivities as plants are growing naturally no protected area and species weredisturbed during the fieldwork voucher specimens were prepared identified following themethod as used in the flora of pakistan verified from pakistan plant database ppd andsubmitted in botanical garden herbarium of the university of balochistan quetta furthermore records were also available in the open herbarium for future reference wwwopenherbarium fresh aerial parts of nine plants fruits of two species of tribulus dryaerial parts of thirteen plants and ten seeds were collected from different ecological zones ofbalochistan table for biochemical profiling fresh plant samples were transported and kepttable list of the selected taxa with geographical coordinates of the collection sites and voucher specimen™s numbervoucher nosite of collectionelevation meter above sea levelpart usedds dapquetta000208mushkafdap fapquetta000090dsquetta000157dap fapdap fapquetta000218quetta000207quettanushkipanjgursibids dap fapquetta000222nushki stoneds dap fapquetta000221nushki janglat areads dapds dapds dapds dapquetta000220quetta000219panjgurpanjgurquetta000224hingol national parkquetta000216ds dap fs fapquetta000215ds dap fs fapquetta000085ds dap fapquetta000209ds dap fapquetta000002panjgurnushkiquettasibiquettas noplant namez propinquumz fabagoz eurypterumt simplexf indicaf bruguierif olivierif bruguierif paulayanaf bruguierit macropterust pentandrus var pterophorust terrestrist pentandrusp harmalaplant codezpzfzetsfifbnfofbpfpfbhtmtpptttpph““““““““““dry seeds ds dry aerial parts dap fresh seeds fs fresh aerial parts fap peganum harmala ph tribulus terrestris tt t pentandrustp tmacropterus t m t pentandrus var pterophorus tpp t pentandrus tp zygophyllum fabago zf z propinquum zp z eurypterum ze tetraena simplexts fagonia indica fi f paulayana fp f bruguieri fbp f bruguierifbn f bruguieri fbh f olivieri fo vouchers submitted in open herbariumwwwopenherbarium list verified by wwwtropicos101371 pone0231612t001 one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantsat ºc samples were collected shade dried and stored at room temperature experimentswere conducted at plant breeding and genetics division mab lab nuclear institute foragriculture and biology niab faisalabad pakistanchemicals and reagents phosphate phosphate sodium chloride acetone sodium carbonate were purchased from acros anics usa methanol meoh dithiothreitoldtt ethyline diamine tetra acetic acid edta 2propanol nitro blue tetrazolium chloride nbt guaiacol αnaphthyl acetate solution fast blue bb xylenol orange disodium saltsulfuric acid h2so4 sodium chloride nacl ferrous ammonium sulphate glycerol odianisidine potassium acetate αamylase enzyme 5dinitrosalicylic acid bovine serum albuminhydrochloric acid hcl hydrogen peroxide h2o2 sodium acetate glacial acetic acid aluminium chloride alcl2 sodium phosphate and ™azinobis3ethylbenzothiazoline6sulfonic acid abts were purchased from sigmaaldrich merck germany polyvinylpyrrolidone pvpp starch and acetic acid were purchased from bioworld genelinx international inc usa folinciocalteu fc reagent bradford dye sodium dodecyl sulfatesds were purchased from thermo fisher scientific uk 6dichloroindophenol dcipwas purchased from millipore sigma usextraction of antioxidant enzymesphosphate buffer mm ph was used for plants extraction g of each sample wassubjected in ml phosphate buffer to ground further the mixture was centrifuged at×g min 4ºc now the supernatant of this extracted plant material used to performfurther phytochemical activities all the data were taken in replicates of threesuperoxide dismutase sod assaythe method of was used to determine sod activity by homogenizing the fresh aerial partsand fruits of selected taxa in phosphate buffer mm ph edta mm and dtt mm following the procedure of and was further analyzed by assessing its property to stopthe photochemical reduction of nitroblue tetrazolium as explicated by one unit of sodactivity was demarcated as the amount of enzyme causing inhibition of photochemicalreduction of nitroblue tetrazolium absorption was measured in double beam spectrophotometer hitachi u2800peroxidase pod assaythe assessment of pod activity was carried out using method with minor changes thehomogenized mixture of the aerial parts and fruits prepared in ml phosphate buffer mm ph edta mm and dtt mm the assay solution contained μl distilledwater phosphate buffer mm ph guaiacol mm h2o2 mm and μl enzymeextract the addition of enzyme extract initiated the reaction at nm absorbance raise wasnoted at interval of sec absorbance change of minˆ’ was demarcated as one unit podactivity enzyme activity was expressed on the basis of fresh sample weightcatalase cat assaycatalase activity was measured by homogenizing the aerial parts and fruit samples prepared inphosphate buffer mm ph edta mm and dtt mm cat was assessedaccording to the method used by the activity was measured in a solution containing mm h2o2 and ml enzyme extract at nm decrease in absorbance was recorded after one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantsinterval of seconds change in absorbance of per min defined cat activity of one unitenzyme activity was expressed on a fresh weight basisascorbate peroxidase apx assaythe assessment of apx activity was carried out following the method used by sampleswere extracted in phosphate buffer mm ph the measurement of apx the assaybuffer contained potassium phosphate buffer mm ph edta m ascorbate mm ml of h2o2 and μl supernatant at nm absorbance decrease was noted afterevery thirty seconds to estimate the oxidation rate of ascorbic acid hydrolytic enzymesesterase activity the αesterase was determined by using the method as suggested by αnaphthyl acetate was used as substrates the reaction mixture contained mm αnaphthyl acetate mm acetone and phosphate buffer m ph and enzymeextract the mixture obtained was incubated for min at 27ºc in dark after min ml ofstaining solution was added fast blue bb and sds with ratio of and again incubated in the dark for min at ºc absorbance at nm was measured for αnaphtholproduced using standard curve enzyme activity was αnaphthol produced in μm minˆ’ g wtalphaamylase activity a modified method for alphaamylase activity was followed forall plant samples as described by other biochemical parameterstotal oxidant status tos the method of was used to determined tos the assayis established on ferrous ion oxidation into ferric ion the presence of oxidants in the samplein acidic medium and ferric ion measurement produced by xylenol orange was measuredobserved the assay is based on two mixtures r1 stock xylenol orange solution 038g in500μl of 25mm h2so4 04g nacl μl glycerol and volume up to 50ml with 25mmh2so4 sample extract and r2 g odianisidine g ferrous ammonium sulphateii absorption measured at 560nm after minutes by using a spectrophotometerpigment analysis the concentration of lycopene chlorophyll a and b total chlorophylland carotenoids were examined by the method of samples g were ground in acetone and centrifuged at g for min absorbance measured at and nm byusing a spectrophotometer partiallytotal phenolic contents tpc and tannin a micro colorimetric assay was used tomeasure tpc by using folinciocalteu fc reagent with some modifications005 gsample was kept in methanol in dark for hours after hours the supernatant wastaken μl fc reagent and ml sodium carbonate mm were added to it placethis mixture at room temperature for one hour and took reading at nm linear regressionequation was calculated by using a standard curve of gallic acid at different concentrations tomeasure tannin 01g pvpp was added in the above prepared sample vortexed vigorouslyand centrifuged again at g the absorbance was measured at nmdetermination of total flavonoid content the assay was determined by a colorimetricmethod using quercetin as standard take μl sample prepared in methanol extractand phosphate buffer mm ph added μl alcl2 μl potassium acetate1m incubate the mixture at room temperature for minutes and take the reading at nm absorbancetotal antioxidant capacity a modified method of tac was followed as described by due to the presence of antioxidants in the sample abts assay represents a decrease of one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plants2azinobis 3ethylbenzothiazoline6 sulfonate radical cation abts¢ bluegreen in colorinto original abts colorless compound the antioxidants of the sample extract according totheir content decolorize the abts¢ radical cation the reaction mixture contained reagentr1 mixture of sodium acetate buffer solution and glacial acetic acid ph58 sample extractand reagent r2 mixture of sodium phosphate buffer solution glacial acetic acid hydrogenperoxide and abts after min at wavelength of 660nm the absorption of each reactionmixture was measured this analysis used asa ascorbic acid to develop a calibration curvethe results for antioxidant contents found in plant extracts were measured as μm asa equivalent to one gramreducing sugars sugar content assessment of reducing sugars level in the plant samples was determined by dinitrosalicylic acid method proposed by total soluble protein contentprotein estimation of plant samples was based on quantitative protein analysis described by aerial parts and fruits samples were homogenized in potassium phosphate mm ph supernatant 5μl and nacl 01n mixed with10 ml of bradford dye incubate the mixturefor minutes to get a proteindye complex measure the quantity at nm absorbance by aspectrophotometerascorbic acid asa 6dichloroindophenol dcip method hameed wasfollowed for measurement of ascorbic acid which measures reduced vitamin c only in shorteach molecule of ascorbic acid converts a dcip molecule into a reduced 6dichloroindophenol dciph2 and this conversion can be recorded as reduced absorption at nm the calibration curve was drawn with the help of known series of ascorbic acid concentrationsascorbate concentration in unknown sample was found by calculating simple linear regressionequationin vitro antidiabetic activity enzyme αamylase inhibition methodthe in vitro antidiabetic activity was determined by assaying the inhibitory activity of theenzyme αamylase which involves the breakdown of starch to produce glucose in thismethod ml of methanolic extracts of all species were tested separately and thus added to ml of the enzyme αamylase in a testtube and incubated for min at ˚c then ml of starch solution was added into it and again incubated for min at ˚c then ml 5dinitrosalicylic acid reagent was added into it in order to terminate the reaction the reaction mixture was then incubated in a boiling water bath for min and then allowed it to cool at roomtemperature the absorbance of the reaction mixture was then measured at nm in a spectrophotometer the standard control of the reaction without the extract represents the enzyme activity the age inhibition of enzyme activity of αamylase was determined byage inhibition of a 00 amylase¼enzyme activity of control 00 enzyme activity of extractenzyme activity of controlx ¼ results compared with standard drug glucophage metformin martin dow pharmaceutical pak ltdin vitro antiinflammory activity protein denaturation methodthe protein denaturation assay was determined using a modified method as described by briefly the reaction mixture ml ph consisted of ml of bovine serum albumin one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plants aqueous solution and ml of distilled water the ph was adjusted to using a smallamount of n hcl ml of acetone or aqueous extract with final concentrations of to mgml was added to the reaction mixture these were incubated at ˚c for min and thenheated at ˚c for min after cooling the samples ml of phosphate buffer solution ph was added the turbidity was measured by a spectrophotometer at nm for the negative control ml of distilled water and ml of bovine serum albumin were used diclofenac sodium with the final concentration of and μgml was used asreference drug the percentage inhibition of protein denaturation was calculated by using thefollowing formula age inhibition ¼½abs control 00 abs sample�abs controlx ¼ results compared with standard drug diclofenac sodium diclofenac na getz pharmapakistan pvt ltdstatistical analysisdata was recorded in mean ± sem resulting data were analyzed by applying descriptive statistics twoway anova with three replications was used in analyses the significance of datawas tested by analysis of variance and tukey hsd test at p0001using xlstat softwareversion copyright addinsoft “ httpwwwxlstatcomresults and discussionthe present study is based on the analysis of biological activities of extracts from thirteen plantspecies of zygophyllaceae remarkably important angiosperm family with many taxa beingused in folk medicines there is a scarcity of data in the literature about these plants as suchmaking a comparison of the results obtained in the present studies was difficult nonetheless afew papers reported some biological activities of t terrestris p harmala and few species offagonia the present investigation explores the presence of enzymatic constituents such assod pod apx cat esterase alpha amylase nonenzymatic antioxidants and other phytochemicals like asa tos tac tsp tpc tf tannins and pigments selected plants also provided evidence for the antidiabetic and antiinflammatory potential of varying extent in seedsand aerial parts the difference in prooxidants and antioxidants causes oxidative stress andchronic diseases in the body cellular damage results in causing cancer one of the mechanisms behind the antioxidation is free radical scavenging action pod helps in scavenging the reactive oxygen species ros causing cell oxidative injury the highest values ofperoxidase and catalase were depicted in the aerial parts of p harmala ± unitsg fwt and ± unitsg f wt respectively as shown in s1a and s1b fig respectively plantspecies having high antioxidant activities can be utilized for different therapeutic applicationsfor the treatment of oxidative stressinduced diseasesthe highest apx value was recorded in t pentandrus ± unitsg f wt s1d figascorbate peroxidase apx enzyme is crucial for the protection from damage by h2o2 andhydroxyl radicals ¢oh antioxidant enzymes activities namely cat pod and sodwere not tested earlier in selected wild medicinal taxa while few records reported earlier inother wild taxa like rumex obtusifolius a wild medicinal plant and found to have a good antioxidant capacity similarly calamintha officinalis also has potent antioxidants alphaamylase and esterase activities were higher in z fabago ± mgg and ± μmming f wt respectively s1e and s1f fig respectively esterase plays an important role in thedisintegration of natural constituents and industrial pollutants and other toxic chemicals it is one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantsalso beneficial for the production of optically pure compounds perfumes and antioxidants the antioxidant enzyme catalase is present in all animal tissues with its highest activities inliver and red blood cells which defend the tissues from highly reactive hydroxyl radicals bydecomposing hydrogen peroxide decrease amount of catalase causes numerous damages dueto hydrogen peroxide and superoxide radical assimilation superoxide dismutase enzymereferred as the significantly involved in cellular defense therefore it is considered as an indicator of antioxidant capacity the highest value of superoxide dismutase ± unitsgf wt was observed in f indica shown in s1c fig traditionally this plant is used for anticancer treatment and possibly detected high concentrations of sod and tac may be responsiblefor this therapeutic effectmaximum tac was depicted in fresh samples of t simplex ±001μmg f wt followed by f indica ±004μmg f wt shown s2a fig no significant variation wasdetected among all dry aerial parts of selected plants in general maximum tac was detectedin the seeds of z eurypterum ± μmg dry wt followed by the aerial parts of t simplex± μmgdry wt in the seeds of the selected taxa no significant variation wasdetected the maximum tac was in f olivieri ± μmg s wt the second highestvalue was in f bruguieri fbn ± μmg s wt previously the aerial parts of f longispina were reported to be a good source for natural antioxidants previously f cretica wasalso found to have high antioxidant and radical scavenging potential due to the high tpc andtfc f olivieri can serve as a natural source to develop the free radical scavengers beneficial in the prevention of oxidative stress development total flavonoid content tfc in the methanolic extract showed maximum quantity infresh samples of t pentandrus ±49μgml sample shown s2b fig fruit samples freshshowed the highest value of flavonoid and ascorbic acid in t terrestris ± μgml sample and ± μgg f wt respectively in dry aerial parts of t pentandrus flavonoid content gives a maximum amount of tfc ± μg ml sample followed by f bruguieri fbh ± μgml sample while in the seeds of p harmala showed maximum tfc±167μgml sample followed by f bruguieri fbp ± μgml sample flavonoids are considered to be effective free radical scavengers in fruits vegetables and medicinalplants highest ascorbic acid reported in p harmala shown s2c fig ascorbic acid is involvedin a number of physiological processes such as pod and sod the highest flavonoidsand ascorbic acid content in fruits of t terrestris and aerial parts of t pentandrus in the present study validated its traditional medicine use and may be responsible to cure various ailments f olivieri as shown s2c fig that gives the highest amount of tanninstotal soluble protein and reducing sugar were high in fresh aerial parts of p harmalashown in s3a and s3b fig respectively earlier isolated antioxidant protein from p harmala seeds possessed antioxidant activity and this activity was due to the presence of hydrophobic amino acids p harmala is one of the most frequently used medicinal plants to treathypertension and cardiac disease worldwide the maximum total soluble proteins ± mgg dry wt were depicted in dry aerial parts of t simplex no significant variation wasobserved in seed samples the highest value of total soluble proteins ± mgg s wt wasfound in f bruguieri fbn total oxidant status tos was lower in f indica ± μmg f wt shown in s3d figtotal flavonoid content was calculated in and expressed as μgml in methanolic extractusing quercetin as standard table significant difference was observed among all selectedspecies of zygophyllaceae in the aerial parts t pentandrus flavonoid content gives maximumamount of tfc ± μg ml sample followed by f bruguieri fbh ± μgmlsample while seeds of p hermala showed maximum tfc ±167μgml sample one 101371 pone0231612 august one 0ctable phytochemical analysis in the dry aerial parts of the selected taxaselected taxa drytotal flavonoidtotal phenolictannintotal solubleascorbic acidreducingamylasetotal antioxidantbiochemical profile and bioactive potential of few medicinal plantsaerial partscontentcontentsprotein contentcontentsugarsumg dry wtumg drymgg dry wtugg dry wt mgg dry wt mgg dryp harmalat simplexquercetin equlientμgml sample±451bc±2275a±398bcde±5375az propinquum±1467bcd±4575az fabago±239b±2850at pentandrus varpterophorust terrestris±884de±1725a±2465de±1650at pentandrus±1642a±1700af bruguieri fbh±3708b±1670af bruguieri fbp±2456cde±7175awt±1625a±550a±150a±4825a±4125a±1500a±2450a±742a±450a±433bc±4abc±016abc±633a±8d±028cd±667bc±4abc±055ab±333a±135c±028a±033abc±075c±012cd±067bc±175c±02cd±4bc±105c±02cd±979ab±238ab±028cd±1333c±35ab±008dcapacityμmg dry wt±251a±234a±345a±269a±284a±345a±416a±186a±256a±254a±248a±246a±369awt±264a±34a±283a±66a±472a±1132a±962a±1817a±2642a±208a±1245a±151a±132af indica±3321e±5400a±75a±1867abc±85ab±004cdf bruguieri fbn±318cde±1350af paulayana±424e±550af olivieri±1873cd±475a±525a±775a±1275a±2bc±225ab±004cd±1133abc±5ab±016bcd±167ab±275a±02ddata are presented as mean values ± standard error n statistical analysis anova test and tukey hsd the different letters above the values in the same columnindicate significant differences with tolerance values with the same superscript letters in the same column are not significant101371 pone0231612t002followed by f bruguieri fbp ± μgml sample table total phenolic contenttpc was estimated in aerial parts of selected taxa no significant tpc variation was foundamong table in general the highest tpc was depicted in t pentandrus var pterophorus± μmg dry wt followed by f bruguieri fbn ± μmg dry wtseeds of selected plant samples showed significant variation highest tpc was detected in zpropinquum ±775μmg s wt followed by f bruguieri fbn ± μmg swt as shown in table no significant tannin variation was detected among aerial parts aswell as in seeds of all tested taxa table however the highest amount of tannins was estimated in t pentandrus var pterophorus ± μmg dry wt followed by z fabago± μmg dry wt in seeds the highest amount of tannins was estimated in p hermala ± μmg s wt followed by z propinquum ± μmg s wt shown intable ascorbic acid was observed in aerial parts of all selected taxa and significant difference found among the studied taxa table the highest value of asa was found in f olivieri± μgg dry wt followed by f bruguieri fbp asa content in seeds showed no significant variation in general the highest asa content was found in f bruguieri fbh ± μgg s wt given in table alphaamylase activity in dry aerial parts of selected plantswas assessed table no significant variation was found among the taxa of zygophyllaceae one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantstable phytochemical analysis in the seeds of the selected taxaselected taxaseedstotal flavonoidcontentquercetin equlientμgml sampletotal phenoliccontentsumg seed wttannintotal solubleprotein contentascorbic acidcontentreducingsugarsamylaseumg seedwtmgg seed wtugg seed wtmgg seedwtmgg seedwttotal antioxidantcapacityμmg seed wtp harmala±1394a±1350abz propinquum±24727a±775az eurypterum±488a±135abt pentandrus varpterophorust terrestris±3498a±1475ab±7156a±50bct pentandrus±20884a±1325cf bruguieri fbh±1005a±2887af bruguieri fbp±161a±3025bcf bruguieri fbn±878a±1148cf paulayana±14735a±3500abcf olivieri±2518a±300abc±2575a±175a±1000a±550a±2025a±1075a±1111a±675a±352a±650a±2750a±80a±4575a±087c±0a±025a±04ab±2a±05a±004a±1633a±25a±02ab±433a±1525a±012a±2a±4a±02a±2942a±219a±009c±33a±175a±008c±038abc±226bc±208bc±151c±17ab±283ab±1184a±094bc±273a±279a±221a±766a±635a±969a±201a±258a±184a±185a±106ab±251bc±169a±3067a±18a±024bc±467a±25a±008c±038ab±057abc±316a±243adata are presented as mean values ± standard error n statistical analysis anova test and tukey hsd the different letters above the values in the same columnindicate significant differences with tolerance values with the same superscript letters in the same column are not significant101371 pone0231612t003table pigment analysis in the dry aerial parts of the selected taxaselected taxalycopenechlorophyll achlorophyll btotal carotenoidstotal chlorophyllp harmalat simplexz propinquumz fabagot pentandrus var pterophorust terrestrist pentandrusf bruguieri fbhf bruguieri fbpf indicaf bruguieri fbnf paulayanaf olivierimgg dry wt±003ab±013b±003b±061ab±139a±001ab±014ab±034ab±024ab±042ab±394ab±102ab±112abugg dry wt±66ab±596bc±005bc±3171bc±005a±024a±196ab±3396bc±537a±2383ab±10962abc±517abc±6862augg dry wt±05ab0b±116b±1767ab±6301a±161ab±498ab±343b±1183ab±821ab±8196

cancer7551

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: the recent focus is on the analysis of biological activities of extracts from thirteen folk medicinal plants from arid and semiarid zones of balochistan pakistan only a small proportionof them have been scientifically analyzed therefore the present investigation explores thebiochemical and bioactive potential of different plant parts superoxide dismutase wasdetected maximum in fagonia indica ± unitsg ascorbate peroxidase in tribulus pentandrus ± unitsg catalase and peroxidase were higher in peganum harmala ± and ± unitsg respectively maximum esterase and αamylaseactivity was found in zygophyllum fabago ± and ± mgg respectively flavonoid content was high in t pentandrus ± μgml the highest total phenolic content and tannin was revealed in f olivieri ± and ± μmg respectivelythe highest value of ascorbic acid was depicted in f bruguieri fbn ± μgg totalsoluble proteins and reducing sugars were detected higher in p harmala ± and ± mgg respectively the maximum total antioxidant capacity was depicted in tetraenasimplex ± μmg the highest value of lycopene and total carotenoids exhibited int terrestris ± and ± mgg respectively chlorophyll contents were foundmaximum in t pentandrus var pterophorus ± ± and ± uggrespectively all taxa exhibited antiinflammatory activity and antidiabetic potential z eurypterum seeds exhibited the highest antiinflammatory potential along with othertaxa indicated “ activity when compared with the standard drug diclofenac sodium seeds of t pentandrus exhibited the highest antidiabetic activity the othertaxa also exhibited inhibitory activity of αamylase ranging from “ compared withmetformin standard drug phytochemical screening revealed that selected taxaproved to be the potential source of natural antioxidants and could further be explored for invivo studies and utilized in pharmaceutical industries as potent therapeutic agents validatingtheir ethnopharmacological usesa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation ahmed a hameed a saeed s biochemical profile and bioactive potential ofthirteen wild folk medicinal plants frombalochistan pakistan one e0231612101371 pone0231612editor branislav t sˇiler institute for biologicalresearch s stanković university of belgradeserbiareceived march accepted july published august copyright ahmed this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation filesfunding the authors received no specificfunding for this workcompeting interests the authors have declaredthat no competing interests exist one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantsintroductionwild medicinal plants are being utilized as folk medicines globally since long before recordedhistory most of the world™s population depends mainly on these herbal medicines to cure various ailments and to reduce risks of chronic human illness such as inflammation [ ] currently most prevailing and powerful drugs used were derived from medicinal plants [ ]based on their antioxidants originates from their ethnopharmacological utilization manymedicinal plants extracts constituted various types of chemicals like alkaloids flavonoids terpenoids glycosides tannins etc each has a property to control a variety of biological andpharmacological activities such as antimicrobial antiparasitic antidiabetic antioxidantantiinflammatory and anticholinesterase [ ] diabetes mellitus is commonly related tometabolic disorders linked with numerous macro and microvascular problems that acceleratemorbidity and mortality [“] it has also been revealed that oxidative stress rise in free radicals and deterioration of antioxidant defense may mediate the prevalence of diabetesassociated complications in diabetic patients [ ] many degenerative disorders like rheumatoidarthritis joints and shoulder inflammation heart disease muscular inflammation asthmacancer and inflammation of the gastrointestinal tract are commonly related to inflammatoryprocesses [ ]it has been proved that herbal extracts containing phytoantioxidants particularly polyphenols flavonoids tannins and other associated compounds have progressive health effects anddecrease disease risk recently much attention has been paid to reveal the effects of bioactivecompounds and antioxidant potentials of medicinal plants to analyze their pharmacologicalactivities [ ] therefore this study explores and compares nineteen biochemical compounds including five pigments for the first time in thirteen species of zygophyllaceae moreover in vitro antioxidant antidiabetic and anti inflammation activities were testedthe zygophyllaceae consists of diverse habits of wild flora including succulents herbsundershrub shrubs and small trees the habitat of these plants is predominantly desert orsaline areas of temperate and tropical regions around the globe [ ] many taxa of the family are used ethnomedicinally [ ]fagonia l is a genus of wild flowering plants of the family zygophyllaceae having about species all over the world the distribution of the genus includes parts of africa the mediterranean basin asia and parts of the america in pakistan species are reported earlierseven species are found during the present study in southern zones of balochistan provincefagonia species were collected to determine the presence of phytochemical compounds earlier reported phytochemicals were alkaloids coumarins flavonols saponins triterpenoid saponins tannins cardiac glycosides and rosmarinic acid [ ]peganum harmala l earlier in zygophyllaceae now placed in nitraceae is a perennial herbaceous plant usually “ cm tall having yellowishwhite flowers blooming in april to mayand commonly dispersed in many regions of the province earlier commonly known phytochemicals from p harmala are alkaloids harmine harmaline flavonoids and anthraquinones [ ]genus tribulus l is considered as the most complex genus in zygophyllaceae because ofthe enormous number of invalid specific epithets and also of the variations present in variouspopulations in pakistan four species are reported tribulus species contain a number of steroidal saponins which may account for their use in muscle building conditioning and treatment of certain ailments t terristrus is earlier reported for rich phytochemicals steroidalsaponins flavonoids alkaloids and lignan amides two new furostanol glycosides namedtribufurosides i j were isolated from the fruits of t terrestris so far only few species of tribulus have been chemically analyzed one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantszygophyllum linn with about species grows mainly between northern africa and central asia particularly in arid and semiarid areas zygophyllum species have been phytochemically studied leading to the identification of various classes of compounds includingtriterpenes flavonoids saponins sterols simple phenolic compounds and esters themain chemical constituents described from zygophyllum species are zygophyllin quinovicacid and glycosides which have been shown to have antiinflammatory and antipyretic activity z simplex l is an annual succulent halophyte plant it was found that it belongs togenus tetraena a sister to genus zygophyllum and recently reassigned to such genus and couldbe referred as tetraena simplex l beier thulin syn z simplex l zygophyllaceae t simplex syn z simplex l was documented as a traditional remedy for the treatment ofdry scaly patches cleaning the skin and as an analgesic and antiinflammatory agent phytochemically it was reported to contain flavonoids steroids and saponins [ “]materials and methodsplant collectionthe selection of medicinal plants was based on ethnobotanical appraisal wild plants were collected from different areas of balochistan pakistan no specific permissions were required forthese locationsactivities as plants are growing naturally no protected area and species weredisturbed during the fieldwork voucher specimens were prepared identified following themethod as used in the flora of pakistan verified from pakistan plant database ppd andsubmitted in botanical garden herbarium of the university of balochistan quetta furthermore records were also available in the open herbarium for future reference wwwopenherbarium fresh aerial parts of nine plants fruits of two species of tribulus dryaerial parts of thirteen plants and ten seeds were collected from different ecological zones ofbalochistan table for biochemical profiling fresh plant samples were transported and kepttable list of the selected taxa with geographical coordinates of the collection sites and voucher specimen™s numbervoucher nosite of collectionelevation meter above sea levelpart usedds dapquetta000208mushkafdap fapquetta000090dsquetta000157dap fapdap fapquetta000218quetta000207quettanushkipanjgursibids dap fapquetta000222nushki stoneds dap fapquetta000221nushki janglat areads dapds dapds dapds dapquetta000220quetta000219panjgurpanjgurquetta000224hingol national parkquetta000216ds dap fs fapquetta000215ds dap fs fapquetta000085ds dap fapquetta000209ds dap fapquetta000002panjgurnushkiquettasibiquettas noplant namez propinquumz fabagoz eurypterumt simplexf indicaf bruguierif olivierif bruguierif paulayanaf bruguierit macropterust pentandrus var pterophorust terrestrist pentandrusp harmalaplant codezpzfzetsfifbnfofbpfpfbhtmtpptttpph““““““““““dry seeds ds dry aerial parts dap fresh seeds fs fresh aerial parts fap peganum harmala ph tribulus terrestris tt t pentandrustp tmacropterus t m t pentandrus var pterophorus tpp t pentandrus tp zygophyllum fabago zf z propinquum zp z eurypterum ze tetraena simplexts fagonia indica fi f paulayana fp f bruguieri fbp f bruguierifbn f bruguieri fbh f olivieri fo vouchers submitted in open herbariumwwwopenherbarium list verified by wwwtropicos101371 pone0231612t001 one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantsat ºc samples were collected shade dried and stored at room temperature experimentswere conducted at plant breeding and genetics division mab lab nuclear institute foragriculture and biology niab faisalabad pakistanchemicals and reagents phosphate phosphate sodium chloride acetone sodium carbonate were purchased from acros anics usa methanol meoh dithiothreitoldtt ethyline diamine tetra acetic acid edta 2propanol nitro blue tetrazolium chloride nbt guaiacol αnaphthyl acetate solution fast blue bb xylenol orange disodium saltsulfuric acid h2so4 sodium chloride nacl ferrous ammonium sulphate glycerol odianisidine potassium acetate αamylase enzyme 5dinitrosalicylic acid bovine serum albuminhydrochloric acid hcl hydrogen peroxide h2o2 sodium acetate glacial acetic acid aluminium chloride alcl2 sodium phosphate and ™azinobis3ethylbenzothiazoline6sulfonic acid abts were purchased from sigmaaldrich merck germany polyvinylpyrrolidone pvpp starch and acetic acid were purchased from bioworld genelinx international inc usa folinciocalteu fc reagent bradford dye sodium dodecyl sulfatesds were purchased from thermo fisher scientific uk 6dichloroindophenol dcipwas purchased from millipore sigma usextraction of antioxidant enzymesphosphate buffer mm ph was used for plants extraction g of each sample wassubjected in ml phosphate buffer to ground further the mixture was centrifuged at×g min 4ºc now the supernatant of this extracted plant material used to performfurther phytochemical activities all the data were taken in replicates of threesuperoxide dismutase sod assaythe method of was used to determine sod activity by homogenizing the fresh aerial partsand fruits of selected taxa in phosphate buffer mm ph edta mm and dtt mm following the procedure of and was further analyzed by assessing its property to stopthe photochemical reduction of nitroblue tetrazolium as explicated by one unit of sodactivity was demarcated as the amount of enzyme causing inhibition of photochemicalreduction of nitroblue tetrazolium absorption was measured in double beam spectrophotometer hitachi u2800peroxidase pod assaythe assessment of pod activity was carried out using method with minor changes thehomogenized mixture of the aerial parts and fruits prepared in ml phosphate buffer mm ph edta mm and dtt mm the assay solution contained μl distilledwater phosphate buffer mm ph guaiacol mm h2o2 mm and μl enzymeextract the addition of enzyme extract initiated the reaction at nm absorbance raise wasnoted at interval of sec absorbance change of minˆ’ was demarcated as one unit podactivity enzyme activity was expressed on the basis of fresh sample weightcatalase cat assaycatalase activity was measured by homogenizing the aerial parts and fruit samples prepared inphosphate buffer mm ph edta mm and dtt mm cat was assessedaccording to the method used by the activity was measured in a solution containing mm h2o2 and ml enzyme extract at nm decrease in absorbance was recorded after one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantsinterval of seconds change in absorbance of per min defined cat activity of one unitenzyme activity was expressed on a fresh weight basisascorbate peroxidase apx assaythe assessment of apx activity was carried out following the method used by sampleswere extracted in phosphate buffer mm ph the measurement of apx the assaybuffer contained potassium phosphate buffer mm ph edta m ascorbate mm ml of h2o2 and μl supernatant at nm absorbance decrease was noted afterevery thirty seconds to estimate the oxidation rate of ascorbic acid hydrolytic enzymesesterase activity the αesterase was determined by using the method as suggested by αnaphthyl acetate was used as substrates the reaction mixture contained mm αnaphthyl acetate mm acetone and phosphate buffer m ph and enzymeextract the mixture obtained was incubated for min at 27ºc in dark after min ml ofstaining solution was added fast blue bb and sds with ratio of and again incubated in the dark for min at ºc absorbance at nm was measured for αnaphtholproduced using standard curve enzyme activity was αnaphthol produced in μm minˆ’ g wtalphaamylase activity a modified method for alphaamylase activity was followed forall plant samples as described by other biochemical parameterstotal oxidant status tos the method of was used to determined tos the assayis established on ferrous ion oxidation into ferric ion the presence of oxidants in the samplein acidic medium and ferric ion measurement produced by xylenol orange was measuredobserved the assay is based on two mixtures r1 stock xylenol orange solution 038g in500μl of 25mm h2so4 04g nacl μl glycerol and volume up to 50ml with 25mmh2so4 sample extract and r2 g odianisidine g ferrous ammonium sulphateii absorption measured at 560nm after minutes by using a spectrophotometerpigment analysis the concentration of lycopene chlorophyll a and b total chlorophylland carotenoids were examined by the method of samples g were ground in acetone and centrifuged at g for min absorbance measured at and nm byusing a spectrophotometer partiallytotal phenolic contents tpc and tannin a micro colorimetric assay was used tomeasure tpc by using folinciocalteu fc reagent with some modifications005 gsample was kept in methanol in dark for hours after hours the supernatant wastaken μl fc reagent and ml sodium carbonate mm were added to it placethis mixture at room temperature for one hour and took reading at nm linear regressionequation was calculated by using a standard curve of gallic acid at different concentrations tomeasure tannin 01g pvpp was added in the above prepared sample vortexed vigorouslyand centrifuged again at g the absorbance was measured at nmdetermination of total flavonoid content the assay was determined by a colorimetricmethod using quercetin as standard take μl sample prepared in methanol extractand phosphate buffer mm ph added μl alcl2 μl potassium acetate1m incubate the mixture at room temperature for minutes and take the reading at nm absorbancetotal antioxidant capacity a modified method of tac was followed as described by due to the presence of antioxidants in the sample abts assay represents a decrease of one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plants2azinobis 3ethylbenzothiazoline6 sulfonate radical cation abts¢ bluegreen in colorinto original abts colorless compound the antioxidants of the sample extract according totheir content decolorize the abts¢ radical cation the reaction mixture contained reagentr1 mixture of sodium acetate buffer solution and glacial acetic acid ph58 sample extractand reagent r2 mixture of sodium phosphate buffer solution glacial acetic acid hydrogenperoxide and abts after min at wavelength of 660nm the absorption of each reactionmixture was measured this analysis used asa ascorbic acid to develop a calibration curvethe results for antioxidant contents found in plant extracts were measured as μm asa equivalent to one gramreducing sugars sugar content assessment of reducing sugars level in the plant samples was determined by dinitrosalicylic acid method proposed by total soluble protein contentprotein estimation of plant samples was based on quantitative protein analysis described by aerial parts and fruits samples were homogenized in potassium phosphate mm ph supernatant 5μl and nacl 01n mixed with10 ml of bradford dye incubate the mixturefor minutes to get a proteindye complex measure the quantity at nm absorbance by aspectrophotometerascorbic acid asa 6dichloroindophenol dcip method hameed wasfollowed for measurement of ascorbic acid which measures reduced vitamin c only in shorteach molecule of ascorbic acid converts a dcip molecule into a reduced 6dichloroindophenol dciph2 and this conversion can be recorded as reduced absorption at nm the calibration curve was drawn with the help of known series of ascorbic acid concentrationsascorbate concentration in unknown sample was found by calculating simple linear regressionequationin vitro antidiabetic activity enzyme αamylase inhibition methodthe in vitro antidiabetic activity was determined by assaying the inhibitory activity of theenzyme αamylase which involves the breakdown of starch to produce glucose in thismethod ml of methanolic extracts of all species were tested separately and thus added to ml of the enzyme αamylase in a testtube and incubated for min at ˚c then ml of starch solution was added into it and again incubated for min at ˚c then ml 5dinitrosalicylic acid reagent was added into it in order to terminate the reaction the reaction mixture was then incubated in a boiling water bath for min and then allowed it to cool at roomtemperature the absorbance of the reaction mixture was then measured at nm in a spectrophotometer the standard control of the reaction without the extract represents the enzyme activity the age inhibition of enzyme activity of αamylase was determined byage inhibition of a 00 amylase¼enzyme activity of control 00 enzyme activity of extractenzyme activity of controlx ¼ results compared with standard drug glucophage metformin martin dow pharmaceutical pak ltdin vitro antiinflammory activity protein denaturation methodthe protein denaturation assay was determined using a modified method as described by briefly the reaction mixture ml ph consisted of ml of bovine serum albumin one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plants aqueous solution and ml of distilled water the ph was adjusted to using a smallamount of n hcl ml of acetone or aqueous extract with final concentrations of to mgml was added to the reaction mixture these were incubated at ˚c for min and thenheated at ˚c for min after cooling the samples ml of phosphate buffer solution ph was added the turbidity was measured by a spectrophotometer at nm for the negative control ml of distilled water and ml of bovine serum albumin were used diclofenac sodium with the final concentration of and μgml was used asreference drug the percentage inhibition of protein denaturation was calculated by using thefollowing formula age inhibition ¼½abs control 00 abs sample�abs controlx ¼ results compared with standard drug diclofenac sodium diclofenac na getz pharmapakistan pvt ltdstatistical analysisdata was recorded in mean ± sem resulting data were analyzed by applying descriptive statistics twoway anova with three replications was used in analyses the significance of datawas tested by analysis of variance and tukey hsd test at p0001using xlstat softwareversion copyright addinsoft “ httpwwwxlstatcomresults and discussionthe present study is based on the analysis of biological activities of extracts from thirteen plantspecies of zygophyllaceae remarkably important angiosperm family with many taxa beingused in folk medicines there is a scarcity of data in the literature about these plants as suchmaking a comparison of the results obtained in the present studies was difficult nonetheless afew papers reported some biological activities of t terrestris p harmala and few species offagonia the present investigation explores the presence of enzymatic constituents such assod pod apx cat esterase alpha amylase nonenzymatic antioxidants and other phytochemicals like asa tos tac tsp tpc tf tannins and pigments selected plants also provided evidence for the antidiabetic and antiinflammatory potential of varying extent in seedsand aerial parts the difference in prooxidants and antioxidants causes oxidative stress andchronic diseases in the body cellular damage results in causing cancer one of the mechanisms behind the antioxidation is free radical scavenging action pod helps in scavenging the reactive oxygen species ros causing cell oxidative injury the highest values ofperoxidase and catalase were depicted in the aerial parts of p harmala ± unitsg fwt and ± unitsg f wt respectively as shown in s1a and s1b fig respectively plantspecies having high antioxidant activities can be utilized for different therapeutic applicationsfor the treatment of oxidative stressinduced diseasesthe highest apx value was recorded in t pentandrus ± unitsg f wt s1d figascorbate peroxidase apx enzyme is crucial for the protection from damage by h2o2 andhydroxyl radicals ¢oh antioxidant enzymes activities namely cat pod and sodwere not tested earlier in selected wild medicinal taxa while few records reported earlier inother wild taxa like rumex obtusifolius a wild medicinal plant and found to have a good antioxidant capacity similarly calamintha officinalis also has potent antioxidants alphaamylase and esterase activities were higher in z fabago ± mgg and ± μmming f wt respectively s1e and s1f fig respectively esterase plays an important role in thedisintegration of natural constituents and industrial pollutants and other toxic chemicals it is one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantsalso beneficial for the production of optically pure compounds perfumes and antioxidants the antioxidant enzyme catalase is present in all animal tissues with its highest activities inliver and red blood cells which defend the tissues from highly reactive hydroxyl radicals bydecomposing hydrogen peroxide decrease amount of catalase causes numerous damages dueto hydrogen peroxide and superoxide radical assimilation superoxide dismutase enzymereferred as the significantly involved in cellular defense therefore it is considered as an indicator of antioxidant capacity the highest value of superoxide dismutase ± unitsgf wt was observed in f indica shown in s1c fig traditionally this plant is used for anticancer treatment and possibly detected high concentrations of sod and tac may be responsiblefor this therapeutic effectmaximum tac was depicted in fresh samples of t simplex ±001μmg f wt followed by f indica ±004μmg f wt shown s2a fig no significant variation wasdetected among all dry aerial parts of selected plants in general maximum tac was detectedin the seeds of z eurypterum ± μmg dry wt followed by the aerial parts of t simplex± μmgdry wt in the seeds of the selected taxa no significant variation wasdetected the maximum tac was in f olivieri ± μmg s wt the second highestvalue was in f bruguieri fbn ± μmg s wt previously the aerial parts of f longispina were reported to be a good source for natural antioxidants previously f cretica wasalso found to have high antioxidant and radical scavenging potential due to the high tpc andtfc f olivieri can serve as a natural source to develop the free radical scavengers beneficial in the prevention of oxidative stress development total flavonoid content tfc in the methanolic extract showed maximum quantity infresh samples of t pentandrus ±49μgml sample shown s2b fig fruit samples freshshowed the highest value of flavonoid and ascorbic acid in t terrestris ± μgml sample and ± μgg f wt respectively in dry aerial parts of t pentandrus flavonoid content gives a maximum amount of tfc ± μg ml sample followed by f bruguieri fbh ± μgml sample while in the seeds of p harmala showed maximum tfc±167μgml sample followed by f bruguieri fbp ± μgml sample flavonoids are considered to be effective free radical scavengers in fruits vegetables and medicinalplants highest ascorbic acid reported in p harmala shown s2c fig ascorbic acid is involvedin a number of physiological processes such as pod and sod the highest flavonoidsand ascorbic acid content in fruits of t terrestris and aerial parts of t pentandrus in the present study validated its traditional medicine use and may be responsible to cure various ailments f olivieri as shown s2c fig that gives the highest amount of tanninstotal soluble protein and reducing sugar were high in fresh aerial parts of p harmalashown in s3a and s3b fig respectively earlier isolated antioxidant protein from p harmala seeds possessed antioxidant activity and this activity was due to the presence of hydrophobic amino acids p harmala is one of the most frequently used medicinal plants to treathypertension and cardiac disease worldwide the maximum total soluble proteins ± mgg dry wt were depicted in dry aerial parts of t simplex no significant variation wasobserved in seed samples the highest value of total soluble proteins ± mgg s wt wasfound in f bruguieri fbn total oxidant status tos was lower in f indica ± μmg f wt shown in s3d figtotal flavonoid content was calculated in and expressed as μgml in methanolic extractusing quercetin as standard table significant difference was observed among all selectedspecies of zygophyllaceae in the aerial parts t pentandrus flavonoid content gives maximumamount of tfc ± μg ml sample followed by f bruguieri fbh ± μgmlsample while seeds of p hermala showed maximum tfc ±167μgml sample one 101371 pone0231612 august one 0ctable phytochemical analysis in the dry aerial parts of the selected taxaselected taxa drytotal flavonoidtotal phenolictannintotal solubleascorbic acidreducingamylasetotal antioxidantbiochemical profile and bioactive potential of few medicinal plantsaerial partscontentcontentsprotein contentcontentsugarsumg dry wtumg drymgg dry wtugg dry wt mgg dry wt mgg dryp harmalat simplexquercetin equlientμgml sample±451bc±2275a±398bcde±5375az propinquum±1467bcd±4575az fabago±239b±2850at pentandrus varpterophorust terrestris±884de±1725a±2465de±1650at pentandrus±1642a±1700af bruguieri fbh±3708b±1670af bruguieri fbp±2456cde±7175awt±1625a±550a±150a±4825a±4125a±1500a±2450a±742a±450a±433bc±4abc±016abc±633a±8d±028cd±667bc±4abc±055ab±333a±135c±028a±033abc±075c±012cd±067bc±175c±02cd±4bc±105c±02cd±979ab±238ab±028cd±1333c±35ab±008dcapacityμmg dry wt±251a±234a±345a±269a±284a±345a±416a±186a±256a±254a±248a±246a±369awt±264a±34a±283a±66a±472a±1132a±962a±1817a±2642a±208a±1245a±151a±132af indica±3321e±5400a±75a±1867abc±85ab±004cdf bruguieri fbn±318cde±1350af paulayana±424e±550af olivieri±1873cd±475a±525a±775a±1275a±2bc±225ab±004cd±1133abc±5ab±016bcd±167ab±275a±02ddata are presented as mean values ± standard error n statistical analysis anova test and tukey hsd the different letters above the values in the same columnindicate significant differences with tolerance values with the same superscript letters in the same column are not significant101371 pone0231612t002followed by f bruguieri fbp ± μgml sample table total phenolic contenttpc was estimated in aerial parts of selected taxa no significant tpc variation was foundamong table in general the highest tpc was depicted in t pentandrus var pterophorus± μmg dry wt followed by f bruguieri fbn ± μmg dry wtseeds of selected plant samples showed significant variation highest tpc was detected in zpropinquum ±775μmg s wt followed by f bruguieri fbn ± μmg swt as shown in table no significant tannin variation was detected among aerial parts aswell as in seeds of all tested taxa table however the highest amount of tannins was estimated in t pentandrus var pterophorus ± μmg dry wt followed by z fabago± μmg dry wt in seeds the highest amount of tannins was estimated in p hermala ± μmg s wt followed by z propinquum ± μmg s wt shown intable ascorbic acid was observed in aerial parts of all selected taxa and significant difference found among the studied taxa table the highest value of asa was found in f olivieri± μgg dry wt followed by f bruguieri fbp asa content in seeds showed no significant variation in general the highest asa content was found in f bruguieri fbh ± μgg s wt given in table alphaamylase activity in dry aerial parts of selected plantswas assessed table no significant variation was found among the taxa of zygophyllaceae one 101371 pone0231612 august one 0cbiochemical profile and bioactive potential of few medicinal plantstable phytochemical analysis in the seeds of the selected taxaselected taxaseedstotal flavonoidcontentquercetin equlientμgml sampletotal phenoliccontentsumg seed wttannintotal solubleprotein contentascorbic acidcontentreducingsugarsamylaseumg seedwtmgg seed wtugg seed wtmgg seedwtmgg seedwttotal antioxidantcapacityμmg seed wtp harmala±1394a±1350abz propinquum±24727a±775az eurypterum±488a±135abt pentandrus varpterophorust terrestris±3498a±1475ab±7156a±50bct pentandrus±20884a±1325cf bruguieri fbh±1005a±2887af bruguieri fbp±161a±3025bcf bruguieri fbn±878a±1148cf paulayana±14735a±3500abcf olivieri±2518a±300abc±2575a±175a±1000a±550a±2025a±1075a±1111a±675a±352a±650a±2750a±80a±4575a±087c±0a±025a±04ab±2a±05a±004a±1633a±25a±02ab±433a±1525a±012a±2a±4a±02a±2942a±219a±009c±33a±175a±008c±038abc±226bc±208bc±151c±17ab±283ab±1184a±094bc±273a±279a±221a±766a±635a±969a±201a±258a±184a±185a±106ab±251bc±169a±3067a±18a±024bc±467a±25a±008c±038ab±057abc±316a±243adata are presented as mean values ± standard error n statistical analysis anova test and tukey hsd the different letters above the values in the same columnindicate significant differences with tolerance values with the same superscript letters in the same column are not significant101371 pone0231612t003table pigment analysis in the dry aerial parts of the selected taxaselected taxalycopenechlorophyll achlorophyll btotal carotenoidstotal chlorophyllp harmalat simplexz propinquumz fabagot pentandrus var pterophorust terrestrist pentandrusf bruguieri fbhf bruguieri fbpf indicaf bruguieri fbnf paulayanaf olivierimgg dry wt±003ab±013b±003b±061ab±139a±001ab±014ab±034ab±024ab±042ab±394ab±102ab±112abugg dry wt±66ab±596bc±005bc±3171bc±005a±024a±196ab±3396bc±537a±2383ab±10962abc±517abc±6862augg dry wt±05ab0b±116b±1767ab±6301a±161ab±498ab±343b±1183ab±821ab±8196 Answer:

Colon_Cancer

aberrant activation of the wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers the development of inhibitors of wnt signallinghas been the focus of multiple drug discovery programs targeting colorectal cancer andother malignancies associated with aberrant pathway activation however progression ofnew clinical entities targeting the wnt pathway has been slow one challenge lies with thelimited predictive power of 2d cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity in particular the relationship between 2d cancer cell biology and cancer stem cell function is poorly understood by contrast 3d tumour anoidsprovide a platform in which complex cellcell interactions can be studied however complex3d models provide a challenging platform for the quantitative analysis of drug responses oftherapies that have differential effects on tumour cell subpopulations here we generatedtumour anoids from colorectal cancer patients and tested their responses to inhibitors oftankyrase tnksi which are known to modulate wnt signalling using compounds with orders of magnitude difference in cellular mechanistic potency together with imagebasedassays we demonstrate that morphometric analyses can capture subtle alterations in anoid responses to wnt inhibitors that are consistent with activity against a cancer stem cellsubpopulation overall our study highlights the value of phenotypic readouts as a quantitative method to asses druginduced effects in a relevant preclinical modela1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation badder lm hollins aj herpers b yan kewan kb thomas m 3d imaging ofcolorectal cancer anoids identifies responses totankyrase inhibitors one e0235319101371 pone0235319editor ning wei university of pittsburgh unitedstatesreceived november accepted june published august copyright badder this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement components withinthe manuscript denoted as s1 table within thetext are held in a public repository wwwebiacukenadataviewprjeb37607 wheresequencing information can be found other data isall contained within the paper andor supportinginformation filesfunding this work was supported by cancerresearch wales phd studentship lmb thecancer research uk cardiff experimental cancermedicine centres ecmc ajh and cancerresearch uk programme grant c1295a15937h one 101371 pone0235319 august one 0cwwwcancerresearchuk innovate ukgrant ke wwwgovukgovernmentanisationsinnovateuk the funders did not playany role in study design data collection andanalysis the decision to publish or the preparationof the manuscriptcompeting interests lsp is a founder and majorshareholder of ocello bv and ce hpb and dw are employees of merck healthcare kgaa mtand jt are employees of cellesce ltdtcd is adirector of cellesce ltd lmb and dab aresiblings the funder provided support in the formof salaries for authors lsp cehpb dw mtand jt but did not have any additional role in thestudy design data collection and analysis decisionto publish or preparation of the manuscript thespecific roles of these authors are articulated in the˜author contributions™ section this does not alterour adherence to one policies on sharingdata and materialsabbreviations crc colorectal cancer nodscidnonobese diabeticsevere combinedimmunodeficiency odx anoid derivedxenograft parp poly adpribose polymerasestnks tankyrase3d imaging of anoids identifies responses to tankyrase inhibitorsintroductionaberrant canonical wntcatenin signalling as a result of activating mutations within thepathway has a prominent role in the initiation and progression of colorectal cancer crc a number of feedback loops control catenin turnover and wnt activation in the absenceof a wnt ligand the multiprotein catenin destruction complex formed of axin12 adenomatous polyposis coli apc and glycogen synthase kinase gsk3 mark catenin for degradation as a result the accumulation and subsequent translocation of catenin to thenucleus is inhibited preventing the downstream activation of target genes components ofthe destruction complex are also tightly regulated axin1 and axin2 are concentrationlimiting components of the destruction complex levels of axin12 are posttranscriptionally regulated by tankyrases tnks1 and tnks2 members of the poly adpribose polymerasesparp family of enzymes which enhance wnt signalling by targeting axin12 for degradation the inhibition of wnt signalling has been validated as a means of blocking tumour growthin many cancer models [ ] small molecule inhibitors of tnks1 and tnks2 have beenshown to reduce wnt signalling in intestinal cancer cell lines and have been suggested to prevent tumour growth due to their ability to stabilise axin1 and axin2 levels and as a result toinhibit catenin mediated transcription [ “] however the progression of tnksi intoclinical trials has been restricted due to significant issues of intestinal toxicity within in vivomodels emphasising the important role of wnt signalling in adult tissue homeostasis furthermore the tnksi described above reduced colorectal cancer cell numbers in 2dculture but did so with relatively low effect sizes by comparison with their ability to reduce levels of wnttcfdependent transcription in the same cell lines similar partialefficacy cell lineresponses were observed during the development of inhibitors of the cdk8 and cdk19kinases suggesting that growth in 2d cell culture may not be an optimal readout for compounds that are anticipated to target cancer stem cells to date most preclinical in vitro studies have relied on the use of conventional 2d culturesof cell lines whilst cancer cell lines can be usefully used to study pathway deregulation theyfrequently fail to be predictive when used as readouts of antitumour efficacy it is wellrecognised that immortalised 2d cell lines adapted to growth on plastic poorly represent theintricate cellular crosstalk present in tumours in particular they are unable to recapitulatethe intercellular interactions that form cancer stem cell niches and as a consequence suchmodels fail to fully reflect in vivo results3d tumour anoids have been shown to provide a more complex insight into tumourcellcell interactions [ ] and have been recognised as models with the potential to bridgethe gap between in vitro and in vivo preclinical studies anoids that have never beenadapted for growth on plastic have been cultured from multiple tumour types and have beenshown to better represent the genetic diversity of distinct tumour subtypes than 2d cell lines anoids derived from geneticallyengineered mice in which the wnt pathway hadbeen oncogenicallyactivated accurately predicted subsequent in vivo responses to inhibitorsof the cdk8 and cdk19 kinases while 2d cell culture only showed partialefficacy anoids have been further shown to predict patient responses and might in future be used in theclinic in personalised medicine the biological and phenotypic complexity of 3d primary anoid cultures allow in principle the assessment of compound activity against a subset of intercellular signalling that is central to tumour growth however most uses of anoid assays to date have relied on fixedendpoint metabolic assays eg atplevel quantification that aggregates responses in everycell within a population of anoids to fully exploit the potential of anoid assays analysis one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorsplatforms need to yield quantitative data that can clearly represent compoundinduced effectson signalling pathways whose outputs are complex whilst simultaneously maintaining usability in a high throughput formatin this work we generated crc patientderived anoids and studied their responses totankyrase inhibitors tnksi using metabolic end point assays tankyrase inhibition showedpartial efficacy reflecting a limited reduction in the growth of tnksisensitive anoidscloser examination of anoid responses suggested that tnksi altered the ratio of stemliketo differentiated cell populations this subtle effect on the cellular composition of anoidswas best reflected by multiparametric imaging analysis of anoids that was nonetheless compatible with high throughput analysis our findings demonstrate the potential of a phenotypicapproach to assess druginduced phenotypes in a preclinical setting which may be applicableto a wider range of therapeutics that target cancer stem cell biology and nichesmaterials and methodsmaterialscorning growth factorreduced matrigel was purchased from vwr “ cell culturemedia were purchased from invitrogen life technologies and cell culture plastics from nuncunless otherwise stated all tnksi compounds were supplied and synthesized by merckhealthcare kgaa darmstadt germany all stock solutions of compounds were reconstituted in dmso activity of each compound had been assessed prior to shipping using a cellbased immunobead assay to determine tnksi potency based on the ability of the inhibitors tostabilise the axin2 the determined ec50 values of nm nm and nm fig 2awere determined for c1 c2 and c3 respectivelyhuman tissuesurgically resected patient materials were obtained from university hospital of wales by thewales cancer bank with written informed ethical consent from male and female patients years of age above the uk age of valid consent with known or suspected malignant disease and anonymised wcb project reference the wales cancer bank has ethicsapproval as a research tissue bank from the wales research ethics committee reference16wa0256 and is licensed by the human tissue authority under the uk human tissueact to store human tissue taken from the living for research licence these approvals cover the collection of samples including written consent processing andstoring samples across multiple collection and storage sites all patient derived material washandled in concordance with hta regulations histological sections of patient tissue wereimaged at university hospital wales and remained anonymisedanoid culturethe isolation of tumour anoids from patient material was processed as previously describedby sato with some refinements outlined below briefly following surgical resectiontumour samples were obtained and stored in hibernate a medium life technologies at ˚ctissue pieces were dissected to remove connective tissue and chopped to pieces approximately mm in diameter following dissection carcinoma pieces were washed in pbs then digestedenzymatically with collagenase and dispase for minutes at ˚c once digested tissue wastriturated in pbs at room temperature to release cell fragments from tissue cell fragmentswithin the supernatant were then centrifuged for min at rcf at ˚c cell pellets wereresuspended in advanced dmemf12 supplemented with glutamax hepes buffer one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorssolution and penicillinstreptomycin then filtered μm collected tissue fragmentswere counted and plated at a density of cell fragments per μl of growth factorreduced matrigel bd biosciences in well plates nunc following matrigel polymerisation cells were overlaid with μl of either œ or œfull media and replenished every days ˜™ media conditions consisted of advanced dmemf12 supplemented with penicillinstreptomycin 2mm glutamax mm hepes1 x b27 supplement x n2 supplement alllife technologies mm nacetyllcysteine sigmaaldrich the following additionalniche factors were added to generate œfull media ngml mouse recombinant egf lifetechnologies ngml mouse recombinant noggin peprotech rspo1 conditionedmedium wnt3a conditioned medium nm a8301 tocris and μmsb202190 sigma 10μm y27632 tocris was also added to media for the first three days ofculture optimal media was determined for each tumour anoid line on the basis of conditions that favoured the most efficient growth of anoids for subsequent passaging maintenance in culture and analyses the most favourable media condition was used all culture weremaintained in humidified incubators at ˚c co2dna extractiondna extraction from anoid cultures was carried out using a qiaamp dna mini kit qiagen following the manufacturer™s instructions purified dna quality was initially assessedusing a nanodrop1000 thermo fisher patient blood samples were processed by the walescancer bank wcb genomic dna was extracted from ml of whole blood using the chemagicstar automated cell lysis and dna extraction workstation hamilton company housedwithin the all wales medical genetics service awmgs laboratory both dna samples werethen passed to the wales gene park for quality control library preparation and sequencingwhole exome library preparationthe following was carried out by the wales gene park genomic dna was initially quantifiedusing the qubit1 life technologies and the samples were serially diluted to ngμl 50ngof gdna was used as the input and the sequencing libraries were prepared using the illumina1 nextera rapid capture enrichment kit illumina inc the steps included tagmentation of gdna cleanup of the tagmented dna amplification cleanup of the amplified dnahybridisation of probes capture of the hybridized probes second hybridization of probes second capture cleanup of the captured library amplification of enriched library cleanup ofthe enriched library and finally validation of the complete library the manufacturer™s instructions were largely followed with extra quantitation steps prior to the hybridization of theprobes to ensure that approximately ng of each sample was pooled the libraries were validated using the agilent bioanalyser and a highsensitivity kit agilent technologies toascertain the insert size and the qubit1 life technologies was used to perform the fluorometric quantitation following validation the libraries were normalized to nm pooledtogether and clustered on the cbot„¢ following the manufacturer™s recommendations poolswere then sequenced using a 75base pairedend 2x75bp pe dual index read format on theillumina1 hiseq4000 according to the manufacturer™s instructionswhole exome sequencing and mutation callingreads were mapped to the human reference genome grch37 with bwa version and duplicate reads were removed with samtools version base quality score recalibration was performed using gatk™s baserecalibrator gatk version [ ] andgatk™s mutect2 was used to call somatic variants tools found at software one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorsbroadinstitutegatk variants were annotated with variant effect predictor version using a cached version of ensemble v91 a custom perl script was used to extract genes ofinterest from annotated variant files default parameters were used for all software unless otherwise stated all of the calls reported were crosschecked against the cbioportal databasewwwcbioportal and are those found to have been previously described within thecosmic database cancersangeracukcosmic as being œlikely oncogenicimmunohistochemistryformalinfixed paraffin embedded histological sections of patient tissue were processedimaged and analysed by the histopathology unit at university hospital wales for anoidhistology anoids embedded within matrigel were fixed with paraformaldehyde for min at room temperature anoids were then lifted from wells and washed several timeswith pbs prior to embedding within low meltingpoint agarose followed by dehydration paraffin embedding sectioning and hematoxylineosin he staining images were acquiredon an olympus dp26 microscopewestern blot detectionanoids were harvested from x μl blobs of growth factorreduced matrigel using cellrecovery solution corning for hour on ice total protein was extracted from anoids byaddition of μl of lysis buffer to whole anoids m trishcl mm edta vvnp40 ipegal in ddh2o containing 1x phosstop phosphatase inhibitor roche and 1xcomplete protease inhibitor cocktail roche the lysates were centrifuged at rcf for min and proteins harvested in the supernatants protein content was measured using a bcaprotein assay kit pierce samples were resolved on novex nupage “ bistris pagegels and then blotted onto nitrocellulose membranes using the invitrogen iblot dry blottingcassette systemblots were blocked at room temperature for h in wv milk powder in wash buffer trisbuffered saline tbs plus tween primary antibodies were diluted in blocking bufferand incubated overnight at ˚c after washing the blots were incubated with hrpconjugatedsecondary antibodies for h at room temperature bands were visualized using the enhancedchemoluminescence supersignal west dura pierce antibodies and dilutions tnks12santa cruz tnks12 e10 sc365897 axin2 cell signalling technologies 76g6 actin sigma ac74 a2228 antimouse or antirabbit hrpconjugated antibodies ge healthcare anoid viability measurementsanoids in culture were gently dissociated to nearsingle cell populations using tryplelife technologies before resuspension within growth factorreduced matrigel and dispensedinto white clear bottomed well plates in μl matrigel per well cells μl of matrigelfollowing matrigel polymerization a 9point 2fold dilution range of compounds were dilutedin tailored growth medium and dispensed within each well dmso was used as a negative control at a final concentration of following a six day exposure to compounds cell titerglo 3d promega reagent was applied as per the manufacturer™s guidelines for an endpointreadout relative luminescence values were obtained on a bmg fluostar plate reader doseresponse curves and ec50 values were obtained using microsoft excel with xlfit plugin version one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorshigh content fluorescence microscopyanoid lines previously established in culture were split to single cells using tryple lifetechnologies and seeded within growth factorreduced matrigel corning in black clearbottomed well plates at μl per well at a density of cells μl matrigel upon polymerisation of matrigel growth media containing a titration range of individual compounds ordmso controls were added to each well prior to incubation at ˚c anoids were exposedto compounds for a total of six days prior to fixation in a fixand stain solution containing para formaldehyde sigma aldrich triton x100 sigma aldrich 025μm rhodaminephalloidin sigma aldrich and μgml hoechst sigma aldrich in pbs at ˚c for hafter staining plates were washed with pbs and sealed imaging was performed on an imagexpress1 highcontent micro confocal platform molecular devices a total of zstackimages were acquired at μm steps through the focal plane per well of a well plate using a x objective image stacks were captured from both tritc channel ex em and dapi channel ex380 em to detect both rhodaminephalloidin factin andhoechst nucleihigh content image analysisimages obtained using the imagexpress platform were processed for phenotypic analysiswithin ominer„¢ software ocello bv integrated within the knime analytics platformkonstanz germany httpwwwknime as described previously [ ] briefly thissoftware enabled the quantification of zstack images derived from hoechst and rhodaminephalloidin stained anoids information from both the dapi and tritc channels wereused to enhance anoid boundary separation masks generated from both channels facilitated the visualisation of main anoid structure internal morphometries such as individuallumens as well as nuclei per anoid objects that were out of focus were filtered and discarded from analysis morphological features were calculated on the basis of individual anoids and pooled for analysis multiple features extracted included counts total area solidityand branching of nuclei lumen and anoid masksfeature training between the negative control anoids and the high dose tnksi treatedanoids was performed to condense the phenotypic features and calculate the euclidian distance the results are plotted with graphpad prism graphpad software la jolla caresults are shown as means ± standard deviations unless otherwise statedquantitative realtime pcrtotal rna was extracted from anoids resuspended in trizol solution life technologiescontaining μgml glycogen cdna was yielded from purified mrna using improm iireverse transcription kit promega quantitativert pcr was then performed using thesensifast sybr green hirox master mix bioline primers were designed sigmaaldrich and listed in s2 table all samples were measured in triplicate with gene expressionnormalised to gapdh housekeeping gene reactions were measured on a step one plus realtime pcr instrument applied biosystems with relative changes in gene expression calculatedusing the δδct methodwholemount immunofluorescent staininganoids were seeded as single cells in well plates in μl growth factor reduced matrigelat a density of cellsμl following a six day exposure to compounds and control conditions anoids were fixed in pfa for min at room temperature wells were then one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorswashed with pbs containing mm glycine prior to blocking overnight in pbs containing horse serum bovine serum albumin triton x100 tween thenstained with primary antibodies overnight s1 table at ˚c secondary antibodies were thenadded overnight at ˚c prior to counterstaining with hoechst wells were washed with pbsthen imaged on a confocal microscope leica tcs sp2 aobsin vivo assessment of tnks inhibitor efficacyall mice were obtained commercially from charles river and then housed under uk homeoffice regulations the work described here was carried out under home office ppl in vivo anoid engraftment studies also termed œanoid derived xenografts odx wereconducted using immunedeficient nodscid gamma irradiated mice for anoid transplantation anoids were in culture for h from single cells prior to subcutaneous transplantation into the right flank of the animal one injection site per mouse mice were checkedevery days for tumour growth by palpation according to home office regulation protocolpalpable tumours mm were counted for use in kaplanmeier analysis cardiff university™s animal welfare and ethical review body awerb reviewed the plan for animal studiesprior to the grant of the home office license animals in the study described werekilled by cervical dislocation schedule methodresultsestablishment of tumour anoids from crc patient materialsurgicallyresected crc material was isolated from patients under informed consent cellfragments were isolated from tissue and plated within growth factor reduced matrigel within hours following surgery adapting methods previously described by sato two different culture conditions were used as standard œ basal and œfull growth factor richmedium each condition was found to be optimal for a distinct subset of anoids fig 1aand allowed the efficient generation of anoids from what were in some cases limitedtumour cell numbers of collected samples were established in culture a ˜takerate™ of for anoid model derivation anoids were denoted by isolation number ˜iso™recent studies have similarly emphasised the requirement for different media to ensure efficient anoid generation from different patients a subset of anoids was studied in more detail optimal culture conditions were determined on the basis of anoid formation efficiencies and visual inspection of anoid integrity anoid lines showed good growth in basal medium whilst were dependenton the additional inclusion of egf ngml noggin ngml nicotinamide mma8301 nm sb202190 μm as well as wnt3a and rspondin conditioned media the anoid medium combinations shown in fig 1a were used for subsequent studies full exome sequencing showed that the subset of anoids containedcommon genotypes found in other anoid banks fig 1a s1 appendix [ ] in particular of anoid lines harboured mutations in components of the wnt signalling pathwaysuch as apc and catenin ctnnb1 iso was found to harbour a mutation in rnf43which is associated with an increased frizzled fz receptor expression and wnt ligand dependence [ ]crc anoids have previously been shown to display histological features in commonwith the tumours from which they were derived paired histological samples from the anoids described were compared to their parental tumours distinct morphologies wereobserved ranging from lines that preferentially formed single epithelial cell layers with largelumens containing apoptotic cells fig 1b s1 fig to multilayered anoids with small one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorsfig generation and establishment of tumour anoids from crc patient material a summary of parental tumour sites and dukes™ stages anoid mediaconditions and mutation status of anoid lines identified in whole exome sequencing analysis b representative hematoxylineosin he staining of ffpetissue sections from primary colorectal tumour patient material and anoid counterparts anoids were cultured for a minimum of days prior to fixation andembedding qualitative morphological similarities were observed between tumour and anoid pairs scale bar μm101371 pone0235319g001indistinct lumens the anoids frequently retained key histopathological grading featuresthat were present within the original patient tumour epithelium for example iso wasestablished from a mucinous adenocarcinoma and displayed a high percentage of mucinousvacuole structures that resembled that of the matching patient tissue the retention of featurespresent within primary patient tumour tissue showed that many features of tumour morphology could be retained without the need for a stromal compartment one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorstankyrase inhibition altered the growth of patientderived anoidsto study crc anoid responses to a wnt pathway inhibitor we utilised the tankyrase inhibitor tnksi c1 fig 2a after six days of treatment axin2 and tnks12 protein levelswere raised in a number of anoid lines fig 2b tnks normally targets axin2 andtnks12 for degradation and tnksi treatment would be expected to stabilise levels of eachprotein to assess the functional impact of tankyrase inhibition on anoid growth cellviability assays were performed for anoid lines iso iso and iso as a measure oftoxicity in the normal cell setting we also evaluated c1 response in healthy intestinal anoids tumour and normal intestinal anoids were digested to nearsingle cells or mechanically dissociated into small fragments embedded within growthfactor reduced matrigel andoverlaid with growth media containing the tnksi for six days prior to measuring relativeatp levels cell titer glo 3d c1 which has a cellular mechanistic ec50 of 2nm showed partial efficacy in reducing atp levels and markedly different potency in the crc lines fig2c“2g c1 treatment in healthy intestinal anoids showed limited effects on metabolicactivity s3 fig ec50 values in crc lines ranged from 2nm to 1μm but accurate values werehard to establish due to assay variance and also because many cells survived even in the presence of high concentrations of compound leading to reduced assay windows the partial survival of anoid cells after days c1 treatment suggest that longterm axin2 and tnks12biomarker responses might be compromised by alterations in cellular composition immunofluorescent staining showed reduced levels of the proliferation marker ki67 in iso but notiso anoids suggesting that reduced proliferation contributed to the lower atp levelsobserved fig 2h and 2imultiparametric phenotypic profiling of anoids in 3d to classifyresponses to tankyrase inhibitionto better understand the tnksi partial efficacy a novel multiparametric 3d image workflowanalysis was used to characterise the morphological effects of the responses three tankyrase tnks12 small molecule inhibitors were used that differed over orders of magnitudein their potency c1 2nm c2 36nm c3 319nm fig 2a anoids were seeded and treatedas previously for days prior to fixation and staining with rhodaminephalloidin factin andhoechst dna images were acquired using a highcontent widefield fluorescencemicroscope as described previously [ ]to characterise phenotypes ominer„¢ software ocello„¢ was used to extract morphological features from 2d projections of the factin and nucleiderived image stacks to generatemasks of individual anoids internal lumen structures and nuclei image segmentation wasused to distinguish components of both channels and minimise any background signal fig3a s4 fig overall shape and fluorescence intensity of individual anoid structures wereextracted on a wellbywell basis to generate approximately quantified features such features included information in regards to shape size boundaries and branching of anoidlumens and nuclear shapeto identify optimum features that could classify tnksi responses feature training andeuclidian distance calculations were performed on each data set as previously described this reduced features into key differentiating components to generate a measure of distancebetween highest c1 tnksi doses and negative control dmso conditions the featurecollection that describes the compoundinduced morphological change differs per anoidmodel but most frequently included anoid size changes lumen shape and size appearanceof dead cells and changes in nucleus morphology related to growth reduction cell anizationand cell death induction s5 fig one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorsfig assessing the effects of tankyrase inhibition on overall anoid viability a structures of three tankyrase inhibitors msc2524070a c1msc2572070a c2 and msc2501490a c3 with corresponding ec50 values b western blot of eight anoid lines treated with c1 nm and harvested one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorsafter days c representative images of anoids treated with c1 nm or dmso scale bar μm d e f doseresponse curves of anoidstreated with c1 for six days anoids previously established in suitable culture conditions were enzymatically digested to single cells and overlaid with mediacontaining a dose range of drug nm“ nm or a dmso control in media cell titer glo 3d readouts were performed in triplicates for iso d iso e iso f g ec50 values obtained from n experiments data are presented as mean ± sem h i representative immunofluorescentstaining of a proliferation marker in anoids iso h and iso i with differential sensitivity to tnksi were treated with c1 nm or dmso control for days fixed and stained with ki67 arrows indicate proliferating cell within the anoid ki67 positivecells in iso only when treated with c1scale bar μm101371 pone0235319g002the morphometric analysis showed that tnks inhibition resulted in phenotypic alterationsin iso iso iso and iso fig 3b importantly the cellular morphometric responsescorrelated with known cellular ec50 values across a range of potencies compounds c1 c2and c3 that had ec50 values of nm nm and nm table also showed the expectedseparation of ec50 activities in the anoid morphometric assays suggesting that observedfig 3d image analysis demonstrates target affinity of tankyrase inhibitors in anoids a flow diagram of highcontent multiparametric image screen processb dose response curves acquired from high content imaging screen anoids were seeded as single cells in well plates and exposed to

cancer7552

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: aberrant activation of the wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers the development of inhibitors of wnt signallinghas been the focus of multiple drug discovery programs targeting colorectal cancer andother malignancies associated with aberrant pathway activation however progression ofnew clinical entities targeting the wnt pathway has been slow one challenge lies with thelimited predictive power of 2d cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity in particular the relationship between 2d cancer cell biology and cancer stem cell function is poorly understood by contrast 3d tumour anoidsprovide a platform in which complex cellcell interactions can be studied however complex3d models provide a challenging platform for the quantitative analysis of drug responses oftherapies that have differential effects on tumour cell subpopulations here we generatedtumour anoids from colorectal cancer patients and tested their responses to inhibitors oftankyrase tnksi which are known to modulate wnt signalling using compounds with orders of magnitude difference in cellular mechanistic potency together with imagebasedassays we demonstrate that morphometric analyses can capture subtle alterations in anoid responses to wnt inhibitors that are consistent with activity against a cancer stem cellsubpopulation overall our study highlights the value of phenotypic readouts as a quantitative method to asses druginduced effects in a relevant preclinical modela1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation badder lm hollins aj herpers b yan kewan kb thomas m 3d imaging ofcolorectal cancer anoids identifies responses totankyrase inhibitors one e0235319101371 pone0235319editor ning wei university of pittsburgh unitedstatesreceived november accepted june published august copyright badder this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement components withinthe manuscript denoted as s1 table within thetext are held in a public repository wwwebiacukenadataviewprjeb37607 wheresequencing information can be found other data isall contained within the paper andor supportinginformation filesfunding this work was supported by cancerresearch wales phd studentship lmb thecancer research uk cardiff experimental cancermedicine centres ecmc ajh and cancerresearch uk programme grant c1295a15937h one 101371 pone0235319 august one 0cwwwcancerresearchuk innovate ukgrant ke wwwgovukgovernmentanisationsinnovateuk the funders did not playany role in study design data collection andanalysis the decision to publish or the preparationof the manuscriptcompeting interests lsp is a founder and majorshareholder of ocello bv and ce hpb and dw are employees of merck healthcare kgaa mtand jt are employees of cellesce ltdtcd is adirector of cellesce ltd lmb and dab aresiblings the funder provided support in the formof salaries for authors lsp cehpb dw mtand jt but did not have any additional role in thestudy design data collection and analysis decisionto publish or preparation of the manuscript thespecific roles of these authors are articulated in the˜author contributions™ section this does not alterour adherence to one policies on sharingdata and materialsabbreviations crc colorectal cancer nodscidnonobese diabeticsevere combinedimmunodeficiency odx anoid derivedxenograft parp poly adpribose polymerasestnks tankyrase3d imaging of anoids identifies responses to tankyrase inhibitorsintroductionaberrant canonical wntcatenin signalling as a result of activating mutations within thepathway has a prominent role in the initiation and progression of colorectal cancer crc a number of feedback loops control catenin turnover and wnt activation in the absenceof a wnt ligand the multiprotein catenin destruction complex formed of axin12 adenomatous polyposis coli apc and glycogen synthase kinase gsk3 mark catenin for degradation as a result the accumulation and subsequent translocation of catenin to thenucleus is inhibited preventing the downstream activation of target genes components ofthe destruction complex are also tightly regulated axin1 and axin2 are concentrationlimiting components of the destruction complex levels of axin12 are posttranscriptionally regulated by tankyrases tnks1 and tnks2 members of the poly adpribose polymerasesparp family of enzymes which enhance wnt signalling by targeting axin12 for degradation the inhibition of wnt signalling has been validated as a means of blocking tumour growthin many cancer models [ ] small molecule inhibitors of tnks1 and tnks2 have beenshown to reduce wnt signalling in intestinal cancer cell lines and have been suggested to prevent tumour growth due to their ability to stabilise axin1 and axin2 levels and as a result toinhibit catenin mediated transcription [ “] however the progression of tnksi intoclinical trials has been restricted due to significant issues of intestinal toxicity within in vivomodels emphasising the important role of wnt signalling in adult tissue homeostasis furthermore the tnksi described above reduced colorectal cancer cell numbers in 2dculture but did so with relatively low effect sizes by comparison with their ability to reduce levels of wnttcfdependent transcription in the same cell lines similar partialefficacy cell lineresponses were observed during the development of inhibitors of the cdk8 and cdk19kinases suggesting that growth in 2d cell culture may not be an optimal readout for compounds that are anticipated to target cancer stem cells to date most preclinical in vitro studies have relied on the use of conventional 2d culturesof cell lines whilst cancer cell lines can be usefully used to study pathway deregulation theyfrequently fail to be predictive when used as readouts of antitumour efficacy it is wellrecognised that immortalised 2d cell lines adapted to growth on plastic poorly represent theintricate cellular crosstalk present in tumours in particular they are unable to recapitulatethe intercellular interactions that form cancer stem cell niches and as a consequence suchmodels fail to fully reflect in vivo results3d tumour anoids have been shown to provide a more complex insight into tumourcellcell interactions [ ] and have been recognised as models with the potential to bridgethe gap between in vitro and in vivo preclinical studies anoids that have never beenadapted for growth on plastic have been cultured from multiple tumour types and have beenshown to better represent the genetic diversity of distinct tumour subtypes than 2d cell lines anoids derived from geneticallyengineered mice in which the wnt pathway hadbeen oncogenicallyactivated accurately predicted subsequent in vivo responses to inhibitorsof the cdk8 and cdk19 kinases while 2d cell culture only showed partialefficacy anoids have been further shown to predict patient responses and might in future be used in theclinic in personalised medicine the biological and phenotypic complexity of 3d primary anoid cultures allow in principle the assessment of compound activity against a subset of intercellular signalling that is central to tumour growth however most uses of anoid assays to date have relied on fixedendpoint metabolic assays eg atplevel quantification that aggregates responses in everycell within a population of anoids to fully exploit the potential of anoid assays analysis one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorsplatforms need to yield quantitative data that can clearly represent compoundinduced effectson signalling pathways whose outputs are complex whilst simultaneously maintaining usability in a high throughput formatin this work we generated crc patientderived anoids and studied their responses totankyrase inhibitors tnksi using metabolic end point assays tankyrase inhibition showedpartial efficacy reflecting a limited reduction in the growth of tnksisensitive anoidscloser examination of anoid responses suggested that tnksi altered the ratio of stemliketo differentiated cell populations this subtle effect on the cellular composition of anoidswas best reflected by multiparametric imaging analysis of anoids that was nonetheless compatible with high throughput analysis our findings demonstrate the potential of a phenotypicapproach to assess druginduced phenotypes in a preclinical setting which may be applicableto a wider range of therapeutics that target cancer stem cell biology and nichesmaterials and methodsmaterialscorning growth factorreduced matrigel was purchased from vwr “ cell culturemedia were purchased from invitrogen life technologies and cell culture plastics from nuncunless otherwise stated all tnksi compounds were supplied and synthesized by merckhealthcare kgaa darmstadt germany all stock solutions of compounds were reconstituted in dmso activity of each compound had been assessed prior to shipping using a cellbased immunobead assay to determine tnksi potency based on the ability of the inhibitors tostabilise the axin2 the determined ec50 values of nm nm and nm fig 2awere determined for c1 c2 and c3 respectivelyhuman tissuesurgically resected patient materials were obtained from university hospital of wales by thewales cancer bank with written informed ethical consent from male and female patients years of age above the uk age of valid consent with known or suspected malignant disease and anonymised wcb project reference the wales cancer bank has ethicsapproval as a research tissue bank from the wales research ethics committee reference16wa0256 and is licensed by the human tissue authority under the uk human tissueact to store human tissue taken from the living for research licence these approvals cover the collection of samples including written consent processing andstoring samples across multiple collection and storage sites all patient derived material washandled in concordance with hta regulations histological sections of patient tissue wereimaged at university hospital wales and remained anonymisedanoid culturethe isolation of tumour anoids from patient material was processed as previously describedby sato with some refinements outlined below briefly following surgical resectiontumour samples were obtained and stored in hibernate a medium life technologies at ˚ctissue pieces were dissected to remove connective tissue and chopped to pieces approximately mm in diameter following dissection carcinoma pieces were washed in pbs then digestedenzymatically with collagenase and dispase for minutes at ˚c once digested tissue wastriturated in pbs at room temperature to release cell fragments from tissue cell fragmentswithin the supernatant were then centrifuged for min at rcf at ˚c cell pellets wereresuspended in advanced dmemf12 supplemented with glutamax hepes buffer one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorssolution and penicillinstreptomycin then filtered μm collected tissue fragmentswere counted and plated at a density of cell fragments per μl of growth factorreduced matrigel bd biosciences in well plates nunc following matrigel polymerisation cells were overlaid with μl of either œ or œfull media and replenished every days ˜™ media conditions consisted of advanced dmemf12 supplemented with penicillinstreptomycin 2mm glutamax mm hepes1 x b27 supplement x n2 supplement alllife technologies mm nacetyllcysteine sigmaaldrich the following additionalniche factors were added to generate œfull media ngml mouse recombinant egf lifetechnologies ngml mouse recombinant noggin peprotech rspo1 conditionedmedium wnt3a conditioned medium nm a8301 tocris and μmsb202190 sigma 10μm y27632 tocris was also added to media for the first three days ofculture optimal media was determined for each tumour anoid line on the basis of conditions that favoured the most efficient growth of anoids for subsequent passaging maintenance in culture and analyses the most favourable media condition was used all culture weremaintained in humidified incubators at ˚c co2dna extractiondna extraction from anoid cultures was carried out using a qiaamp dna mini kit qiagen following the manufacturer™s instructions purified dna quality was initially assessedusing a nanodrop1000 thermo fisher patient blood samples were processed by the walescancer bank wcb genomic dna was extracted from ml of whole blood using the chemagicstar automated cell lysis and dna extraction workstation hamilton company housedwithin the all wales medical genetics service awmgs laboratory both dna samples werethen passed to the wales gene park for quality control library preparation and sequencingwhole exome library preparationthe following was carried out by the wales gene park genomic dna was initially quantifiedusing the qubit1 life technologies and the samples were serially diluted to ngμl 50ngof gdna was used as the input and the sequencing libraries were prepared using the illumina1 nextera rapid capture enrichment kit illumina inc the steps included tagmentation of gdna cleanup of the tagmented dna amplification cleanup of the amplified dnahybridisation of probes capture of the hybridized probes second hybridization of probes second capture cleanup of the captured library amplification of enriched library cleanup ofthe enriched library and finally validation of the complete library the manufacturer™s instructions were largely followed with extra quantitation steps prior to the hybridization of theprobes to ensure that approximately ng of each sample was pooled the libraries were validated using the agilent bioanalyser and a highsensitivity kit agilent technologies toascertain the insert size and the qubit1 life technologies was used to perform the fluorometric quantitation following validation the libraries were normalized to nm pooledtogether and clustered on the cbot„¢ following the manufacturer™s recommendations poolswere then sequenced using a 75base pairedend 2x75bp pe dual index read format on theillumina1 hiseq4000 according to the manufacturer™s instructionswhole exome sequencing and mutation callingreads were mapped to the human reference genome grch37 with bwa version and duplicate reads were removed with samtools version base quality score recalibration was performed using gatk™s baserecalibrator gatk version [ ] andgatk™s mutect2 was used to call somatic variants tools found at software one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorsbroadinstitutegatk variants were annotated with variant effect predictor version using a cached version of ensemble v91 a custom perl script was used to extract genes ofinterest from annotated variant files default parameters were used for all software unless otherwise stated all of the calls reported were crosschecked against the cbioportal databasewwwcbioportal and are those found to have been previously described within thecosmic database cancersangeracukcosmic as being œlikely oncogenicimmunohistochemistryformalinfixed paraffin embedded histological sections of patient tissue were processedimaged and analysed by the histopathology unit at university hospital wales for anoidhistology anoids embedded within matrigel were fixed with paraformaldehyde for min at room temperature anoids were then lifted from wells and washed several timeswith pbs prior to embedding within low meltingpoint agarose followed by dehydration paraffin embedding sectioning and hematoxylineosin he staining images were acquiredon an olympus dp26 microscopewestern blot detectionanoids were harvested from x μl blobs of growth factorreduced matrigel using cellrecovery solution corning for hour on ice total protein was extracted from anoids byaddition of μl of lysis buffer to whole anoids m trishcl mm edta vvnp40 ipegal in ddh2o containing 1x phosstop phosphatase inhibitor roche and 1xcomplete protease inhibitor cocktail roche the lysates were centrifuged at rcf for min and proteins harvested in the supernatants protein content was measured using a bcaprotein assay kit pierce samples were resolved on novex nupage “ bistris pagegels and then blotted onto nitrocellulose membranes using the invitrogen iblot dry blottingcassette systemblots were blocked at room temperature for h in wv milk powder in wash buffer trisbuffered saline tbs plus tween primary antibodies were diluted in blocking bufferand incubated overnight at ˚c after washing the blots were incubated with hrpconjugatedsecondary antibodies for h at room temperature bands were visualized using the enhancedchemoluminescence supersignal west dura pierce antibodies and dilutions tnks12santa cruz tnks12 e10 sc365897 axin2 cell signalling technologies 76g6 actin sigma ac74 a2228 antimouse or antirabbit hrpconjugated antibodies ge healthcare anoid viability measurementsanoids in culture were gently dissociated to nearsingle cell populations using tryplelife technologies before resuspension within growth factorreduced matrigel and dispensedinto white clear bottomed well plates in μl matrigel per well cells μl of matrigelfollowing matrigel polymerization a 9point 2fold dilution range of compounds were dilutedin tailored growth medium and dispensed within each well dmso was used as a negative control at a final concentration of following a six day exposure to compounds cell titerglo 3d promega reagent was applied as per the manufacturer™s guidelines for an endpointreadout relative luminescence values were obtained on a bmg fluostar plate reader doseresponse curves and ec50 values were obtained using microsoft excel with xlfit plugin version one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorshigh content fluorescence microscopyanoid lines previously established in culture were split to single cells using tryple lifetechnologies and seeded within growth factorreduced matrigel corning in black clearbottomed well plates at μl per well at a density of cells μl matrigel upon polymerisation of matrigel growth media containing a titration range of individual compounds ordmso controls were added to each well prior to incubation at ˚c anoids were exposedto compounds for a total of six days prior to fixation in a fixand stain solution containing para formaldehyde sigma aldrich triton x100 sigma aldrich 025μm rhodaminephalloidin sigma aldrich and μgml hoechst sigma aldrich in pbs at ˚c for hafter staining plates were washed with pbs and sealed imaging was performed on an imagexpress1 highcontent micro confocal platform molecular devices a total of zstackimages were acquired at μm steps through the focal plane per well of a well plate using a x objective image stacks were captured from both tritc channel ex em and dapi channel ex380 em to detect both rhodaminephalloidin factin andhoechst nucleihigh content image analysisimages obtained using the imagexpress platform were processed for phenotypic analysiswithin ominer„¢ software ocello bv integrated within the knime analytics platformkonstanz germany httpwwwknime as described previously [ ] briefly thissoftware enabled the quantification of zstack images derived from hoechst and rhodaminephalloidin stained anoids information from both the dapi and tritc channels wereused to enhance anoid boundary separation masks generated from both channels facilitated the visualisation of main anoid structure internal morphometries such as individuallumens as well as nuclei per anoid objects that were out of focus were filtered and discarded from analysis morphological features were calculated on the basis of individual anoids and pooled for analysis multiple features extracted included counts total area solidityand branching of nuclei lumen and anoid masksfeature training between the negative control anoids and the high dose tnksi treatedanoids was performed to condense the phenotypic features and calculate the euclidian distance the results are plotted with graphpad prism graphpad software la jolla caresults are shown as means ± standard deviations unless otherwise statedquantitative realtime pcrtotal rna was extracted from anoids resuspended in trizol solution life technologiescontaining μgml glycogen cdna was yielded from purified mrna using improm iireverse transcription kit promega quantitativert pcr was then performed using thesensifast sybr green hirox master mix bioline primers were designed sigmaaldrich and listed in s2 table all samples were measured in triplicate with gene expressionnormalised to gapdh housekeeping gene reactions were measured on a step one plus realtime pcr instrument applied biosystems with relative changes in gene expression calculatedusing the δδct methodwholemount immunofluorescent staininganoids were seeded as single cells in well plates in μl growth factor reduced matrigelat a density of cellsμl following a six day exposure to compounds and control conditions anoids were fixed in pfa for min at room temperature wells were then one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorswashed with pbs containing mm glycine prior to blocking overnight in pbs containing horse serum bovine serum albumin triton x100 tween thenstained with primary antibodies overnight s1 table at ˚c secondary antibodies were thenadded overnight at ˚c prior to counterstaining with hoechst wells were washed with pbsthen imaged on a confocal microscope leica tcs sp2 aobsin vivo assessment of tnks inhibitor efficacyall mice were obtained commercially from charles river and then housed under uk homeoffice regulations the work described here was carried out under home office ppl in vivo anoid engraftment studies also termed œanoid derived xenografts odx wereconducted using immunedeficient nodscid gamma irradiated mice for anoid transplantation anoids were in culture for h from single cells prior to subcutaneous transplantation into the right flank of the animal one injection site per mouse mice were checkedevery days for tumour growth by palpation according to home office regulation protocolpalpable tumours mm were counted for use in kaplanmeier analysis cardiff university™s animal welfare and ethical review body awerb reviewed the plan for animal studiesprior to the grant of the home office license animals in the study described werekilled by cervical dislocation schedule methodresultsestablishment of tumour anoids from crc patient materialsurgicallyresected crc material was isolated from patients under informed consent cellfragments were isolated from tissue and plated within growth factor reduced matrigel within hours following surgery adapting methods previously described by sato two different culture conditions were used as standard œ basal and œfull growth factor richmedium each condition was found to be optimal for a distinct subset of anoids fig 1aand allowed the efficient generation of anoids from what were in some cases limitedtumour cell numbers of collected samples were established in culture a ˜takerate™ of for anoid model derivation anoids were denoted by isolation number ˜iso™recent studies have similarly emphasised the requirement for different media to ensure efficient anoid generation from different patients a subset of anoids was studied in more detail optimal culture conditions were determined on the basis of anoid formation efficiencies and visual inspection of anoid integrity anoid lines showed good growth in basal medium whilst were dependenton the additional inclusion of egf ngml noggin ngml nicotinamide mma8301 nm sb202190 μm as well as wnt3a and rspondin conditioned media the anoid medium combinations shown in fig 1a were used for subsequent studies full exome sequencing showed that the subset of anoids containedcommon genotypes found in other anoid banks fig 1a s1 appendix [ ] in particular of anoid lines harboured mutations in components of the wnt signalling pathwaysuch as apc and catenin ctnnb1 iso was found to harbour a mutation in rnf43which is associated with an increased frizzled fz receptor expression and wnt ligand dependence [ ]crc anoids have previously been shown to display histological features in commonwith the tumours from which they were derived paired histological samples from the anoids described were compared to their parental tumours distinct morphologies wereobserved ranging from lines that preferentially formed single epithelial cell layers with largelumens containing apoptotic cells fig 1b s1 fig to multilayered anoids with small one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorsfig generation and establishment of tumour anoids from crc patient material a summary of parental tumour sites and dukes™ stages anoid mediaconditions and mutation status of anoid lines identified in whole exome sequencing analysis b representative hematoxylineosin he staining of ffpetissue sections from primary colorectal tumour patient material and anoid counterparts anoids were cultured for a minimum of days prior to fixation andembedding qualitative morphological similarities were observed between tumour and anoid pairs scale bar μm101371 pone0235319g001indistinct lumens the anoids frequently retained key histopathological grading featuresthat were present within the original patient tumour epithelium for example iso wasestablished from a mucinous adenocarcinoma and displayed a high percentage of mucinousvacuole structures that resembled that of the matching patient tissue the retention of featurespresent within primary patient tumour tissue showed that many features of tumour morphology could be retained without the need for a stromal compartment one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorstankyrase inhibition altered the growth of patientderived anoidsto study crc anoid responses to a wnt pathway inhibitor we utilised the tankyrase inhibitor tnksi c1 fig 2a after six days of treatment axin2 and tnks12 protein levelswere raised in a number of anoid lines fig 2b tnks normally targets axin2 andtnks12 for degradation and tnksi treatment would be expected to stabilise levels of eachprotein to assess the functional impact of tankyrase inhibition on anoid growth cellviability assays were performed for anoid lines iso iso and iso as a measure oftoxicity in the normal cell setting we also evaluated c1 response in healthy intestinal anoids tumour and normal intestinal anoids were digested to nearsingle cells or mechanically dissociated into small fragments embedded within growthfactor reduced matrigel andoverlaid with growth media containing the tnksi for six days prior to measuring relativeatp levels cell titer glo 3d c1 which has a cellular mechanistic ec50 of 2nm showed partial efficacy in reducing atp levels and markedly different potency in the crc lines fig2c“2g c1 treatment in healthy intestinal anoids showed limited effects on metabolicactivity s3 fig ec50 values in crc lines ranged from 2nm to 1μm but accurate values werehard to establish due to assay variance and also because many cells survived even in the presence of high concentrations of compound leading to reduced assay windows the partial survival of anoid cells after days c1 treatment suggest that longterm axin2 and tnks12biomarker responses might be compromised by alterations in cellular composition immunofluorescent staining showed reduced levels of the proliferation marker ki67 in iso but notiso anoids suggesting that reduced proliferation contributed to the lower atp levelsobserved fig 2h and 2imultiparametric phenotypic profiling of anoids in 3d to classifyresponses to tankyrase inhibitionto better understand the tnksi partial efficacy a novel multiparametric 3d image workflowanalysis was used to characterise the morphological effects of the responses three tankyrase tnks12 small molecule inhibitors were used that differed over orders of magnitudein their potency c1 2nm c2 36nm c3 319nm fig 2a anoids were seeded and treatedas previously for days prior to fixation and staining with rhodaminephalloidin factin andhoechst dna images were acquired using a highcontent widefield fluorescencemicroscope as described previously [ ]to characterise phenotypes ominer„¢ software ocello„¢ was used to extract morphological features from 2d projections of the factin and nucleiderived image stacks to generatemasks of individual anoids internal lumen structures and nuclei image segmentation wasused to distinguish components of both channels and minimise any background signal fig3a s4 fig overall shape and fluorescence intensity of individual anoid structures wereextracted on a wellbywell basis to generate approximately quantified features such features included information in regards to shape size boundaries and branching of anoidlumens and nuclear shapeto identify optimum features that could classify tnksi responses feature training andeuclidian distance calculations were performed on each data set as previously described this reduced features into key differentiating components to generate a measure of distancebetween highest c1 tnksi doses and negative control dmso conditions the featurecollection that describes the compoundinduced morphological change differs per anoidmodel but most frequently included anoid size changes lumen shape and size appearanceof dead cells and changes in nucleus morphology related to growth reduction cell anizationand cell death induction s5 fig one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorsfig assessing the effects of tankyrase inhibition on overall anoid viability a structures of three tankyrase inhibitors msc2524070a c1msc2572070a c2 and msc2501490a c3 with corresponding ec50 values b western blot of eight anoid lines treated with c1 nm and harvested one 101371 pone0235319 august one 0c3d imaging of anoids identifies responses to tankyrase inhibitorsafter days c representative images of anoids treated with c1 nm or dmso scale bar μm d e f doseresponse curves of anoidstreated with c1 for six days anoids previously established in suitable culture conditions were enzymatically digested to single cells and overlaid with mediacontaining a dose range of drug nm“ nm or a dmso control in media cell titer glo 3d readouts were performed in triplicates for iso d iso e iso f g ec50 values obtained from n experiments data are presented as mean ± sem h i representative immunofluorescentstaining of a proliferation marker in anoids iso h and iso i with differential sensitivity to tnksi were treated with c1 nm or dmso control for days fixed and stained with ki67 arrows indicate proliferating cell within the anoid ki67 positivecells in iso only when treated with c1scale bar μm101371 pone0235319g002the morphometric analysis showed that tnks inhibition resulted in phenotypic alterationsin iso iso iso and iso fig 3b importantly the cellular morphometric responsescorrelated with known cellular ec50 values across a range of potencies compounds c1 c2and c3 that had ec50 values of nm nm and nm table also showed the expectedseparation of ec50 activities in the anoid morphometric assays suggesting that observedfig 3d image analysis demonstrates target affinity of tankyrase inhibitors in anoids a flow diagram of highcontent multiparametric image screen processb dose response curves acquired from high content imaging screen anoids were seeded as single cells in well plates and exposed to Answer:

Colon_Cancer

"dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genes”i reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236338copyright dwivedi this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [“] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used œhousekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [“] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employed“i cv �xsx where �x and σx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isn™t a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of “pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and “pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the ™ cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence ™ ™fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm ˚camplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco„¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco„¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks “ curls were deparaffinized inxylene at ˚c followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol„¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers™ instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at ˚c for minutes followed by amplification for cycles“denaturation at ˚c for sec amplification at ˚c for sec and extension at ˚c for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq sampleˆ’mean cq ntc and relative expression aseˆ’δcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithms“genorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and “pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [“] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression “ across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkin™s bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation “ and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of œadjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [“] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgs“ptcd2 ppp1r3b andfbxw9 have “ sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting “ original draft sreejeta mondal sujan k dharwriting “ review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct “ 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb “ pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep “ 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for œdoubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesrearrangements haematologica jun “ 103324haematol2018 pmid baro´ c espinet b salido m garcı´a m sa´nchez b florensa l cryptic ighbcl2 rearrangementswith variant fish patterns in follicular lymphoma leuk res feb “ 101016jleukres201009011 pmid hofman p heeke s alixpanabières c pantel k liquid biopsy in the era of immunooncology is itready for primetime use for cancer patients suppressed immune microenviron repert brain metastases from patients with resected nsclc “fatani s h mukhtar m h ali a s correlation between serum antiapoptotic bcl2 level and its immunohistochemical expression in relation to apoptosis in gastric cancer j mol biomark diagn albitar m zijun xy wang y manman d tzankov a visco c myc and bcl2 mrna expressionas determined by ngs predicts survival in dlbcl in gcb but not in abc subgroup blood nov 134supplement_15092“ derenzini e rossi a agostinelli c rossi m melle f motta g integration of nanostring profilingand functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement “zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may “101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep “ 101038leu2016382 pmid bomben r ferrero s d™agaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr “ 103324haematol2017184325pmid dheda k huggett jf chang js kim lu

cancer7553

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genes”i reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236338copyright dwivedi this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [“] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used œhousekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [“] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employed“i cv �xsx where �x and σx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isn™t a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of “pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and “pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the ™ cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence ™ ™fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm ˚camplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco„¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco„¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks “ curls were deparaffinized inxylene at ˚c followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol„¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers™ instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at ˚c for minutes followed by amplification for cycles“denaturation at ˚c for sec amplification at ˚c for sec and extension at ˚c for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq sampleˆ’mean cq ntc and relative expression aseˆ’δcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithms“genorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and “pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [“] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression “ across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkin™s bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation “ and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of œadjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [“] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgs“ptcd2 ppp1r3b andfbxw9 have “ sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting “ original draft sreejeta mondal sujan k dharwriting “ review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct “ 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb “ pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep “ 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for œdoubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesrearrangements haematologica jun “ 103324haematol2018 pmid baro´ c espinet b salido m garcı´a m sa´nchez b florensa l cryptic ighbcl2 rearrangementswith variant fish patterns in follicular lymphoma leuk res feb “ 101016jleukres201009011 pmid hofman p heeke s alixpanabières c pantel k liquid biopsy in the era of immunooncology is itready for primetime use for cancer patients suppressed immune microenviron repert brain metastases from patients with resected nsclc “fatani s h mukhtar m h ali a s correlation between serum antiapoptotic bcl2 level and its immunohistochemical expression in relation to apoptosis in gastric cancer j mol biomark diagn albitar m zijun xy wang y manman d tzankov a visco c myc and bcl2 mrna expressionas determined by ngs predicts survival in dlbcl in gcb but not in abc subgroup blood nov 134supplement_15092“ derenzini e rossi a agostinelli c rossi m melle f motta g integration of nanostring profilingand functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement “zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may “101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep “ 101038leu2016382 pmid bomben r ferrero s d™agaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr “ 103324haematol2017184325pmid dheda k huggett jf chang js kim lu Answer:

Colon_Cancer

cancer is the second leading cause of death in the united states cancer screenings candetect precancerous cells and allow for earlier diagnosis and treatment our purpose was tobetter understand risk factors for cancer screenings and assess the effect of cancer screenings on changes of cardiovascular health cvh measures before and after cancer screenings among patientsmethodswe used the guideline advantage tga”american heart association ambulatory qualityclinical data registry of electronic health record data n patients to investigateassociations between timeseries cvh measures and receipt of breast cervical and coloncancer screenings long shortterm memory lstm neural networks was employed to predict receipt of cancer screenings we also compared the distributions of cvh factorsbetween patients who received cancer screenings and those who did not finally we examined and quantified changes in cvh measures among the screened and nonscreenedgroupsresultsmodel performance was evaluated by the area under the receiver operator curve aurocthe average auroc of curves was for breast for cervical and for coloncancer screening distribution comparison found that screened patients had a higher prevalence of poor cvh categories cvh submetrics were improved for patients after cancerscreeningsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation guo a drake bf khan ym langabeer iijr foraker re timeseries cardiovascularrisk factors and receipt of screening for breastcervical and colon cancer the guidelineadvantage one e0236836 101371 pone0236836editor antonio palazo´nbru universidad miguelhernandez de elche spainreceived april accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236836copyright guo this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement the data are ownedby a third party and the authors do not havepermission to share the data requesting access tothe guideline advantage tga data must be done one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsby contacting the american heart association viaemail qualityresearchheart the python coderelated to the analyses can be found in githubrepository githubcomaixiaguopythoncodefunding the authors received no specificfunding for this workcompeting interests the authors have declaredthat no competing interests existdeep learning algorithm could be used to investigate the associations between timeseriescvh measures and cancer screenings in an ambulatory population patients with moreadverse cvh profiles tend to be screened for cancers and cancer screening may alsoprompt favorable changes in cvh cancer screenings may increase patient cvh healththus potentially decreasing burden of disease and costs for the health system eg cardiovascular diseases and cancersintroductioncancer is the second leading cause of death for both men and women in the united statesus breast cancer is the second leading cause of cancer death among women colorectal cancer ranks second among men and third among women while cervical cancer ranksas a major cause of cancer death among women regular cancer screenings for breast cervical and colorectal cancers can help to diagnose cancers early and reduce cancer deaths for example in the past years the number of deaths caused by cervical cancer has significantly decreased thanks to pap tests which can find abnormal cervical cells before they turn tocancer similarly colonoscopy removes noncancerous colon polyps before becomingmalignant and regular mammography screening can identify breast cancer in an earliermore treatable stage thus breast cancer screening bcs cervical cancer screening cecsand colorectal cancer screening cocs are very important for early detection and treatmentfactors associated with cancer screenings include demographic factors health insurancecoverage education level smoking status obesity and cholesterol testing for example receiptof mammography is associated with modifiable factors such as weight smoking and other lifestyle factors [“] receipt of cecs is associated with healthier weight lower cardiovascular disease occurrence and lower cholesterol some studies suggest that smokingsedentary lifestyle high body mass index and high comorbidity are associated with a higherpercentage of cocs participation [“] traditionally data for such studies originate fromquestionnaires claims data and telephone surveys and statistical analysis methods such aslogistic regression models are applied to examine the associations between the risk factors andcancer screenings electronic health records ehr contain longitudinal healthcare information and data including diagnoses medications procedures lab tests and images andtherefore can be used to discover new patterns and relationships from the rich data deeplearning algorithms have been widely and successfully used in bioinformatics and healthcarefields as they can effectively capture features and patterns in longitudinal data in this study we investigated associations between longitudinal cvh risk factors and thereceipt of cancer screenings using ehr data by the long shortterm memory lstm model we then studied the distribution of cvh factors between patients who did and did notreceive cancer screenings to further investigate the associations finally we compared measures of cvh longitudinally within those who did and did not receive screening to betterunderstand the effect of cancer screenings on cvh measuresmaterials and methodsethics statementall the data were fully anonymized before we accessed them our study was approved by theinstitutional review board at the washington university school of medicine in st louis we one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsobtained a written acknowledgement of proprietary rights and nondisclosure and data useagreement from the american heart association the washington university_nda_dua_contractid 158065_20190426_kdata source and study populationthe guideline advantage tga is a clinical data registry established in by the americancancer society the american diabetes association and the american heart associationaha ehr data has been collected from over clinics across the us by the tga totrack and monitor disease management and outpatient preventative care we used longitudinal tga data to predict three types of cancer screenings among unique patientswe used a 6year range “ to identify female patients in the “ year oldage group who received bcs female patients in the “ year old age group who receivedcecs and patients in the “ year old age group who received cocs if patientsreceived multiple types of cancer screening we only considered the first using the same criteria for gender and age we randomly selected a comparison group of patients who did notreceive cancer screenings for bcs for cecs and for cocswe utilized the following cvh measures defined by the aha smoking status body massindex bmi blood pressure bp hemoglobin a1c a1c and cholesterol lowdensitylipoprotein ldl in our dataset we then classified them into three categories ideal intermediate or poor according to table we utilized the multum drug database as a template to convert the drug names in our dataset to their corresponding drug classes thelevenshtein distance algorithm was employed for the conversion by comparing the drugnames in our dataset to the multum drug database template the conversion was consideredsuccessful and medications were considered as treatments for bp a1c or ldl table if thedistance between the two compared strings was less than five all cvh measurements prior tothe date of cancer screening were considered in the analysis for those who received screeningand all cvh measurements in the data set were considered in the analysis for those who didnot receive screeningfor the primary analysis we selected patients who had at least one measure of cvh for bcs for cecs and for cocs in the comparison groups there were availabledata for bcs cecs and cocsstatistical analysiswe first studied the lstm prediction of cancer screening from timeseries cvh factors wedivided each cvh factor into its submetric of œideal œintermediate or œpoor according totable for example if a patient had a measure of œideal blood pressure then that featuretable measures of cvh which are available in the tga adapted from lloydjones poor healthintermediate healthideal healthhealth behaviorssmoking statusyesformer � monthsbody mass index� kgm2“ kgm2health factorsnever or quit months kgm2ldl� mgdl“ mgdl or treated to goal mgdlblood pressurefasting plasmaglucosesystolic � mm hg or diastolic � mmhgsystolic “ mm hg or diastolic “ mm hg or treated togoalsystolic mm hgdiastolic mm hg� mgdl“ mgdl or treated to goal mgdl101371 pone0236836t001 one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningswas called blood pressure ideal all features were then embedded to a 32dimensional vectorspace by word2vec for each type of cancer screenings the python genism word2vecmodel used the following hyperparameters size embedding dimension was window themaximum distance between a target word and all words around it was min_count theminimum number of words counted when training the model was sg the training algorithm was cbow the continuous bag of words time information for each measure wasadded and was calculated by the difference in days between each visit date and the most recentvisit date thus each feature was associated with its own time point in the unit of daysthe resulting embedded vectors and associated time points were fed to the lstm modeldue to the comparison group being much larger than the number of patients with cancerscreening we randomly selected patients for bcs patients for cecs and patientsfor cocs and repeated this process for times to account for the imbalance betweenscreened and unscreened groups each time the data set for each type of cancer screening wassplit into a training data set and a test data set we trained the lstm model onthe training data and tested the trained model on the test data we utilized the average of thearea under the receiver operator curve auroc to evaluate the performance of our lstmmodel for each type of cancer evaluatedour lstm model comprised an input layer one hidden layer with dimensions andan output layer the hyperparameter used in the model was as follows a sigmoid function wasused as the activation function in the output layer a binary crossentropy was used as the lossfunction adam optimizer was used to optimize the model with a minibatch size of sampleswe then investigated whether distributions of cvh“counts and percentages for each submetric“differed between patients who did and who did not receive cancer screenings by chisquared test finally we studied changes in cvh factors within screening group for the samepatients who received screening and for those who did not within screening group we compared cvh measures from before and on the day of the screening to the cvh measures collected after the screening for the patients who did not receive screening we compared cvhmeasures before and after the midpoint of the visit dates if patients only had a single visitthen they were not included in the before and after analysis analyses were conducted by usingthe libraries of scikitlearn scipy matplotlib with python version in resultsthe majority of our study population was white with a mean of age of approximately yearsfor bcs years for cecs and years for cocs table the nonwhite study populationwas predominantly africanamerican the average number of measures avg amongpatients who received screening was higher than that of patients who were not screened forexample the average number of bp measurements for patients with bcs was for cecsand for cocs compared to for bcs for cecs and for cocs for patients who werenot screenedfig displays the performance of lstm cancer screening predictions in terms of repeated aurocs for each type of screening the average auroc of curves was forbcs for cecs and for cocstable lists the numbers and proportions of patients in ideal intermediate and poor categories for each submetric for the comparison between patients who received cancer screeningand those who did not we applied a chisquared test to check if the frequencies herepercentages between screening groups were significantly different from one other within eachcvh submetric as shown in table patients who received cancer screening had a higher one 101371 pone0236836 august one 0ctable characteristics [mean sd or n ] of the study population by receipt of cancer screeningcardiovascular risk factors and receipt of cancer screeningscancer screeningsbcsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cecsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cocsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n n n n yesnon � n n avg avg avg avg avg avg � the percentages may not add up to due to rounding101371 pone0236836t002prevalence of poor a1c for bcs for cecs and for cocs compared topatients who did not receive screening for bcs for cecs and for cocsfig shows changes in cvh submetrics within the same patient screening groups fig a“2c show the changes in cvh submetrics for the patients who were screened while fig2e and 2f show the changes in cvh for patients who were not screened one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the area under the curve auc are shown for lstm cancer screening predictions from timeseries cvh factors which were repeated times withdifferent comparison patients for bcs a cecs b and cocs c101371 pone0236836g001from the first column of fig we can see that the prevalence of œpoor submetricsdecreased after cancer screenings for example all five submetrics improved after bcs fig a while bp and a1c improved after cecs fig 2b and bp a1c and smoking improvedafter cocs fig 2c notably for the prevalence of poor a1c decreased for all patients whoreceived cancer screenings in bcs in cecs and in cocs on the other handfrom the second column of fig we can see that the prevalence of œpoor a1c increased forall comparison patientsdiscussionin this study we demonstrated associations between timeseries cvh risk factor measuresand receipt of three types of cancer screenings ie breast cervical and colon cancer screenings by using a nationally representative dataset“tga data the tga data enabled us toexamine multiple sites cvh submetrics and types of cancer screenings using advanced deeplearning models an advantage of our study was that all cvh submetrics were investigatedsimultaneously for an association with different cancer screenings on a unique nationallyrepresentative dataset of patients ie the large tga data set which contains longitudinaltable comparison cvh factors between patients with cancer screening or without [n ]patients with bcs n chisquared pvalueidealintermediatepoorpatients without bcs n idealintermediatepoorpatients with cecs n chisquared pvalueidealintermediatepoorpatients without cecs n idealintermediatepoorpatients with cocs n chisquared pvalueidealintermediatepoorpatients without cocs n idealintermediatepoorbmi bmi bmi 101371 pone0236836t003bp bp bp a1c a1c a1c ldl ldl ldl smoking smoking smoking one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the plots of percentages for poor cvh factors for the same patients before and after time points of cancer screening for patients with screeningsa“c and before and after middle time points for patients without cancer screenings d“f the first row is for bcs second row is for cecs andthe third is for cocs101371 pone0236836g002cvh measurements and cancer screening patterns from more than different clinics in theusthe comparison of different cvh measure distributions between patients who receivedcancer screenings and those who did not showed that patients with poorer cvh especiallypoor a1c were more likely to receive cancer screenings specifically patients with poorera1c were more likely to receive cancer screenings some recent studies have showed that individuals with diabetes had higher incidence of certain cancers and also were more likely tobe diagnosed with advancedstage tumors [“] thus providers might be more likely torecommend patients with diabetes to uptake cancer screenings for early prevention of developing cancers which may lead to more individuals with diabetes to participate in cancerscreeningsmoreover we investigated the effects of cancer screenings on the changes of cvh measuresof the patients to better understand if the screenings had potential associations with theimprovement of cvh measures our results indicated that patients who received cancerscreenings appeared to have better control of cvh factors especially a1c than patients whodid not receive cancer screenings specifically a1c levels were improved after patientsreceived any type of screening while a1c levels worsened among patients who did not receivecancer screening a similar trend could be observed for bmi it became better after patientsreceived any type of screening while bmi became worse among patients without bcs orcocs levels of bp were improved after patients received bcs or cocs screenings and worsened among patients without bcs or cocs poor levels of ldl decreased among patientsafter receipt of bcs and among those without bcs however ldl improvements were muchgreater among patients after receipt of bcs decrease in ldl than those without bcs decrease in ldl after receipt of bcs and cocs current smoking declined comparedto the increase observed among those without the screeningsin summary our analyses showed that patients with poor cvh measures were more likelyto receive cancer screenings patients with receipt of cancer screenings appeared to haveimproved cvh measures after the screening as compared to before one possible reason forthis was that patients might receive more attention and through care from providers to detectand manage cvh by virtue of reviewing cancer screening and other risk factor data at thepopulation level better cvh is associated with a lower risk of cardiovascular disease cvdand cancers thus cancer screenings may indirectly decrease burden and cost on thehealth system eg cvd and cancers by improving patient cvh healthlimitationsthere were some limitations in our analyses we used values of auroc to evaluate associations between timeseries cvh measurements and receipt of cancer screenings higherauroc values indicated stronger associations between predictors and the binary outcomes however our observed auroc values were relatively low and thus have limited clinicalutility at this time cancer screenings are potentially affected by cvh and other factors weacknowledge that we had relatively few patients with receipt of cancer screening specificallythere were relatively few patients who received cancer screenings compared to patients whodid not within the same age and gender groups this limitation likely affected the accuracy of one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsour prediction models the prediction accuracy of our models could be improved if morepatients in our data set had received cancer screeningswe demonstrated that deep learning lstm models can effectively predict the associationsbetween timeseries cvh measures and receipt of cancer screening poor cvh especiallypoor a1c may prompt providers to recommend cancer screening for their patients andpatients who received cancer screening may also receive better care for andor have improvedselfmanagement of cvh especially a1c overall these findings suggest that unhealthierpatients are screened for cancers and that cancer screening may also prompt favorablechanges in cvhauthor contributionsconceptualization randi e forakerformal analysis aixia guosupervision randi e forakerwriting “ original draft aixia guowriting “ review editing bettina f drake yosef m khan james r langabeer ii randi eforakerwwwmedicalnewstodaycoms282929phpreferences humphrey ll helfand m chan bks woolf sh breast cancer screening a summary of the evidencefor the us preventive services task force annals of internal medicine 107326000348191375_part_120020903000012 american cancer society cancer facts figures am cancer soc 10 pmid wwwcdcgovcancercervicalstatisticsindexhtmwwwcdcgovcancerdcpcpreventionscreeninghtmwwwcdcgovcancercervicalstatisticsindexhtm1 edwards qt li ax pike mc kolonel ln ursin g henderson be ethnic differences in the use ofregular mammography the multiethnic cohort breast cancer res treat 101007s1054900800497 pmid bynum jpw braunstein jb sharkey p haddad k wu aw the influence of health status age andrace on screening mammography in elderly women arch intern med 101001archinte165182083 pmid lipscombe ll hux je booth gl reduced screening mammography among women with diabetesarch intern med 101001archinte165182090 pmid berz d sikov w colvin g weitzen s œweighing in on screening mammography breast cancer restreat 101007s105490080037y pmid cook nr rosner ba hankinson se colditz ga mammographic screening and risk factors for breastcancer am j epidemiol 101093ajekwp304 pmid fontaine kr heo m allison db body weight and cancer screening among women j womenshealth gend based med 101089152460901300233939 pmid hsia j kemper e kiefe c zapka j sofaer s pettinger m the importance of health insurance asa determinant of cancer screening evidence from the women™s health initiative prev med baltim 101006pmed20000697 pmid hueston w stiles m the papanicolaou smear as a sentinel screening test for health screening inwomen arch intern med “ pmid one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings robb ka miles a wardle j demographic and psychosocial factors associated with perceived risk forcolorectal cancer cancer epidemiology biomarkers and prevention robb ka miles a wardle j perceived risk of colorectal cancer sources of risk judgments cancer epidemiol biomarkers prev 10115810559965epi060151 pmid gimeno garca az factors influencing colorectal cancer screening participation gastroenterologyresearch and practice 1011552012483417 pmid jensen pb jensen lj brunak s mining electronic health records towards better research applications and clinical care nature reviews genetics 101038nrg3208 pmid goodfellow i bengio y courville a deep learning mit press 101533 rajkomar a oren e chen k dai am hajaj n hardt m scalable and accurate deep learning withelectronic health records npj digit med 101038s4174601800291 pmid hochreiter s s long shortterm memory neural comput “ bufalino v bauman ma shubrook jh evolution of œthe guideline advantage lessons learnedfrom the front lines of outpatient performance measurement ca cancer j clin “103322caac21233 pmid wwwscrippssparkleassetsdocumentsheart_rhythm_factspdf shickel b tighe pj bihorac a rashidi p deep ehr a survey of recent advances in deep learningtechniques for electronic health record ehr analysis ieee j biomed heal informatics 101109jbhi20172767063 pmid levenshtein vi binary codes capable of correcting deletions insertions and reversals sov phys dokl citeulikeid311174lloydjones dm hong y labarthe d mozaffarian d appel lj van horn l defining and settingnational goals for cardiovascular health promotion and disease reduction circulation “ 101161circulationaha109192703 pmid mikolov t corrado g chen k dean j word2vec proc int conf learn represent iclr kingma dp ba j adam a method for stochastic optimization corr 2015abs14126 pearson k on the criterion that a given system of deviations from the probable in the case of a correlated system of variables is such that it can be reasonably supposed to have arisen from random sampling philos mag “tsilidis kk kasimis jc lopez ds ntzani ee ioannidis jpa type diabetes and cancer umbrellareview of metaanalyses of observationlal studies bmj online 101136bmjg7607 pmid lipscombe ll fischer hd austin pc fu l jaakkimainen rl ginsburg o the associationbetween diabetes and breast cancer stage at diagnosis a populationbased study breast cancer restreat 101007s1054901533235 pmid bhatia d lega ic wu w lipscombe ll breast cervical and colorectal cancer screening in adults withdiabetes a systematic review and metaanalysis diabetologia 101007s00125 pmid wilkinson je culpepper l associations between colorectal cancer screening and glycemic control inpeople with diabetes boston massachusetts “ prev chronic dis wang yq wang cf zhu l yuan h wu lx chen zh ideal cardiovascular health and the subclinicalimpairments of cardiovascular diseases a crosssectional study in central south china bmc cardiovasc disord 101186s1287201706979 pmid foraker re abdelrasoul m kuller lh jackson rd van horn l seguin ra cardiovascularhealth and incident cardiovascular disease and cancer the women™s health initiative am j prev med “ 101016jamepre201507039 pmid yin j using the roc curve to measure association and evaluate prediction accuracy for a binary outcome biometrics biostat int j 1015406bbij20170500134 one 101371 pone0236836 august one 0c'

cancer7554

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: cancer is the second leading cause of death in the united states cancer screenings candetect precancerous cells and allow for earlier diagnosis and treatment our purpose was tobetter understand risk factors for cancer screenings and assess the effect of cancer screenings on changes of cardiovascular health cvh measures before and after cancer screenings among patientsmethodswe used the guideline advantage tga”american heart association ambulatory qualityclinical data registry of electronic health record data n patients to investigateassociations between timeseries cvh measures and receipt of breast cervical and coloncancer screenings long shortterm memory lstm neural networks was employed to predict receipt of cancer screenings we also compared the distributions of cvh factorsbetween patients who received cancer screenings and those who did not finally we examined and quantified changes in cvh measures among the screened and nonscreenedgroupsresultsmodel performance was evaluated by the area under the receiver operator curve aurocthe average auroc of curves was for breast for cervical and for coloncancer screening distribution comparison found that screened patients had a higher prevalence of poor cvh categories cvh submetrics were improved for patients after cancerscreeningsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation guo a drake bf khan ym langabeer iijr foraker re timeseries cardiovascularrisk factors and receipt of screening for breastcervical and colon cancer the guidelineadvantage one e0236836 101371 pone0236836editor antonio palazo´nbru universidad miguelhernandez de elche spainreceived april accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236836copyright guo this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement the data are ownedby a third party and the authors do not havepermission to share the data requesting access tothe guideline advantage tga data must be done one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsby contacting the american heart association viaemail qualityresearchheart the python coderelated to the analyses can be found in githubrepository githubcomaixiaguopythoncodefunding the authors received no specificfunding for this workcompeting interests the authors have declaredthat no competing interests existdeep learning algorithm could be used to investigate the associations between timeseriescvh measures and cancer screenings in an ambulatory population patients with moreadverse cvh profiles tend to be screened for cancers and cancer screening may alsoprompt favorable changes in cvh cancer screenings may increase patient cvh healththus potentially decreasing burden of disease and costs for the health system eg cardiovascular diseases and cancersintroductioncancer is the second leading cause of death for both men and women in the united statesus breast cancer is the second leading cause of cancer death among women colorectal cancer ranks second among men and third among women while cervical cancer ranksas a major cause of cancer death among women regular cancer screenings for breast cervical and colorectal cancers can help to diagnose cancers early and reduce cancer deaths for example in the past years the number of deaths caused by cervical cancer has significantly decreased thanks to pap tests which can find abnormal cervical cells before they turn tocancer similarly colonoscopy removes noncancerous colon polyps before becomingmalignant and regular mammography screening can identify breast cancer in an earliermore treatable stage thus breast cancer screening bcs cervical cancer screening cecsand colorectal cancer screening cocs are very important for early detection and treatmentfactors associated with cancer screenings include demographic factors health insurancecoverage education level smoking status obesity and cholesterol testing for example receiptof mammography is associated with modifiable factors such as weight smoking and other lifestyle factors [“] receipt of cecs is associated with healthier weight lower cardiovascular disease occurrence and lower cholesterol some studies suggest that smokingsedentary lifestyle high body mass index and high comorbidity are associated with a higherpercentage of cocs participation [“] traditionally data for such studies originate fromquestionnaires claims data and telephone surveys and statistical analysis methods such aslogistic regression models are applied to examine the associations between the risk factors andcancer screenings electronic health records ehr contain longitudinal healthcare information and data including diagnoses medications procedures lab tests and images andtherefore can be used to discover new patterns and relationships from the rich data deeplearning algorithms have been widely and successfully used in bioinformatics and healthcarefields as they can effectively capture features and patterns in longitudinal data in this study we investigated associations between longitudinal cvh risk factors and thereceipt of cancer screenings using ehr data by the long shortterm memory lstm model we then studied the distribution of cvh factors between patients who did and did notreceive cancer screenings to further investigate the associations finally we compared measures of cvh longitudinally within those who did and did not receive screening to betterunderstand the effect of cancer screenings on cvh measuresmaterials and methodsethics statementall the data were fully anonymized before we accessed them our study was approved by theinstitutional review board at the washington university school of medicine in st louis we one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsobtained a written acknowledgement of proprietary rights and nondisclosure and data useagreement from the american heart association the washington university_nda_dua_contractid 158065_20190426_kdata source and study populationthe guideline advantage tga is a clinical data registry established in by the americancancer society the american diabetes association and the american heart associationaha ehr data has been collected from over clinics across the us by the tga totrack and monitor disease management and outpatient preventative care we used longitudinal tga data to predict three types of cancer screenings among unique patientswe used a 6year range “ to identify female patients in the “ year oldage group who received bcs female patients in the “ year old age group who receivedcecs and patients in the “ year old age group who received cocs if patientsreceived multiple types of cancer screening we only considered the first using the same criteria for gender and age we randomly selected a comparison group of patients who did notreceive cancer screenings for bcs for cecs and for cocswe utilized the following cvh measures defined by the aha smoking status body massindex bmi blood pressure bp hemoglobin a1c a1c and cholesterol lowdensitylipoprotein ldl in our dataset we then classified them into three categories ideal intermediate or poor according to table we utilized the multum drug database as a template to convert the drug names in our dataset to their corresponding drug classes thelevenshtein distance algorithm was employed for the conversion by comparing the drugnames in our dataset to the multum drug database template the conversion was consideredsuccessful and medications were considered as treatments for bp a1c or ldl table if thedistance between the two compared strings was less than five all cvh measurements prior tothe date of cancer screening were considered in the analysis for those who received screeningand all cvh measurements in the data set were considered in the analysis for those who didnot receive screeningfor the primary analysis we selected patients who had at least one measure of cvh for bcs for cecs and for cocs in the comparison groups there were availabledata for bcs cecs and cocsstatistical analysiswe first studied the lstm prediction of cancer screening from timeseries cvh factors wedivided each cvh factor into its submetric of œideal œintermediate or œpoor according totable for example if a patient had a measure of œideal blood pressure then that featuretable measures of cvh which are available in the tga adapted from lloydjones poor healthintermediate healthideal healthhealth behaviorssmoking statusyesformer � monthsbody mass index� kgm2“ kgm2health factorsnever or quit months kgm2ldl� mgdl“ mgdl or treated to goal mgdlblood pressurefasting plasmaglucosesystolic � mm hg or diastolic � mmhgsystolic “ mm hg or diastolic “ mm hg or treated togoalsystolic mm hgdiastolic mm hg� mgdl“ mgdl or treated to goal mgdl101371 pone0236836t001 one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningswas called blood pressure ideal all features were then embedded to a 32dimensional vectorspace by word2vec for each type of cancer screenings the python genism word2vecmodel used the following hyperparameters size embedding dimension was window themaximum distance between a target word and all words around it was min_count theminimum number of words counted when training the model was sg the training algorithm was cbow the continuous bag of words time information for each measure wasadded and was calculated by the difference in days between each visit date and the most recentvisit date thus each feature was associated with its own time point in the unit of daysthe resulting embedded vectors and associated time points were fed to the lstm modeldue to the comparison group being much larger than the number of patients with cancerscreening we randomly selected patients for bcs patients for cecs and patientsfor cocs and repeated this process for times to account for the imbalance betweenscreened and unscreened groups each time the data set for each type of cancer screening wassplit into a training data set and a test data set we trained the lstm model onthe training data and tested the trained model on the test data we utilized the average of thearea under the receiver operator curve auroc to evaluate the performance of our lstmmodel for each type of cancer evaluatedour lstm model comprised an input layer one hidden layer with dimensions andan output layer the hyperparameter used in the model was as follows a sigmoid function wasused as the activation function in the output layer a binary crossentropy was used as the lossfunction adam optimizer was used to optimize the model with a minibatch size of sampleswe then investigated whether distributions of cvh“counts and percentages for each submetric“differed between patients who did and who did not receive cancer screenings by chisquared test finally we studied changes in cvh factors within screening group for the samepatients who received screening and for those who did not within screening group we compared cvh measures from before and on the day of the screening to the cvh measures collected after the screening for the patients who did not receive screening we compared cvhmeasures before and after the midpoint of the visit dates if patients only had a single visitthen they were not included in the before and after analysis analyses were conducted by usingthe libraries of scikitlearn scipy matplotlib with python version in resultsthe majority of our study population was white with a mean of age of approximately yearsfor bcs years for cecs and years for cocs table the nonwhite study populationwas predominantly africanamerican the average number of measures avg amongpatients who received screening was higher than that of patients who were not screened forexample the average number of bp measurements for patients with bcs was for cecsand for cocs compared to for bcs for cecs and for cocs for patients who werenot screenedfig displays the performance of lstm cancer screening predictions in terms of repeated aurocs for each type of screening the average auroc of curves was forbcs for cecs and for cocstable lists the numbers and proportions of patients in ideal intermediate and poor categories for each submetric for the comparison between patients who received cancer screeningand those who did not we applied a chisquared test to check if the frequencies herepercentages between screening groups were significantly different from one other within eachcvh submetric as shown in table patients who received cancer screening had a higher one 101371 pone0236836 august one 0ctable characteristics [mean sd or n ] of the study population by receipt of cancer screeningcardiovascular risk factors and receipt of cancer screeningscancer screeningsbcsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cecsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n cocsdemographicsage mean std yearwhite race n nonwhite race n unknown race n cvh factors mean std avg measuresa1cldl mgdlbmi kgm2systolic blood pressure sbp mmhgdiastolic blood pressure dbp mmhgcurrent smoking n n n n yesnon � n n avg avg avg avg avg avg � the percentages may not add up to due to rounding101371 pone0236836t002prevalence of poor a1c for bcs for cecs and for cocs compared topatients who did not receive screening for bcs for cecs and for cocsfig shows changes in cvh submetrics within the same patient screening groups fig a“2c show the changes in cvh submetrics for the patients who were screened while fig2e and 2f show the changes in cvh for patients who were not screened one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the area under the curve auc are shown for lstm cancer screening predictions from timeseries cvh factors which were repeated times withdifferent comparison patients for bcs a cecs b and cocs c101371 pone0236836g001from the first column of fig we can see that the prevalence of œpoor submetricsdecreased after cancer screenings for example all five submetrics improved after bcs fig a while bp and a1c improved after cecs fig 2b and bp a1c and smoking improvedafter cocs fig 2c notably for the prevalence of poor a1c decreased for all patients whoreceived cancer screenings in bcs in cecs and in cocs on the other handfrom the second column of fig we can see that the prevalence of œpoor a1c increased forall comparison patientsdiscussionin this study we demonstrated associations between timeseries cvh risk factor measuresand receipt of three types of cancer screenings ie breast cervical and colon cancer screenings by using a nationally representative dataset“tga data the tga data enabled us toexamine multiple sites cvh submetrics and types of cancer screenings using advanced deeplearning models an advantage of our study was that all cvh submetrics were investigatedsimultaneously for an association with different cancer screenings on a unique nationallyrepresentative dataset of patients ie the large tga data set which contains longitudinaltable comparison cvh factors between patients with cancer screening or without [n ]patients with bcs n chisquared pvalueidealintermediatepoorpatients without bcs n idealintermediatepoorpatients with cecs n chisquared pvalueidealintermediatepoorpatients without cecs n idealintermediatepoorpatients with cocs n chisquared pvalueidealintermediatepoorpatients without cocs n idealintermediatepoorbmi bmi bmi 101371 pone0236836t003bp bp bp a1c a1c a1c ldl ldl ldl smoking smoking smoking one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsfig the plots of percentages for poor cvh factors for the same patients before and after time points of cancer screening for patients with screeningsa“c and before and after middle time points for patients without cancer screenings d“f the first row is for bcs second row is for cecs andthe third is for cocs101371 pone0236836g002cvh measurements and cancer screening patterns from more than different clinics in theusthe comparison of different cvh measure distributions between patients who receivedcancer screenings and those who did not showed that patients with poorer cvh especiallypoor a1c were more likely to receive cancer screenings specifically patients with poorera1c were more likely to receive cancer screenings some recent studies have showed that individuals with diabetes had higher incidence of certain cancers and also were more likely tobe diagnosed with advancedstage tumors [“] thus providers might be more likely torecommend patients with diabetes to uptake cancer screenings for early prevention of developing cancers which may lead to more individuals with diabetes to participate in cancerscreeningsmoreover we investigated the effects of cancer screenings on the changes of cvh measuresof the patients to better understand if the screenings had potential associations with theimprovement of cvh measures our results indicated that patients who received cancerscreenings appeared to have better control of cvh factors especially a1c than patients whodid not receive cancer screenings specifically a1c levels were improved after patientsreceived any type of screening while a1c levels worsened among patients who did not receivecancer screening a similar trend could be observed for bmi it became better after patientsreceived any type of screening while bmi became worse among patients without bcs orcocs levels of bp were improved after patients received bcs or cocs screenings and worsened among patients without bcs or cocs poor levels of ldl decreased among patientsafter receipt of bcs and among those without bcs however ldl improvements were muchgreater among patients after receipt of bcs decrease in ldl than those without bcs decrease in ldl after receipt of bcs and cocs current smoking declined comparedto the increase observed among those without the screeningsin summary our analyses showed that patients with poor cvh measures were more likelyto receive cancer screenings patients with receipt of cancer screenings appeared to haveimproved cvh measures after the screening as compared to before one possible reason forthis was that patients might receive more attention and through care from providers to detectand manage cvh by virtue of reviewing cancer screening and other risk factor data at thepopulation level better cvh is associated with a lower risk of cardiovascular disease cvdand cancers thus cancer screenings may indirectly decrease burden and cost on thehealth system eg cvd and cancers by improving patient cvh healthlimitationsthere were some limitations in our analyses we used values of auroc to evaluate associations between timeseries cvh measurements and receipt of cancer screenings higherauroc values indicated stronger associations between predictors and the binary outcomes however our observed auroc values were relatively low and thus have limited clinicalutility at this time cancer screenings are potentially affected by cvh and other factors weacknowledge that we had relatively few patients with receipt of cancer screening specificallythere were relatively few patients who received cancer screenings compared to patients whodid not within the same age and gender groups this limitation likely affected the accuracy of one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screeningsour prediction models the prediction accuracy of our models could be improved if morepatients in our data set had received cancer screeningswe demonstrated that deep learning lstm models can effectively predict the associationsbetween timeseries cvh measures and receipt of cancer screening poor cvh especiallypoor a1c may prompt providers to recommend cancer screening for their patients andpatients who received cancer screening may also receive better care for andor have improvedselfmanagement of cvh especially a1c overall these findings suggest that unhealthierpatients are screened for cancers and that cancer screening may also prompt favorablechanges in cvhauthor contributionsconceptualization randi e forakerformal analysis aixia guosupervision randi e forakerwriting “ original draft aixia guowriting “ review editing bettina f drake yosef m khan james r langabeer ii randi eforakerwwwmedicalnewstodaycoms282929phpreferences humphrey ll helfand m chan bks woolf sh breast cancer screening a summary of the evidencefor the us preventive services task force annals of internal medicine 107326000348191375_part_120020903000012 american cancer society cancer facts figures am cancer soc 10 pmid wwwcdcgovcancercervicalstatisticsindexhtmwwwcdcgovcancerdcpcpreventionscreeninghtmwwwcdcgovcancercervicalstatisticsindexhtm1 edwards qt li ax pike mc kolonel ln ursin g henderson be ethnic differences in the use ofregular mammography the multiethnic cohort breast cancer res treat 101007s1054900800497 pmid bynum jpw braunstein jb sharkey p haddad k wu aw the influence of health status age andrace on screening mammography in elderly women arch intern med 101001archinte165182083 pmid lipscombe ll hux je booth gl reduced screening mammography among women with diabetesarch intern med 101001archinte165182090 pmid berz d sikov w colvin g weitzen s œweighing in on screening mammography breast cancer restreat 101007s105490080037y pmid cook nr rosner ba hankinson se colditz ga mammographic screening and risk factors for breastcancer am j epidemiol 101093ajekwp304 pmid fontaine kr heo m allison db body weight and cancer screening among women j womenshealth gend based med 101089152460901300233939 pmid hsia j kemper e kiefe c zapka j sofaer s pettinger m the importance of health insurance asa determinant of cancer screening evidence from the women™s health initiative prev med baltim 101006pmed20000697 pmid hueston w stiles m the papanicolaou smear as a sentinel screening test for health screening inwomen arch intern med “ pmid one 101371 pone0236836 august one 0ccardiovascular risk factors and receipt of cancer screenings robb ka miles a wardle j demographic and psychosocial factors associated with perceived risk forcolorectal cancer cancer epidemiology biomarkers and prevention robb ka miles a wardle j perceived risk of colorectal cancer sources of risk judgments cancer epidemiol biomarkers prev 10115810559965epi060151 pmid gimeno garca az factors influencing colorectal cancer screening participation gastroenterologyresearch and practice 1011552012483417 pmid jensen pb jensen lj brunak s mining electronic health records towards better research applications and clinical care nature reviews genetics 101038nrg3208 pmid goodfellow i bengio y courville a deep learning mit press 101533 rajkomar a oren e chen k dai am hajaj n hardt m scalable and accurate deep learning withelectronic health records npj digit med 101038s4174601800291 pmid hochreiter s s long shortterm memory neural comput “ bufalino v bauman ma shubrook jh evolution of œthe guideline advantage lessons learnedfrom the front lines of outpatient performance measurement ca cancer j clin “103322caac21233 pmid wwwscrippssparkleassetsdocumentsheart_rhythm_factspdf shickel b tighe pj bihorac a rashidi p deep ehr a survey of recent advances in deep learningtechniques for electronic health record ehr analysis ieee j biomed heal informatics 101109jbhi20172767063 pmid levenshtein vi binary codes capable of correcting deletions insertions and reversals sov phys dokl citeulikeid311174lloydjones dm hong y labarthe d mozaffarian d appel lj van horn l defining and settingnational goals for cardiovascular health promotion and disease reduction circulation “ 101161circulationaha109192703 pmid mikolov t corrado g chen k dean j word2vec proc int conf learn represent iclr kingma dp ba j adam a method for stochastic optimization corr 2015abs14126 pearson k on the criterion that a given system of deviations from the probable in the case of a correlated system of variables is such that it can be reasonably supposed to have arisen from random sampling philos mag “tsilidis kk kasimis jc lopez ds ntzani ee ioannidis jpa type diabetes and cancer umbrellareview of metaanalyses of observationlal studies bmj online 101136bmjg7607 pmid lipscombe ll fischer hd austin pc fu l jaakkimainen rl ginsburg o the associationbetween diabetes and breast cancer stage at diagnosis a populationbased study breast cancer restreat 101007s1054901533235 pmid bhatia d lega ic wu w lipscombe ll breast cervical and colorectal cancer screening in adults withdiabetes a systematic review and metaanalysis diabetologia 101007s00125 pmid wilkinson je culpepper l associations between colorectal cancer screening and glycemic control inpeople with diabetes boston massachusetts “ prev chronic dis wang yq wang cf zhu l yuan h wu lx chen zh ideal cardiovascular health and the subclinicalimpairments of cardiovascular diseases a crosssectional study in central south china bmc cardiovasc disord 101186s1287201706979 pmid foraker re abdelrasoul m kuller lh jackson rd van horn l seguin ra cardiovascularhealth and incident cardiovascular disease and cancer the women™s health initiative am j prev med “ 101016jamepre201507039 pmid yin j using the roc curve to measure association and evaluate prediction accuracy for a binary outcome biometrics biostat int j 1015406bbij20170500134 one 101371 pone0236836 august one 0c' Answer:

Colon_Cancer

the development of fungal fruiting bodies from a hyphal thallus is inducible under low temperature cold stress the molecular mechanism has been subject to surprisingly few studies analysis of gene expression level has become an important means to study genefunction and its regulation mechanism but identification of reference genes rgs stabilityunder cold stress have not been reported in famous medicinal mushroomforming fungi cordyceps militaris herein candidate rgs had been systematically validated under coldstress in c militaris three different algorithms genorm normfinder and bestkeeper wereapplied to evaluate the expression stability of the rgs our results showed that ubc andubq were the most stable rgs for cold treatments in short and long periods respectively rgs ubc and pp2a and rgs ubq tub and cyp were the suitable rgs for coldtreatments in short and long periods respectively moreover target genes twocomponentsystem histidine kinase genes were selected to validate the most and least stable rgsunder cold treatment which indicated that use of unstable expressed genes as rgs leadsto biased results our results provide a good starting point for accurate reverse transcriptasequantitative polymerase chain reaction normalization by using ubc and ubq in c militarisunder cold stress and better support for understanding the mechanism of response to coldstress and fruiting body formation in c militaris and other mushroomforming fungi in futureresearcha1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation liu yn liu by ma yc yang hl liugq analysis of reference genes stabilityand histidine kinase expression under cold stressin cordyceps militaris one e0236898101371 pone0236898editor shwet kamal icardirectorate ofmushroom research indiareceived april accepted july published august copyright liu this is an open access distributed under the terms of the creativecommons attribution license which permitsunrestricted use distribution and reproduction inany medium provided the original author andsource are crediteddata availability statement all relevant data arewithin the paper and its supporting informationfilesfunding this work was supported by the nationalnatural science foundation of china no and the natural sciencefoundation of hunan province no 2020jj5972the scientific research fund of hunan provincialeducation department china no 17k106 and18b167 the national key r d programs ofintergovernmental international science andtechnology cooperation no 2017yfe0108100introductioncordyceps militaris a famous traditional chinese medicine has been used as a healthy food fora long time in china the c militaris fruiting body has antiinflammatory antitumor antiinfluenza virus and radioprotection functions methods for commercial production of fruiting bodies of this fungus have been established in artificial media [ ] or withinsects such as silkworm bombyx mori pupae but the molecular mechanism of fruitingbody formation is not well understood the essential role of light in fruiting body development one 101371 pone0236898 august one 0cthe project of international cooperation base ofscience and technology innovation on forestresource biotechnology of hunan province no2018wk4008 and aid program for science andtechnology innovative research team in highereducational instituions of hunan province thefunders had no role in the study design datacollection and analysis decision to publish orpreparation of the manuscriptcompeting interests the authors have declaredthat no competing interests existstable reference genes in cordyceps militaris for cold stressin c militaris was demonstrated in previous study inactivation of a bluelight receptene white collar1 wc1 resulted in thicker aerial hyphae disordered development in c militaris further study showed that the fruiting body primordia could form in the drosophilaarabidopsis synechocystis and human dash type cryptochromes crys gene mutantstrains but the fruiting bodies were unable to elongate normally and crydash and cmwc1exhibited interdependent expression under light in c militaris compared to light temperature is another pivotal factor influencing fruiting body formation from a hyphal thallus which represents a transition from simple to complex multicellularity the fruiting body formation of various mushrooms depends on low temperaturecold stress induction for example the fruiting body development was induced by shiftingthe cultivation condition ˚c for mycelia to lower temperature ˚c in flammulina velutipes in pleurotus ostreatus the mycelia were cultured at ˚c for days in a sawdustmedium and then the temperature was lowered to ˚c to induce fruiting body development in c militaris after the mycelia were cultured under static conditions at ˚c on potatodextrose broth medium for days the mycelia were injected into pupae and then the inoculated pupae were kept at ˚c to induce the fruiting bodies however the molecular mechanism of mycelial development to fruiting body under cold induction has been subject tosurprisingly few studies which will seriously restrict the further development of commercialproduction of mushrooms fruiting bodiestwocomponent signal transduction is commonly used by eubacteria archea and eukaryotes as a stimulus“response coupling mechanism to sense and respond to changes in many different environmental conditions especially temperature sensing they consist of ahistidine kinase hk and a response regulator the histidine kinase desk from bacillus subtilis is mechanistically the best understood after a temperature downshift desk gets autophosphorylated at the conserved histidine residue and donates the phosphate group to theconserved aspartate residue of desr the phosphorylated desr activates the transcription ofthe des gene however little is known the role of hk genes in cold stress response andfruiting body formation in c militaris and other mushroomforming fungidue to its high sensitivity specificity and reliable quantification realtime quantitativepolymerase chain reaction qrtpcr is an important tool to understand the complex signaling networks when an anism is submitted to different stimuli [ ] however the resultsof qrtpcr are often inaccurate because of the occurrence of errors in the primer specificitycomplementary dna cdna synthesis and pcr amplification therefore qrtpcrmust be normalized using reference genes that show a stable expression however validationof suitable rgs for expression analysis under cold stress have not been conducted in c militaris and many other mushroomforming fungi in this study we systematically identified candidate rgs in c militaris that were measured using qrtpcr three different algorithmsgenorm normfinder and bestkeeper were applied to evaluate the expression stability of the candidate rgs under cold treatment in c militaris moreover the relative expression levelsof twocomponentsystem hk genes were analyzed under cold stress conditions with threedifferent normalization strategies the rgs screened out in this paper could provide robustness to study the regulatory mechanism of cold induced fruiting body formation from myceliain c militaris and other mushroomforming fungimaterials and methodsstrains and culture conditionsa laboratory and commercial strain of c militaris cgmcc from china generalmicrobiological culture collection center was used the c militaris was cultured at ˚c in one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressdark with an artificial medium containing g rice g powder of silkworm pupae and25ml nutrient solution glucose g kh2po4 g mgso4 g ammonium citrate g peptone g vitamin b1 mg and 1000ml distilled watercold stressafter cultured for days at ˚c in dark the c militaris mycelia were transferred to ˚c indark for different treatments under cold stress for different time periods including short periods and 8hours and long periods and 8d after treatmentmycelia samples were immediately frozen in liquid nitrogen and stored at ˆ’Ëšc in a deepfreezer until rna isolation untreated mycelia samples were used as the control each treatment was repeated for timesrna isolation and cdna synthesisrna isolation and cdna synthesis were performed as previously described method briefly g mycelia were collected and subsequently were disrupted under liquid nitrogenconditions the rna isolation kit takara china was used to extract total rna treatedwith dnase i to avoid genomic dna contamination μg total rna of each sample wasadded to μl reverse transcription reaction system to synthesize cdna the cdna was subjected to 10fold serial dilution for determining the amplification efficiency of qrtpcr uponcold treatmentsrealtime quantitative polymerase chain reaction conditionsqrtpcr experiments were performed using a previously described method briefly allgenes were amplified by initial heating at ˚c for min followed by cycles of ˚c for s ˚c for s and ˚c for s at the final amplification cycle the specificity of pcr reactions was checked through the use of melting curve analysis “Ëšc in increments of ˚cevery s negative controls were included to ensure the suitability of the assay conditionseach experiment described above was repeated independently at least for timesselection of candidate reference genesin this study genes namely act tub ubc ef1α gapdh pp2a ubq pgk rpsfbox cyp and gtpb were selected as candidate rgs our gene panel contained traditional rgs such as tubulin tub actin act polyubiquitin ubq glyceraldehyde3phosphate dehydrogenase gapdh and translation elongation factor1α ef1α geneschosen on the basis of stable expression in several rna sequencing and quantitative pcrexperiments such as cyclophilin cyp and ubiquitinconjugating enzyme ubc and novel stable rgs in cold stress such as phosphoglycerate kinase pgk and serinethreonine protein phosphatase 2a pp2a details of these genes are provided in table these genes were annotated with a c militaris genome database national center for biotechnology information accession gse28001 and aevu00000000 and used to design primers the specific primers for twocomponentsystem histidine kinase hk genes were listed ins1 tabledata analysis to select the internal reference genethe expression stability of the candidate rgs was analyzed using genorm normfinder and bestkeeper as previously described one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable details on primers used for quantitative reverse transcriptase polymerase chain reaction analysisgeneacttububcef1αannotationaccession noactin cytoskeleton proteinxm_0066695521tubulinxm_0066692031ubiquitinconjugating enzymexm_0066722771elongation factor 1alphaxm_0066659681gapdhglyceraldehyde 3phosphate dehydrogenasexm_0066696971pp2aubqpgkrpsfboxcypgtpbserinethreonine protein phosphatase 2axm_0066730331polyubiquitinxm_0066724691phosphoglycerate kinasexm_0066734081ribosomal protein s25xm_0066653991fbox proteinxm_0066728101cyclophilinxm_0066747781gtpbinding proteinxm_0066746481primer sequences ™™fcaacaacttcctgacgggcrtccttgggcttctgctgacfatgtcgttcgtcgtgaggragagtggcgttgtagggtfaccattgacacgagccagttrgcccatgtaagcctcctcaftatcggaactgtgcctgtrcgttaccacgacggatttfcatccactcctacactgctacrctcaagacgaacagtcaggtfcctcctacagtcgtcatcagcragaaatgtcaaagcgagaftcaaagaagataatggtaacgrgtatgggttctcggaaaggtfgctcaagcccgtcgtttcrccctcctcctcaatgtggfaagtggtctaagggcaaggrttctcctccaggtcggtaafccgatgacaacgacagcgacrgtagttgaccgtggagatgtfttttccgccttattccaccrtccagagcatcaaatccctftaagaagcccaagaagaaaargtcccacaggttcagcgtf forward r reverse101371 pone0236898t001amplification size bpefficiency abbreviationsact actin cytoskeleton protein cdna complementary dna cq quantification cycle cvcoefficient of variation cyp cyclophilin ef1α elongation factor 1alpha fbox fbox protein gapdh glyceraldehyde 3phosphate dehydrogenase gtpb gtpbinding protein hkhistidine kinase m value measurement value pgk phosphoglycerate kinase pp2a serinethreonine protein phosphatase 2a rgs reference genes rps ribosomal protein s25qrtpcr realtime quantitative polymerase chain reaction sd standard deviation tubtubulin ubc ubiquitinconjugating enzyme ubq polyubiquitin vnvn1 pairwisevariationresultsexpression profiling of candidate reference genes in c militaris genes namely act tub ubc ef1α gapdh pp2a ubq pgk rps fbox cyp andgtpb were selected as candidate rgs to determine the most stable rgs under cold treatments we first calculated the pcr efficiency of twelve pairs of primers according to the previously reported method as shown in table qrtpcr efficiency of the genes rangedfrom cyp to ef1α which fell within the acceptable range the quantification cycle cq values of the genes exhibited a high variation ranging and shown in fig the cq values ranged from to and to of short periods fig 1a and long one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig the range of quantification cycle cq values of the candidate reference genes in c militaris under cold stress for short a and long periods b101371 pone0236898g001periods fig 1b respectively rps and pp2a was the most and least transcribed respectivelyacross all the tested samples fig 1a and 1b three different algorithms genorm normfinder and bestkeeper were used to analyze the expression stability of the genes in the nextsectiongenorm analysisthe genorm algorithm calculates the expression stability factor m the m value is defined asthe average pairwise variation of a particular gene with all other reference genes within a givengroup of cdna samples where a low value represents stable and a high value represents unstable expression as determined by genorm fig expression stability m values of the genes both in short and long periods were within the acceptable range m the mvalue ranged from pgk to ubc and pp2a in short periods fig 2a and pgk to tub and ef1α in long periods fig 2b the genes were ranked from thehighest m value least stable to the lowest m value most stable pgk act gapdh tubef1α fbox rps ubq cyp gtpb ubc and pp2a in short periods fig 2a pgk ubcact fbox rps gtpb pp2a cyp gapdh ubq tub and ef1α in long periods fig 2bfig genorm analysis of the average expression stability values m of the candidate reference genes under cold stress for short a and long periods b a higherm value indicates more unstable expression101371 pone0236898g002 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable gene expression stability under short time cold stress ranked by genorm normfinder and bestkeepergenesubcpp2agtpbcypubqrpsfboxef1αtubgapdhactpgkgenormnormfindermean rankabestkeeperm valuerank orderstability valuerank ordercvsd101371 pone0236898t002normfinder analysisnext the expression stability of the candidate rgs was analyzed using normfinder to confirm the genorm results in short periods ubc and ubq were the most stable their stabilityvalues were and respectively table in long periods cyp and ubq were themost highly ranked their stability values were and respectively table combining the results of genorm and normfinder analysis ubc followed by pp2a gtpb and ubqwere the most stable rgs in short periods table mean rank ubq followed by tub cypand gapdh were the most stable rgs in long periods table mean rankoptimal number of reference gene for normalization across theexperimental setsnext we determine the minimal number of genes for qrtpcr normalization by estimatedpairwise variation vnvn1 in genorm below which was a proposed cutoff vnvn1value adding an additional rg is not required according to this principle the vnvn1value was calculated in short periods the v23 value was therefore ubctable gene expression stability under long time cold stress ranked by genorm normfinder and bestkeepergenestubef1αubqgapdhcyppp2agtpbrpsfboxactubcpgkgenormnormfindermean rankabestkeeperm valuerank orderstability valuerank ordercvsd101371 pone0236898t003 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig genorm analysis of the pairwise variation v of the candidate reference genes under cold stress for short a and long periods b the pairwise variation vnvn1 measured the effect of adding additional reference genes on the normalization factor for cold stress treatment in c militaris101371 pone0236898g003together with pp2a would be sufficient for purpose fig 3a and table in long periodscompared with v23 v34 value was suggesting that three rgs ubqtub and cyp were identified as available for normalization fig 3b and table bestkeeper analysiswe further used bestkeeper to analyze the stability of candidate rgs by calculating thestandard deviation sd and the coefficient of variation cv of their cq values it has beenreported that any studied gene showing a sd in expression lower than can be considered stable and the most stably expressed gene exhibiting the lowest cv as shown in table inshort periods the top two stable genes ubc and pp2a with lower cv values and respectively and the sd values of ubc and pp2a were and respectively in long periods the top three stable genes ubq tub and cyp with lower cv values and respectively and the sd values of ubq tub and cyp were and respectively table these bestkeeper results also suggested that ubcpp2a and ubqtubcyp were stable in short and long periods respectivelytaken together our results suggesting that two rgs ubcpp2a and three rgs ubqtubcyp were identified as available for qrtpcr normalization under cold incubation duringshort and long cold periods respectively in c militarisquantitative effects of best and least ranked reference genes on target geneexpressionto validate the utility of stable rgs on gene expression analysis three different strategies theleast stable rg pgk the best ranked rg and multiple stable rgs were selected to normalizethe expression of target gene in short and long periods treatments respectively using pgkubc and ubcpp2a for short periods rg and pgk ubq and ubqtubcyp for long periods rg hk genes in cold stress response were used as target genes based on the annotationwith c militaris genome database national center for biotechnology information accessiongse28001 and aevu00000000 genes cmhk19 s1 table were identified as hk homologous genes normalization of the hk genes using the three different strategies showed that asignificant increase in transcription of hk2hk3hk6 and hk1hk3 hk5 were observed one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig relative expression levels of hk genes during cold stress conditions using the least stable gene best rankedgene or multiple stable reference genes for normalization the least stable reference gene pgk best rankedreference gene ubc and multiple reference genes ubcpp2a were used for normalization to analysis of hk2 a hk3b and hk6 c expression levels in short periods the least stable reference gene pgk best ranked reference geneubq and multiple reference genes ubqtubcyp were used for normalization to analysis of hk1 d hk3 e andhk5 f expression levels in long periods the average ct value of multiple reference genes is used to analysis of hkgene expression levels in the strategy of multiple reference genes were used for normalization101371 pone0236898g004under cold incubation during short and long cold periods respectively fig the geneexpression levels of other hk genes were not shown when using the best ranked ubc or themultiple stable ubcpp2a as the rgs in short periods the upregulated trends for hk2 hk3and hk6 genes were consistent and the peak points were observed at h fig 4a“4c whenusing the best ranked ubq or the multiple stable ubqtubcyp as the rgs in long periodsthe upregulated trends for hk1 hk3 and hk5 genes were consistent and the peak points wereobserved at d fig 4d“4f respectively however using the least ranked pgk as the rg inshort periods the peak points were observed at h of hk2 hk3 hk6 respectively fig4a“4c in addition when pgk was applied as the rg in our experiment the transcriptionpatterns of hk genes were higher than those using the best ranked rg and multiple stable rgsfig 4a“4f which indicated that normalization using the least stable pgk as rg resulted inan overestimated relative expression level of the target genes thus our experimental resultssuggest that it is feasible to use any one or more of ubcpp2a and ubqtubcyp genes asthe normalization gene under cold incubation during short and long cold periods respectivelywith c militarisdiscussioncompared with microarray and rna sequencing qrtpcr technique has the advantages ofhigh accuracy high sensitivity good repeatability and low cost for quantifying gene expression one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable the screening results of candidate reference genes of different species under cold stressgenepp2acyppgkef1αactgapdhubcspeciesananas comosuselymus sibiricusananas comosusbeauveria bassianahordeum vulgareatropa belladonnahemarthria compressa and h altissimamorchella sppbeauveria bassianaelymus sibiricusmorchella spparabidopsis pumilaarabidopsis pumila101371 pone0236898t004expression stability stableunstablerefexpression stability in this studystablestablestablestableunstablestablestableunstablestablestableunstablestablestablechen zhang chen zhou cai li lin stable short periodsstable long periodsunstable short and long periodsunstable short periodszhang unstable short and long periodszhou zhang zhang unstable short and long periodsjin jin stable short periods unstable long periods[ ] to ensure the accuracy of qrtpcr results it is necessary to analyze the stability ofinternal rg under a specific experimental condition a previous study in c militaris showedthat polymerase ii large subunit rpb1 gene was the best rg during all developmental stagesexamined while the most common reference genes actin and tub were not suitable internalcontrols in addition the actin also was not suitable rgs under developmental stagestrain and nutrient source in lentinula edodes however suitable rgs for expressionanalysis under cold stress have not been reported in c militaris in this study the expressionstability of candidate rgs in c militaris was systematically analyzed by genorm normfinder and bestkeeper under the cold treatment our results showed that ubc and ubq wereidentified as the most suitable for qrtpcr normalization under cold incubation during shortand long cold periods respectively while the act a traditionally rg was not suitable coldstress in c militaris therefore we suggest to use two or more rgs rpb1 ubc and ubq tostudy the expression levels of the fruiting body developmental genes in c militaris in the followup studya large number of studies have been conducted to identify the suitable rgs under coldtreatment conditions among different species but the most stable rgs are diverse table inpineapple ananas comosus l pp2a and cyp were the stable rgs under cold stress andin hulless barley hordeum vulgare l var nudum hook f pgk was not stable rg undercold stress which were consistent with our results however in hemarthria compressaand h altissima leaf tissue ef1α was the most stable gene under cold stress and inatropa belladonna pgk was a reliable gene for normalizing gene expression under cold stressconditions which were not consistent with our results in entomopathogenic fungusbeauveria bassiana both act and cyp were the most stably expressed gene sets under a variety of stress conditions including cold however in our study act was not stable inour experimental conditions in another mushroomforming ascomycota true morelsmorchella spp two candidate internal control genes act and gapdh were not reliablegene for normalizing gene expression under cold stress conditions which was similar toour findings an interesting result is that in arabidopsis pumila both ubc and gapdhunder cold stress were the most stable rgs table but in our result ubc and gapdhwere stable and not stable rgs in short periods respectively the similar results were alsofound in siberian wild rye elymus sibiricus that pp2a and act presented the highest degreeof expression stability for cold stress but in our result only pp2a was stable rg while one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressact was found to be unstable the more interesting result is that in our study ubc was thestable rg in short periods but not the stable rg in long periods all of these results indicatethat the expression stability of the same gene differs in different species moreover the expression stability of the same gene is also not the same under the same treatment at different timesin arabidopsis thaliana arabidopsis histidine kinase ahk2 ahk3 and coldinducibletype a arabidopsis response regulators arrs play roles in cold signaling in additionstresssensing in fungi also depends on a signaling cascade comprised of a twocomponentphosphorylation relay plus a subsequent map kinase cascade to trigger gene expression moreover twocomponent pathways are important determinants of pathogenicity in animalpathogens such as candida albicans cryptococcus neoformans and plant pathogensincluding fusarium oxysporum tomato monilinia fructicola brown rot of stone fruit and botrytis cinerea bean tomato and apple in this study the relative expressionlevels of twocomponentsystem hk genes were analyzed under cold stress conditions withthree different normalization strategies the results showed that hk genes were significantlyupregulated after cold stress using stable rgs in normalization however an overestimatedrelative expression level in the hk genes transcription was obtained from qrtpcr usingpgk in normalization fig the relative expression level of target gene is overestimated dueto use unstable gene normalization is also found in other studies [ ] these results indicated that using unstable rgs for qrtpcr normalization will lead to inconsistent resultsmoreover our results suggested that the upregulated hk genes participated in response tocold stress of c militaris however whether or how these hk genes involved in coldinducedfruiting body formation of c militaris needs further studyconclusionswe systematic validated candidate rgs under cold incubation during short and long coldperiods respectively in c militaris our results showed that two rgs ubcpp2a and threergs ubqtubcyp were identified as available for qrtpcr normalization under cold incubation during short and long cold periods respectively in addition our results indicate thatfailure to statistically validate rgs leads to inconsistent results moreover the role of upregulated hk genes in coldinduced fruiting body formation in c militaris needs further studyall in all our results provide a good starting point for accurate qrtpcr normalization in cmilitaris under cold stress and better support for understanding the mechanism of response tocold stress and fruiting body formation in c militaris and other mushroomforming fungi infuture researchsupporting informations1 table details on primers of twocomponentsystem histidine kinas used for qrtpcranalysisdocacknowledgmentsthe authors thank jing zhang yanghong zhang and feifei xue for their help during carryingout the experiments the authors thank xiaoxiao lu and jiashun zhang for their help in dataanalysis we would like to thank muling shi for english language editingauthor contributionsconceptualization yongnan liu biyang liu one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfunding acquisition gaoqiang liuvalidation youchu ma gaoqiang liuwriting “ review editing hailong yangreferences won sy park eh antiinflammatory and related pharmacological activities of cultured mycelia andfruiting bodies of cordyceps militaris j ethnopharmacol “ 101016jjep200410009 pmid jin c kim g choi y induction of apoptosis by aqueous extract of cordyceps militaris through activation of caspases and inactivation of akt in human breast cancer mdamb231 cells “lee hh park h sung gh lee k lee t lee i antiinfluenza effect of cordyceps militaris throughimmunomodulation in a dba2 mouse model of microbiology “ 101007s1227501443000 wos000339600200010 pmid jeong mh park ys jeong dh lee cg kim js oh sj in vitro evaluation of cordyceps militarisas a potential radioprotective agent international of molecular medicine “103892ijmm20141901 wos000344423800020 pmid xie cy gu zx fan gj gu fr han yb chen zg production of cordycepin and mycelia by submerged fermentation of cordyceps militaris in mixture natural culture appl biochem biotechnol “ 101007s1201000985672 pmid zheng zl huang ch cao l xie ch han rh agrobacterium tumefaciensmediated transformation asa tool for insertional mutagenesis in medicinal fungus cordyceps militaris fungal bioluk “ 101016jfunbio201012011 wos000289023800008 pmid hong ip kang pd kim ky nam sh lee my choi ys fruit body formation on silkworm by cordyceps militaris mycobiology “ 104489myco2010382128pmid pubmed central pmcid pmc3741563tiantian l caihong d tao y junde s effects of blue light on the growth and bioactive compoundproduction of cordyceps militaris mycosystema yang t guo mm yang hj guo sp dong ch the bluelight receptor cmwc1 mediates fruit bodydevelopment and secondary metabolism in cordyceps militaris appl microbiol biot “ 101007s0025301570476 wos000368103200019 pmid wang f song x dong x zhang j dong c dashtype cryptochromes regulate fruiting body development and secondary metabolism differently than cmwc1 in the fungus cordyceps militaris applmicrobiol biot “ 101007s0025301782767wos000402008300025 pmid krizsan k almasi e merenyi z sahu n viragh m koszo t transcriptomic atlas of mushroomdevelopment reveals conserved genes behind complex multicellularity in fungi p natl acad sci usa “ 101073pnas1817822116 wos000463936900040 pmid wu t hu c xie b zhang l yan s wang w a single transcription factor pdd1 determines development and yield of winter mushroom flammulina velutipes appl environ microbiol 101128aem0173519 pmid sunagawa m magae y isolation of genes differentially expressed during the fruit body development ofpleurotus ostreatus by differential display of rapd fems microbiol lett “ 101016jfemsle200504018 pmid bourret rb silversmith re twocomponent signal transduction annual review of biochemistry “ aguilar ps hernandezarriaga am cybulski le erazo ac mendoza d de molecular basis of thermosensing a twocomponent signal transduction thermometer in bacillus subtilis embo “ vandesompele j de preter k pattyn f poppe b van roy n de paepe a accurate normalization of realtime quantitative rtpcr data by geometric averaging of multiple internal control genesgenome biol 37research0034 101186gb200237research0034 pmid pubmed central pmcid pmc126239 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stress bustin sa benes v nolan t pfaffl mw quantitative realtime rtpcr”a perspective j mol endocrinol “ 101677jme101755 pmid huggett j dheda k bustin s zumla a realtime rtpcr normalisation strategies and considerations genes immunity “lu x liu y zhao l liu y zhao m selection of reliable reference genes for qrtpcr during methyljasmonate salicylic acid and hydrogen peroxide treatments in ganoderma lucidum world j microbiolbiotechnol 101007s112740182476x pmid han bin yang zheng samma kaleem m systematic validation of candidate reference genes forqrtpcr normalization under iron deficiency in arabidopsis biometals “ 101007s1053401396235 pmid gutierrez n gimenez mj palomino c avila cm assessment of candi

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You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: the development of fungal fruiting bodies from a hyphal thallus is inducible under low temperature cold stress the molecular mechanism has been subject to surprisingly few studies analysis of gene expression level has become an important means to study genefunction and its regulation mechanism but identification of reference genes rgs stabilityunder cold stress have not been reported in famous medicinal mushroomforming fungi cordyceps militaris herein candidate rgs had been systematically validated under coldstress in c militaris three different algorithms genorm normfinder and bestkeeper wereapplied to evaluate the expression stability of the rgs our results showed that ubc andubq were the most stable rgs for cold treatments in short and long periods respectively rgs ubc and pp2a and rgs ubq tub and cyp were the suitable rgs for coldtreatments in short and long periods respectively moreover target genes twocomponentsystem histidine kinase genes were selected to validate the most and least stable rgsunder cold treatment which indicated that use of unstable expressed genes as rgs leadsto biased results our results provide a good starting point for accurate reverse transcriptasequantitative polymerase chain reaction normalization by using ubc and ubq in c militarisunder cold stress and better support for understanding the mechanism of response to coldstress and fruiting body formation in c militaris and other mushroomforming fungi in futureresearcha1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation liu yn liu by ma yc yang hl liugq analysis of reference genes stabilityand histidine kinase expression under cold stressin cordyceps militaris one e0236898101371 pone0236898editor shwet kamal icardirectorate ofmushroom research indiareceived april accepted july published august copyright liu this is an open access distributed under the terms of the creativecommons attribution license which permitsunrestricted use distribution and reproduction inany medium provided the original author andsource are crediteddata availability statement all relevant data arewithin the paper and its supporting informationfilesfunding this work was supported by the nationalnatural science foundation of china no and the natural sciencefoundation of hunan province no 2020jj5972the scientific research fund of hunan provincialeducation department china no 17k106 and18b167 the national key r d programs ofintergovernmental international science andtechnology cooperation no 2017yfe0108100introductioncordyceps militaris a famous traditional chinese medicine has been used as a healthy food fora long time in china the c militaris fruiting body has antiinflammatory antitumor antiinfluenza virus and radioprotection functions methods for commercial production of fruiting bodies of this fungus have been established in artificial media [ ] or withinsects such as silkworm bombyx mori pupae but the molecular mechanism of fruitingbody formation is not well understood the essential role of light in fruiting body development one 101371 pone0236898 august one 0cthe project of international cooperation base ofscience and technology innovation on forestresource biotechnology of hunan province no2018wk4008 and aid program for science andtechnology innovative research team in highereducational instituions of hunan province thefunders had no role in the study design datacollection and analysis decision to publish orpreparation of the manuscriptcompeting interests the authors have declaredthat no competing interests existstable reference genes in cordyceps militaris for cold stressin c militaris was demonstrated in previous study inactivation of a bluelight receptene white collar1 wc1 resulted in thicker aerial hyphae disordered development in c militaris further study showed that the fruiting body primordia could form in the drosophilaarabidopsis synechocystis and human dash type cryptochromes crys gene mutantstrains but the fruiting bodies were unable to elongate normally and crydash and cmwc1exhibited interdependent expression under light in c militaris compared to light temperature is another pivotal factor influencing fruiting body formation from a hyphal thallus which represents a transition from simple to complex multicellularity the fruiting body formation of various mushrooms depends on low temperaturecold stress induction for example the fruiting body development was induced by shiftingthe cultivation condition ˚c for mycelia to lower temperature ˚c in flammulina velutipes in pleurotus ostreatus the mycelia were cultured at ˚c for days in a sawdustmedium and then the temperature was lowered to ˚c to induce fruiting body development in c militaris after the mycelia were cultured under static conditions at ˚c on potatodextrose broth medium for days the mycelia were injected into pupae and then the inoculated pupae were kept at ˚c to induce the fruiting bodies however the molecular mechanism of mycelial development to fruiting body under cold induction has been subject tosurprisingly few studies which will seriously restrict the further development of commercialproduction of mushrooms fruiting bodiestwocomponent signal transduction is commonly used by eubacteria archea and eukaryotes as a stimulus“response coupling mechanism to sense and respond to changes in many different environmental conditions especially temperature sensing they consist of ahistidine kinase hk and a response regulator the histidine kinase desk from bacillus subtilis is mechanistically the best understood after a temperature downshift desk gets autophosphorylated at the conserved histidine residue and donates the phosphate group to theconserved aspartate residue of desr the phosphorylated desr activates the transcription ofthe des gene however little is known the role of hk genes in cold stress response andfruiting body formation in c militaris and other mushroomforming fungidue to its high sensitivity specificity and reliable quantification realtime quantitativepolymerase chain reaction qrtpcr is an important tool to understand the complex signaling networks when an anism is submitted to different stimuli [ ] however the resultsof qrtpcr are often inaccurate because of the occurrence of errors in the primer specificitycomplementary dna cdna synthesis and pcr amplification therefore qrtpcrmust be normalized using reference genes that show a stable expression however validationof suitable rgs for expression analysis under cold stress have not been conducted in c militaris and many other mushroomforming fungi in this study we systematically identified candidate rgs in c militaris that were measured using qrtpcr three different algorithmsgenorm normfinder and bestkeeper were applied to evaluate the expression stability of the candidate rgs under cold treatment in c militaris moreover the relative expression levelsof twocomponentsystem hk genes were analyzed under cold stress conditions with threedifferent normalization strategies the rgs screened out in this paper could provide robustness to study the regulatory mechanism of cold induced fruiting body formation from myceliain c militaris and other mushroomforming fungimaterials and methodsstrains and culture conditionsa laboratory and commercial strain of c militaris cgmcc from china generalmicrobiological culture collection center was used the c militaris was cultured at ˚c in one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressdark with an artificial medium containing g rice g powder of silkworm pupae and25ml nutrient solution glucose g kh2po4 g mgso4 g ammonium citrate g peptone g vitamin b1 mg and 1000ml distilled watercold stressafter cultured for days at ˚c in dark the c militaris mycelia were transferred to ˚c indark for different treatments under cold stress for different time periods including short periods and 8hours and long periods and 8d after treatmentmycelia samples were immediately frozen in liquid nitrogen and stored at ˆ’Ëšc in a deepfreezer until rna isolation untreated mycelia samples were used as the control each treatment was repeated for timesrna isolation and cdna synthesisrna isolation and cdna synthesis were performed as previously described method briefly g mycelia were collected and subsequently were disrupted under liquid nitrogenconditions the rna isolation kit takara china was used to extract total rna treatedwith dnase i to avoid genomic dna contamination μg total rna of each sample wasadded to μl reverse transcription reaction system to synthesize cdna the cdna was subjected to 10fold serial dilution for determining the amplification efficiency of qrtpcr uponcold treatmentsrealtime quantitative polymerase chain reaction conditionsqrtpcr experiments were performed using a previously described method briefly allgenes were amplified by initial heating at ˚c for min followed by cycles of ˚c for s ˚c for s and ˚c for s at the final amplification cycle the specificity of pcr reactions was checked through the use of melting curve analysis “Ëšc in increments of ˚cevery s negative controls were included to ensure the suitability of the assay conditionseach experiment described above was repeated independently at least for timesselection of candidate reference genesin this study genes namely act tub ubc ef1α gapdh pp2a ubq pgk rpsfbox cyp and gtpb were selected as candidate rgs our gene panel contained traditional rgs such as tubulin tub actin act polyubiquitin ubq glyceraldehyde3phosphate dehydrogenase gapdh and translation elongation factor1α ef1α geneschosen on the basis of stable expression in several rna sequencing and quantitative pcrexperiments such as cyclophilin cyp and ubiquitinconjugating enzyme ubc and novel stable rgs in cold stress such as phosphoglycerate kinase pgk and serinethreonine protein phosphatase 2a pp2a details of these genes are provided in table these genes were annotated with a c militaris genome database national center for biotechnology information accession gse28001 and aevu00000000 and used to design primers the specific primers for twocomponentsystem histidine kinase hk genes were listed ins1 tabledata analysis to select the internal reference genethe expression stability of the candidate rgs was analyzed using genorm normfinder and bestkeeper as previously described one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable details on primers used for quantitative reverse transcriptase polymerase chain reaction analysisgeneacttububcef1αannotationaccession noactin cytoskeleton proteinxm_0066695521tubulinxm_0066692031ubiquitinconjugating enzymexm_0066722771elongation factor 1alphaxm_0066659681gapdhglyceraldehyde 3phosphate dehydrogenasexm_0066696971pp2aubqpgkrpsfboxcypgtpbserinethreonine protein phosphatase 2axm_0066730331polyubiquitinxm_0066724691phosphoglycerate kinasexm_0066734081ribosomal protein s25xm_0066653991fbox proteinxm_0066728101cyclophilinxm_0066747781gtpbinding proteinxm_0066746481primer sequences ™™fcaacaacttcctgacgggcrtccttgggcttctgctgacfatgtcgttcgtcgtgaggragagtggcgttgtagggtfaccattgacacgagccagttrgcccatgtaagcctcctcaftatcggaactgtgcctgtrcgttaccacgacggatttfcatccactcctacactgctacrctcaagacgaacagtcaggtfcctcctacagtcgtcatcagcragaaatgtcaaagcgagaftcaaagaagataatggtaacgrgtatgggttctcggaaaggtfgctcaagcccgtcgtttcrccctcctcctcaatgtggfaagtggtctaagggcaaggrttctcctccaggtcggtaafccgatgacaacgacagcgacrgtagttgaccgtggagatgtfttttccgccttattccaccrtccagagcatcaaatccctftaagaagcccaagaagaaaargtcccacaggttcagcgtf forward r reverse101371 pone0236898t001amplification size bpefficiency abbreviationsact actin cytoskeleton protein cdna complementary dna cq quantification cycle cvcoefficient of variation cyp cyclophilin ef1α elongation factor 1alpha fbox fbox protein gapdh glyceraldehyde 3phosphate dehydrogenase gtpb gtpbinding protein hkhistidine kinase m value measurement value pgk phosphoglycerate kinase pp2a serinethreonine protein phosphatase 2a rgs reference genes rps ribosomal protein s25qrtpcr realtime quantitative polymerase chain reaction sd standard deviation tubtubulin ubc ubiquitinconjugating enzyme ubq polyubiquitin vnvn1 pairwisevariationresultsexpression profiling of candidate reference genes in c militaris genes namely act tub ubc ef1α gapdh pp2a ubq pgk rps fbox cyp andgtpb were selected as candidate rgs to determine the most stable rgs under cold treatments we first calculated the pcr efficiency of twelve pairs of primers according to the previously reported method as shown in table qrtpcr efficiency of the genes rangedfrom cyp to ef1α which fell within the acceptable range the quantification cycle cq values of the genes exhibited a high variation ranging and shown in fig the cq values ranged from to and to of short periods fig 1a and long one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig the range of quantification cycle cq values of the candidate reference genes in c militaris under cold stress for short a and long periods b101371 pone0236898g001periods fig 1b respectively rps and pp2a was the most and least transcribed respectivelyacross all the tested samples fig 1a and 1b three different algorithms genorm normfinder and bestkeeper were used to analyze the expression stability of the genes in the nextsectiongenorm analysisthe genorm algorithm calculates the expression stability factor m the m value is defined asthe average pairwise variation of a particular gene with all other reference genes within a givengroup of cdna samples where a low value represents stable and a high value represents unstable expression as determined by genorm fig expression stability m values of the genes both in short and long periods were within the acceptable range m the mvalue ranged from pgk to ubc and pp2a in short periods fig 2a and pgk to tub and ef1α in long periods fig 2b the genes were ranked from thehighest m value least stable to the lowest m value most stable pgk act gapdh tubef1α fbox rps ubq cyp gtpb ubc and pp2a in short periods fig 2a pgk ubcact fbox rps gtpb pp2a cyp gapdh ubq tub and ef1α in long periods fig 2bfig genorm analysis of the average expression stability values m of the candidate reference genes under cold stress for short a and long periods b a higherm value indicates more unstable expression101371 pone0236898g002 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable gene expression stability under short time cold stress ranked by genorm normfinder and bestkeepergenesubcpp2agtpbcypubqrpsfboxef1αtubgapdhactpgkgenormnormfindermean rankabestkeeperm valuerank orderstability valuerank ordercvsd101371 pone0236898t002normfinder analysisnext the expression stability of the candidate rgs was analyzed using normfinder to confirm the genorm results in short periods ubc and ubq were the most stable their stabilityvalues were and respectively table in long periods cyp and ubq were themost highly ranked their stability values were and respectively table combining the results of genorm and normfinder analysis ubc followed by pp2a gtpb and ubqwere the most stable rgs in short periods table mean rank ubq followed by tub cypand gapdh were the most stable rgs in long periods table mean rankoptimal number of reference gene for normalization across theexperimental setsnext we determine the minimal number of genes for qrtpcr normalization by estimatedpairwise variation vnvn1 in genorm below which was a proposed cutoff vnvn1value adding an additional rg is not required according to this principle the vnvn1value was calculated in short periods the v23 value was therefore ubctable gene expression stability under long time cold stress ranked by genorm normfinder and bestkeepergenestubef1αubqgapdhcyppp2agtpbrpsfboxactubcpgkgenormnormfindermean rankabestkeeperm valuerank orderstability valuerank ordercvsd101371 pone0236898t003 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig genorm analysis of the pairwise variation v of the candidate reference genes under cold stress for short a and long periods b the pairwise variation vnvn1 measured the effect of adding additional reference genes on the normalization factor for cold stress treatment in c militaris101371 pone0236898g003together with pp2a would be sufficient for purpose fig 3a and table in long periodscompared with v23 v34 value was suggesting that three rgs ubqtub and cyp were identified as available for normalization fig 3b and table bestkeeper analysiswe further used bestkeeper to analyze the stability of candidate rgs by calculating thestandard deviation sd and the coefficient of variation cv of their cq values it has beenreported that any studied gene showing a sd in expression lower than can be considered stable and the most stably expressed gene exhibiting the lowest cv as shown in table inshort periods the top two stable genes ubc and pp2a with lower cv values and respectively and the sd values of ubc and pp2a were and respectively in long periods the top three stable genes ubq tub and cyp with lower cv values and respectively and the sd values of ubq tub and cyp were and respectively table these bestkeeper results also suggested that ubcpp2a and ubqtubcyp were stable in short and long periods respectivelytaken together our results suggesting that two rgs ubcpp2a and three rgs ubqtubcyp were identified as available for qrtpcr normalization under cold incubation duringshort and long cold periods respectively in c militarisquantitative effects of best and least ranked reference genes on target geneexpressionto validate the utility of stable rgs on gene expression analysis three different strategies theleast stable rg pgk the best ranked rg and multiple stable rgs were selected to normalizethe expression of target gene in short and long periods treatments respectively using pgkubc and ubcpp2a for short periods rg and pgk ubq and ubqtubcyp for long periods rg hk genes in cold stress response were used as target genes based on the annotationwith c militaris genome database national center for biotechnology information accessiongse28001 and aevu00000000 genes cmhk19 s1 table were identified as hk homologous genes normalization of the hk genes using the three different strategies showed that asignificant increase in transcription of hk2hk3hk6 and hk1hk3 hk5 were observed one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig relative expression levels of hk genes during cold stress conditions using the least stable gene best rankedgene or multiple stable reference genes for normalization the least stable reference gene pgk best rankedreference gene ubc and multiple reference genes ubcpp2a were used for normalization to analysis of hk2 a hk3b and hk6 c expression levels in short periods the least stable reference gene pgk best ranked reference geneubq and multiple reference genes ubqtubcyp were used for normalization to analysis of hk1 d hk3 e andhk5 f expression levels in long periods the average ct value of multiple reference genes is used to analysis of hkgene expression levels in the strategy of multiple reference genes were used for normalization101371 pone0236898g004under cold incubation during short and long cold periods respectively fig the geneexpression levels of other hk genes were not shown when using the best ranked ubc or themultiple stable ubcpp2a as the rgs in short periods the upregulated trends for hk2 hk3and hk6 genes were consistent and the peak points were observed at h fig 4a“4c whenusing the best ranked ubq or the multiple stable ubqtubcyp as the rgs in long periodsthe upregulated trends for hk1 hk3 and hk5 genes were consistent and the peak points wereobserved at d fig 4d“4f respectively however using the least ranked pgk as the rg inshort periods the peak points were observed at h of hk2 hk3 hk6 respectively fig4a“4c in addition when pgk was applied as the rg in our experiment the transcriptionpatterns of hk genes were higher than those using the best ranked rg and multiple stable rgsfig 4a“4f which indicated that normalization using the least stable pgk as rg resulted inan overestimated relative expression level of the target genes thus our experimental resultssuggest that it is feasible to use any one or more of ubcpp2a and ubqtubcyp genes asthe normalization gene under cold incubation during short and long cold periods respectivelywith c militarisdiscussioncompared with microarray and rna sequencing qrtpcr technique has the advantages ofhigh accuracy high sensitivity good repeatability and low cost for quantifying gene expression one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable the screening results of candidate reference genes of different species under cold stressgenepp2acyppgkef1αactgapdhubcspeciesananas comosuselymus sibiricusananas comosusbeauveria bassianahordeum vulgareatropa belladonnahemarthria compressa and h altissimamorchella sppbeauveria bassianaelymus sibiricusmorchella spparabidopsis pumilaarabidopsis pumila101371 pone0236898t004expression stability stableunstablerefexpression stability in this studystablestablestablestableunstablestablestableunstablestablestableunstablestablestablechen zhang chen zhou cai li lin stable short periodsstable long periodsunstable short and long periodsunstable short periodszhang unstable short and long periodszhou zhang zhang unstable short and long periodsjin jin stable short periods unstable long periods[ ] to ensure the accuracy of qrtpcr results it is necessary to analyze the stability ofinternal rg under a specific experimental condition a previous study in c militaris showedthat polymerase ii large subunit rpb1 gene was the best rg during all developmental stagesexamined while the most common reference genes actin and tub were not suitable internalcontrols in addition the actin also was not suitable rgs under developmental stagestrain and nutrient source in lentinula edodes however suitable rgs for expressionanalysis under cold stress have not been reported in c militaris in this study the expressionstability of candidate rgs in c militaris was systematically analyzed by genorm normfinder and bestkeeper under the cold treatment our results showed that ubc and ubq wereidentified as the most suitable for qrtpcr normalization under cold incubation during shortand long cold periods respectively while the act a traditionally rg was not suitable coldstress in c militaris therefore we suggest to use two or more rgs rpb1 ubc and ubq tostudy the expression levels of the fruiting body developmental genes in c militaris in the followup studya large number of studies have been conducted to identify the suitable rgs under coldtreatment conditions among different species but the most stable rgs are diverse table inpineapple ananas comosus l pp2a and cyp were the stable rgs under cold stress andin hulless barley hordeum vulgare l var nudum hook f pgk was not stable rg undercold stress which were consistent with our results however in hemarthria compressaand h altissima leaf tissue ef1α was the most stable gene under cold stress and inatropa belladonna pgk was a reliable gene for normalizing gene expression under cold stressconditions which were not consistent with our results in entomopathogenic fungusbeauveria bassiana both act and cyp were the most stably expressed gene sets under a variety of stress conditions including cold however in our study act was not stable inour experimental conditions in another mushroomforming ascomycota true morelsmorchella spp two candidate internal control genes act and gapdh were not reliablegene for normalizing gene expression under cold stress conditions which was similar toour findings an interesting result is that in arabidopsis pumila both ubc and gapdhunder cold stress were the most stable rgs table but in our result ubc and gapdhwere stable and not stable rgs in short periods respectively the similar results were alsofound in siberian wild rye elymus sibiricus that pp2a and act presented the highest degreeof expression stability for cold stress but in our result only pp2a was stable rg while one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressact was found to be unstable the more interesting result is that in our study ubc was thestable rg in short periods but not the stable rg in long periods all of these results indicatethat the expression stability of the same gene differs in different species moreover the expression stability of the same gene is also not the same under the same treatment at different timesin arabidopsis thaliana arabidopsis histidine kinase ahk2 ahk3 and coldinducibletype a arabidopsis response regulators arrs play roles in cold signaling in additionstresssensing in fungi also depends on a signaling cascade comprised of a twocomponentphosphorylation relay plus a subsequent map kinase cascade to trigger gene expression moreover twocomponent pathways are important determinants of pathogenicity in animalpathogens such as candida albicans cryptococcus neoformans and plant pathogensincluding fusarium oxysporum tomato monilinia fructicola brown rot of stone fruit and botrytis cinerea bean tomato and apple in this study the relative expressionlevels of twocomponentsystem hk genes were analyzed under cold stress conditions withthree different normalization strategies the results showed that hk genes were significantlyupregulated after cold stress using stable rgs in normalization however an overestimatedrelative expression level in the hk genes transcription was obtained from qrtpcr usingpgk in normalization fig the relative expression level of target gene is overestimated dueto use unstable gene normalization is also found in other studies [ ] these results indicated that using unstable rgs for qrtpcr normalization will lead to inconsistent resultsmoreover our results suggested that the upregulated hk genes participated in response tocold stress of c militaris however whether or how these hk genes involved in coldinducedfruiting body formation of c militaris needs further studyconclusionswe systematic validated candidate rgs under cold incubation during short and long coldperiods respectively in c militaris our results showed that two rgs ubcpp2a and threergs ubqtubcyp were identified as available for qrtpcr normalization under cold incubation during short and long cold periods respectively in addition our results indicate thatfailure to statistically validate rgs leads to inconsistent results moreover the role of upregulated hk genes in coldinduced fruiting body formation in c militaris needs further studyall in all our results provide a good starting point for accurate qrtpcr normalization in cmilitaris under cold stress and better support for understanding the mechanism of response tocold stress and fruiting body formation in c militaris and other mushroomforming fungi infuture researchsupporting informations1 table details on primers of twocomponentsystem histidine kinas used for qrtpcranalysisdocacknowledgmentsthe authors thank jing zhang yanghong zhang and feifei xue for their help during carryingout the experiments the authors thank xiaoxiao lu and jiashun zhang for their help in dataanalysis we would like to thank muling shi for english language editingauthor contributionsconceptualization yongnan liu biyang liu one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfunding acquisition gaoqiang liuvalidation youchu ma gaoqiang liuwriting “ review editing hailong yangreferences won sy park eh antiinflammatory and related pharmacological activities of cultured mycelia andfruiting bodies of cordyceps militaris j ethnopharmacol “ 101016jjep200410009 pmid jin c kim g choi y induction of apoptosis by aqueous extract of cordyceps militaris through activation of caspases and inactivation of akt in human breast cancer mdamb231 cells “lee hh park h sung gh lee k lee t lee i antiinfluenza effect of cordyceps militaris throughimmunomodulation in a dba2 mouse model of microbiology “ 101007s1227501443000 wos000339600200010 pmid jeong mh park ys jeong dh lee cg kim js oh sj in vitro evaluation of cordyceps militarisas a potential radioprotective agent international of molecular medicine “103892ijmm20141901 wos000344423800020 pmid xie cy gu zx fan gj gu fr han yb chen zg production of cordycepin and mycelia by submerged fermentation of cordyceps militaris in mixture natural culture appl biochem biotechnol “ 101007s1201000985672 pmid zheng zl huang ch cao l xie ch han rh agrobacterium tumefaciensmediated transformation asa tool for insertional mutagenesis in medicinal fungus cordyceps militaris fungal bioluk “ 101016jfunbio201012011 wos000289023800008 pmid hong ip kang pd kim ky nam sh lee my choi ys fruit body formation on silkworm by cordyceps militaris mycobiology “ 104489myco2010382128pmid pubmed central pmcid pmc3741563tiantian l caihong d tao y junde s effects of blue light on the growth and bioactive compoundproduction of cordyceps militaris mycosystema yang t guo mm yang hj guo sp dong ch the bluelight receptor cmwc1 mediates fruit bodydevelopment and secondary metabolism in cordyceps militaris appl microbiol biot “ 101007s0025301570476 wos000368103200019 pmid wang f song x dong x zhang j dong c dashtype cryptochromes regulate fruiting body development and secondary metabolism differently than cmwc1 in the fungus cordyceps militaris applmicrobiol biot “ 101007s0025301782767wos000402008300025 pmid krizsan k almasi e merenyi z sahu n viragh m koszo t transcriptomic atlas of mushroomdevelopment reveals conserved genes behind complex multicellularity in fungi p natl acad sci usa “ 101073pnas1817822116 wos000463936900040 pmid wu t hu c xie b zhang l yan s wang w a single transcription factor pdd1 determines development and yield of winter mushroom flammulina velutipes appl environ microbiol 101128aem0173519 pmid sunagawa m magae y isolation of genes differentially expressed during the fruit body development ofpleurotus ostreatus by differential display of rapd fems microbiol lett “ 101016jfemsle200504018 pmid bourret rb silversmith re twocomponent signal transduction annual review of biochemistry “ aguilar ps hernandezarriaga am cybulski le erazo ac mendoza d de molecular basis of thermosensing a twocomponent signal transduction thermometer in bacillus subtilis embo “ vandesompele j de preter k pattyn f poppe b van roy n de paepe a accurate normalization of realtime quantitative rtpcr data by geometric averaging of multiple internal control genesgenome biol 37research0034 101186gb200237research0034 pmid pubmed central pmcid pmc126239 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stress bustin sa benes v nolan t pfaffl mw quantitative realtime rtpcr”a perspective j mol endocrinol “ 101677jme101755 pmid huggett j dheda k bustin s zumla a realtime rtpcr normalisation strategies and considerations genes immunity “lu x liu y zhao l liu y zhao m selection of reliable reference genes for qrtpcr during methyljasmonate salicylic acid and hydrogen peroxide treatments in ganoderma lucidum world j microbiolbiotechnol 101007s112740182476x pmid han bin yang zheng samma kaleem m systematic validation of candidate reference genes forqrtpcr normalization under iron deficiency in arabidopsis biometals “ 101007s1053401396235 pmid gutierrez n gimenez mj palomino c avila cm assessment of candi Answer:

Colon_Cancer

curative therapeutic options for a number of immunological disorders remain to be established and approaches for identifying drug candidates are relatively limited furthermorephenotypic screening methods using induced pluripotent stem cell ipscderived immunecells or hematopoietic cells need improvement in the present study using immortalizedmonocytic cell lines derived from ipscs we developed a highthroughput screening htssystem to detect compounds that inhibit il1 secretion and nlrp3 inflammasome activation from activated macrophages the ipscs were generated from a patient with neonatalonset multisystem inflammatory disease nomid as a model of a constitutively activatednlrp3 inflammasome hts of compounds including fdaapproved drugs and compounds with known bioactivity identified compounds as predominantly il1 inhibitorssince these compounds are known inflammasome inhibitors or derivatives of these resultsprove the validity of our hts system which can be a versatile platform for identifying drugcandidates for immunological disorders associated with monocytic lineage cellsintroductionone of the main cell types affected by immunological disorders are white blood cells such aslymphocytes monocytes and neutrophils although our understanding of the cellular pathophysiology of immunological disorders has greatly benefited from in vitro studies usingpatientderived primary hematopoietic cells or in vivo animal models these approaches haveseveral limitations patientderived hematopoietic cells cannot be obtained in sufficient quantities and their in vitro phenotypes can be affected by the in vivo conditions of the patient suchas the cytokine milieu or the administration of therapeutic agents while animal models haveprovided important insights into these disorders species differences in the immunologicala1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation seki r ohta a niwa a sugimine y naitoh nakahata t induced pluripotentstem cellderived monocytic cell lines from anomid patient serve as a screening platform formodulating nlrp3 inflammasome activity one e0237030 101371 pone0237030editor xiaoping bao purdue university unitedstatesreceived march accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237030copyright seki this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfunding this work was supported by the grant forthe core center for ips cell research of researchcenter network for realization of regenerativemedicine from the japan agency for medicalresearch and development amed [tn and mks] the program for intractable diseases researchutilizing diseasespecific ips cells of amed and [tn and mks]practical research project for allergic diseasesand immunology research on allergic diseasesand immunology of amed [mks]practical research project for rareintractablediseases of amed [mks] and thedevelopment causes discrepancies in the function and phenotype of the immune cells [“]overall highthroughput screening hts of therapeutic compounds using patientderivedcells or animal models is usually not feasiblethe establishment of disease or patientspecific induced pluripotent stem cells ipscs has led to the development of a new field of disease modeling owing to their pluripotencyand capacity for selfrenewal ipscs can function as an unlimited source of patientderivedsomatic cells and progenitor cells ipscs have also been used as a source of phenotypebasedhts [“] however several roadblocks remain for ipscbased hts as follows obtaining alarge number of differentiated progenies from pscs is cost and laborintensive and theyield and function of the differentiated cells often vary among clones and experimentalbatchesjapan society for the promotion of science jspswe have established ipscs from patients with autoinflammatory syndromes including neokakenhi grant number [mks]nippon shinyaku co ltd provided support inthe form of salaries to the authors [rs and hn]the specific roles of rs and hn are articulated inthe ˜author contributions™ section the funders hadno role in study design data collection andanalysis decision to publish or preparation of themanuscriptcompeting interests rs and hn are employeesof nippon shinyaku co ltd this employmentdoes not alter our adherence to one policieson sharing data and materialsnatalonset multisystem inflammatory disease nomid also known as chronic infantile neurological cutaneous and articular [cinca] syndrome nakajonishimura syndrome and blau syndrome for disease modeling in these studies ipscderived myeloid cells wereimmortalized by transducing lentiviral vectors that encoded myc bmi1 and mdm2 anddisease phenotypes were recapitulated in vitro thus ipscderived immortalized myeloid celllines ipsmls can be expanded from one experimental batch with reduced financial and laborcosts they also can be stored and differentiated into terminally differentiated progenies therefore ipsmls can overcome the roadblocks associated with ipscbased hts nomid is the most severe form of cryopyrinassociated periodic syndrome caps anautoinflammatory disease caused by heterozygous mutations in the nlrp3 gene [ ]nacht lrr and pyd domainscontaining protein nlrp3 is expressed mainly in myelomonocytic lineage cells and acts as a sensor of cellular stress induced by various pathogens andsterile stimuli in normal macrophages a œpriming stimulus such as lipopolysaccharidelps induces the expression of nlrp3 and prointerleukin il1 an inactive form of theproinflammatory cytokine il1 then an œactivating stimulus such as adenosine triphosphate atp enhances the assembly of a protein complex known as nlrp3 inflammasomethis inflammasome contains the protease caspase1 which processes proil1 to the matureform on the other hand lps stimulation of monocytic cells obtained from untreated capspatients induces robust il1 secretion without secondary activating signals due to autoactivation of nlrp3 inflammasome indeed antiil1 therapy for caps patients has beenproven effective [ ] however antiil1 therapy has several weak points the efficacy ofantiil1 therapy is often inadequate for patients with severe phenotypes il1 maturation is mediated not only by nlrp3 inflammasome but also other inflammasomes and proteases [ ] thus a complete blockade of il1 may result in excessive immunosuppressionmoreover the cost and lifelong injection of biologics worsen the patients™ quality of life therefore other therapeutic approaches such as the direct inhibition of nlrp3 inflammasomeactivity are under considerationnlrp3 inflammasome is an attractive drug target because nlrp3 inflammasome activation is associated with the pathogenesis of various chronic inflammatory conditions recently several selective nlrp3 inhibitors entered the clinical phase their chemicalstructures are undisclosed but presumed to be sulfonylureas or their derivatives mcc950 asulfonylureabased potent selective inhibitor of nlrp3 inflammasome activation wasalso recently identified as a direct nlrp3 inhibitor by binding to the walker b atphydrolysismotif of the nacht domain [ ] given that capsrelated mutations frequently occursin the nacht domain it is not surprising that most capsrelated nlrp3 mutantsescape efficient mcc950 inhibition therefore novel nlrp3 inflammasome inhibitorseffective for diseases including caps are sought one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningin the present study we developed an hts system to identify compounds that regulate theactivity of nlrp3 inflammasome using ipscs generated from a nomid patient we established ipsmls which we used as a prototype of nlrp3activated immune cells by immortalizing ipscderived monocytic progenitor cells we conducted hts of approximately compounds and validated their inhibitory effect on the secretion of il1resultsfunctional monocytic cells on feeder and serumfree monolayer culturewe previously reported that macrophages derived from ipscs carrying diseaseassociatedmutations in the nlrp3 gene showed excessive secretion of il1 without secondary signals these macrophages were considered functional based on compatible appearances withprimary macrophages under an electron microscope the secretion of proinflammatory cytokines and the phagocytosis of listeria monocytogenes macrophages in that study wereobtained via a differentiation protocol using op9 feeder layers recently we have updated ourmonocytemacrophage differentiation system to a defined condition without feeder cells andserums which can also produce functional macrophages we therefore examined whethermonocytes obtained with the feeder and serumfree monolayer differentiation system exhibited a similar in vitro phenotypefor this a nlrp3mutated ipsc clone derived from a nomid patient with a somaticnlrp3y570c mutation was differentiated into monocyticlineage cells since thenlrp3 mutation was a somatic mosaicism the nlrp3nonmutated wildtype clonederived from the same donor was used as an isogenic control ipsc line the differentiatedcells showed a mononuclear and slightly foamy appearance consistent with the appearance ofin vitro differentiated monocytes fig 1a and expressed the hematopoietic cell marker cd45the myeloid cell marker cd11b and the monocytic cell marker cd14 fig 1bwe collected floating monocytic cells from the culture supernatant and evaluated theircytokine production we used lps as a priming signal and silica as a secondary inflammasomeactivating signal as expected the wildtype ipscderived monocytes required twosequential signals to secrete il1 fig 1c while the monocytes carrying the nlrp3 mutationshowed excessive il1 secretion without a secondary signal fig 1d both clones showedrobust secretion of il6 confirming the appropriate downstream signal transduction of lpsstimulation fig 1c and 1d overall the monocytic cells differentiated under feeder andserumfree condition showed the abnormal il1 secretion associated with the in vitro phenotype of nomidestablishment of ipsmls from monocytic progenitor cellswe next established an ipsml line from the ipscderived monocytic cells for this we recovered monocytic lineage cells and introduced lentiviral vectors encoding myc bmi1 andmdm2 after “ days culture in the presence of macrophage colonystimulating factormcsf the cells started to continuously proliferate in an exponential manner fig 2a theipsmls showed a small mononuclear appearance consistent with the appearance of monocyticlineage cells fig 2b the integration of transgenes into the genome of ipsmls was confirmed fig 2c the ipsmls expressed cd45 cd11b and cd14 similar to the originalipscderived monocytic cells fig 2d karyotype analysis of the ipsml demonstrated thatmost of the cells maintain normal karyotype except for some normal variations s1 tablebecause monocytes are heterogeneous progenitor cells that can differentiate into macrophagesand dendritic cells [ ] they might be composed of a heterogeneous population potentiallymaking their cytokine production insufficient we therefore compared the cytokine profiles of one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig functional monocytic cells on feeder and serumfree monolayer culture a a representative maygiemsa staining image of monocytic cells derived fromipscs b a flow cytometric analysis of monocytic cells the staining profiles of specific antibodies thick lines and isotypematched controls gray areas are showncd il1 and il6 secretion from monocytic cells with wildtype c and mutant d nlrp3 cells were stimulated with lps ngml for hours and silica μgml for an additional hour bars show the mean ± sem of four experiments � p paired ttest101371 pone0237030g001ipsmls and terminallydifferentiated ipsmlderived macrophages the ipsmls carrying anlrp3 mutation nomidmls were differentiated into macrophages mlmps fig 2esince mlmps produced an increased amount of il1 and il6 compared with nomidmlsfig 2f and 2g and showed less variability than ipscderived monocytes fig 2h wedecided to use mlmps to construct our htsestablishment of an hts platform using mlmpsto establish our hts system we differentiated nomidmls into mlmps and disseminatedthem onto 384well plates we added the compounds at the same time as when we disseminated the cells into the culture wells then the mlmps were cultured with appropriate concentrations of compounds for hours in order for the cells to firmly adhere to the plate surfaceand they were stimulated with lps for another hours fig 3a after incubation the supernatant was collected and the relative concentrations of cytokines were measured using ahomogeneous time resolved fluorescence htrf assay in this protocol a caspase1 inhibitor one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig establishment of ipsmls from monocytic cells a cumulative growth curve of ipsmls the number of cells was counted and cumulated in every passageculture the day of lentiviral transduction was regarded as day b a representative maygiemsa staining image of ipsmls c a reverse transcriptionpcr analysisof ipsmls transgenespecific primer pairs were used my myc b bmi1 md mdm2 as a positive control lentiviral expression vectors were used d a flowcytometric analysis of ipsmls the staining profiles of specific antibodies thick lines and isotypematched controls gray areas are shown e a phase contrastimage of mlmps f g il1 f and il6 g secretion from nomidmls and mlmps cells were stimulated with lps ngml for hours the bars show themean ± sem of four experiments �� p paired ttest h the coefficient of variation cv of lpsstimulated ipscderived monocytes fig 1c and 1dnomidmls and mlmps f g101371 pone0237030g002acyvadcho specifically inhibited the secretion of il1 in a dosedependent mannerfig 3b in contrast the proteasome inhibitor mg132 which inhibits the nfκb pathway byblocking the degradation of iκb decreased the production of both il1 and il6 in a one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig establishment of an hts platform using mlmps a schematic illustration of the compound screening b dosedependent inhibition of il1 closedcircles and il6 open circles secretion by acyvadcho mg132 and mcc950 bars show the mean ± sem of five experiments c the quality of the assaysystem was evaluated signaltobackground ratios and z™factors of plates for il1 closed and il6 open are shown d criteria and number of hitcompounds [il1] and [il6] indicate the percent inhibition of il1 and il6 secretion respectively e doseresponse curves of the hit compounds bars showthe mean ± sd of four wells101371 pone0237030g003dosedependent manner fig 3b these data proved the specificity of the hts system wealso evaluated the inhibitory effect of mcc950 mcc950 inhibited the secretion of il1 withan approximate ic50 at μm without inhibiting the secretion of il6 fig 3b overall theipsmlbased cytokine assay in combination with the htrf system successfully detected the one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeninginhibitory effect of previously reported compounds proving the application of our system tohtshts using annotated compoundsto identify compounds that inhibit the activation of mutant nlrp3 we next performed htsusing compounds including fdaapproved drugs and compounds with known bioactivity to evaluate the quality of the assay system the z™factor and signalbackground sb ratio of each plate were calculated using the data from dmso controls with or without stimuli s1 fig regarding il1 the z™factor and sb ratio were consistently high for each384well plate z™factor ± and sb ratio ± mean±se fig 3c since a z™factor over indicates an excellent assay these data proved the appropriateness of our hts system the z™factor and sb ratio for il6 were slightly lower than those for il1 z™factor± and sb ratio ±in the first screening we assessed the inhibitory effects of all compounds influencing cytokine secretion at μm s2 table and s2 fig we first selected the compounds thatinduced a more than reduction in il1 secretion fig 3d of these the compoundsthat also nonspecifically inhibited or enhanced il6 secretion were excluded we adopted amore relaxed criterion for il6 modulators because the z™factors of the il6 assay were lowerthan those of the il1 assay we also excluded duplicate compounds and known steroidswith broad antiinflammatory effects for the remaining compounds we evaluated thereproducibility of the inhibitory effects at different dose twelve compounds showed ic50values at less than μm and predominant il1 inhibition at least at two different dosesfinally seven compounds consistently showed predominant il1 inhibition fig 3e allseven compounds have previously been reported to inhibit nlrp3 inflammasome [“]indicating their inhibitory effects on both wildtype and mutant cellsvalidation of hit compounds with primary human cellswe also wondered if the effect of these compounds could be recapitulated in primary humancells we used primary peripheral blood mononuculear cells pbmcs obtained from healthyvolunteers for the validation to obtain prompt il1 secretion from nonmutant cells westimulated the pbmcs with lps and atp compounds modulating the activity of hsp9017aag and herbimycin a showed a selective inhibitory effect on il1 secretion comparable to the effects seen in mlmps fig on the other hand the remaining compoundsaside from the pancaspase inhibitor zvadfmk inhibited both il1 and il6 fig indicating that they had offtarget effects or their il1 inhibitory effects were nonspecificdiscussionwe established a diseaseassociated phenotypic hts system for nomid using ipsmls ourhts platform showed a stable z™factor and sb ratio for the amount of il1 to avoid falsepositives such as nonspecific inhibitors and cytotoxic compounds we also measured theamount of il6 an inflammasomeindependent cytokine we identified compounds as predominant inhibitors of il1 secretion from among candidates the compounds identified here have already been reported as inhibitors of nlrp3 inflammasome proving thevalidity of our strategy screening a larger number of compounds may help identify novel andmore specific compoundswe could efficiently immortalize monocytic progenitor cells derived from human ipscsmoreover these ipsmls could differentiate into terminally differentiated macrophagesmlmps as previously reported [ ] mlmps showed enhanced cytokine secretion and one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig hit validation with pbmcs dosedependent inhibition of il1 closed circles and il6 open circles secretion from pbmcs of a healthy donor cellswere stimulated with lps ngml for hours and atp mm for an additional hour bars show the mean ± sd of four wells101371 pone0237030g004therefore were deemed suitable for hts in this manner the financial and labor costs of htscan be dramatically decreased our strategy can be applied to other immunological disordersin which monocytemacrophagelineage cells play critical pathological roles in addition theimmortalization of ipscderived hematopoietic cells for hts can be expanded to otherhematopoietic disorders one concern associated with the immortalization of monocytic cellsis that the immortalization can affect the characteristics of the cells especially regarding thecell cycle and death which may affect the immunological functions to avoid any potentialchanges in the cellular phenotype we first expanded the ipsmls generated a larger numberof frozen stocks and then used the same passage number of the stocks for a series of htssessionsmodulating the function of the inflammasome by small compounds is a promising frontierfor controlling diseases associated with inflammasomemediated chronic inflammation somecompounds such as mcc950 have already been shown to be markedly effective without anysignificant side effects in animal models nevertheless identifying more candidates to modulate nlrp3 inflammasome is desirable as mcc950 inefficiently inhibits capsrelated nlrp3mutants interestingly we identified several compounds effective in mutant cells that were previously reported to inhibit the activation of nlrp3 inflammasome indicating that the activation of mutant nlrp3 shares activating signals to some degree with wildtype nlrp3however several compounds that exerted predominantly il1 inhibitory effects on mutantcells were nonselective on pbmcs which could be explained by offtarget effects or false positives offtarget effects including cytotoxicity could be the result of a higher sensitivity bypbmcs to the true positives the clear reason is difficult to elucidate however because of theheterogeneity of the pbmc population on the other hand ipsmls consist of relativelyhomogenous cell populations and may therefore be more useful than primary cells in in vitro one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningexperiments as for nonspecific inhibitors and false positives in hts the upregulation of certain signaling pathways irrelevant to the inflammasome in ipsmls might be one cause cytotoxic compounds may also suppress cytokine secretion in a nonspecific manneradditionally our hts system relies on the amount of secreted cytokines which can varybetween cells and only indirectly describes the inflammasome activity thus a combination ofmeasuring the cytotoxicity and direct detection of the inflammasome activity should improvethe accuracy of our hts systemto conclude although phenotypic screenings have been performed with various cell typesdifferentiated from human pscs the number of screenings with immune cells is still limitedour hts system provides a large number of functional monocytic lineage cells and a versatileplatform for compound screening to modify diseaseassociated phenotypes therefore it canbe used to screen candidate compounds for the treatment of congenital immunological disorders associated with monocytic lineage cellsmaterials and methodsstudy approvalthis study was approved by the ethics committee of kyoto university r0091g0259 andwritten informed consent was obtained from the patient™s guardians in accordance with thedeclaration of helsinkimaintenance and differentiation of human ipscswe previously established ipscs from a nomid patient cira188ai with nlrp3 somaticmosaicism y570c undifferentiated ipscs were maintained on mitotically inactivatedsnl feeder cells with primate es cell medium reprocell japan supplemented with ngml bfgf wako pure chemicals industries japan feeder and serumfree monocytic celldifferentiation was performed in accordance with previously described protocols with somemodifications [ ]lentivirus productionlentiviral constructs encoding myc bmi1 and mdm2 in the csiiefrfa vector and twoplasmids for lentiviral vector packaging pcmvvsvgrsvrev and pcaghivgp werekindly provided by dr satoru senju kumamoto university kumamoto japan and hiroyukimiyoshi riken bioresource center tsukuba japan plasmids were transfected into 293tcells crl3216 atcc usa by lipofection lipofectamine ltx thermo fisher scientificusa and days later viral ps in the culture supernatants were concentrated by centrifugation at rpm for hours at ˚cgeneration of ipsmlsipsmls were generated as previously described with some modifications [ ] briefly onday of the monocytic cell differentiation from ipscs floating cells were collected andinfected with lentiviruses the cells were cultured in stempro34 serumfree medium thermofisher scientific containing mm lglutamine in the presence of gmcsf ngml andmcsf ngml rd systems usa after approximately days proliferating ipsmlsappeared for macrophage differentiation ipsmls within passages after thawing were cultured in rpmi1640 medium sigmaaldrich usa containing fetal bovine serum equitechbio usa and mcsf ngml for days with a medium change on day adherent one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningcells were collected by treatment with accumax innovative cell technologies usa and usedfor the subsequent experimentsmaygiemsa stainingcells were seeded onto glass slides by cytospin thermo fisher scientific and stainedwith maygrunwald and giemsa staining solution merck kgaa germany in accordancewith the manufacturer™s instructions the slides were examined using a biorevo bz9000keyence japan a planapo × objective nikon japan and the bzii viewer software program keyence were used for the image acquisitionflow cytometrycells were treated with fcr blocking reagent miltenyi biotec germany and stained with primary antibodies cd45fitc becton dickinson and company usa cd11bpe andcd14apc beckman coulter usa at dilutions of for negative controls primary antibodies were replaced with mouse igg1 bd dapi sigmaaldrich was used to exclude deadcells the flow cytometric analysis data were collected using a macsquant analyzer miltenyibiotec and analyzed using the flowjo software program treestar usareverse transcriptionpolymerase chain reaction pcrrna samples were prepared using an rneasy mini kit qiagen germany total rna ng was reverse transcribed into cdna using a primescript rt master mix kit takarajapan pcr was performed on a veriti thermal cycler thermo fisher scientific withtakara ex taq hs polymerase using approximately ng cdna the forward primers targeting the coding region of the genes were ™gatcagcaacaaccgaaaat3™ myc™ccattgaattctttgaccagaa3™ bmi1 and ™gctgaagagggctttgatg3™mdm2 the reverse primer targeting wpre was ™gttgcgtcagcaaacacagt3™measurement of cytokinescells were seeded at × cellswell onto a 96well cell culture plate in rpmi1640 mediumcontaining fbs the cells were stimulated with ngml lps from e coli k12 invivogen usa for hours and μgml silica us silica usa for an additional hour aftercentrifugation the supernatants were collected the concentrations of cytokines in the culturesupernatants were analyzed using a flowcytomix kit ebioscience usa and a macsquantanalyzer in accordance with the manufacturer™s instructioncompound screeningcells were seeded at × cellswell onto 384well cell culture plates in rpmi1640 mediumcontaining fbs and compounds four hours later the cells were stimulated with ngmllps for hours after centrifugation the supernatants were transferred to 384well smallvolume white plates using a biomek nxp beckman coulter the relative cytokine levels weremeasured using an htrf kit cisbio bioassays france and a powerscan4 microplatereader ds pharma biomedical japan with excitation at nm and emission at and nm the htrf signals were calculated as the htrf ratio ×em nmem nmand then the percent inhibition was calculated using dmso controls cell seeding and reagentdispensation were performed with a multidrop combi thermo fisher scientific thereagents purchased are as follows acyvadcho mg132 merck kgaa mcc950sigmaaldrich fdaapproved drug library iccb known bioactives library enzo life one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningsciences usa usdrug collection international drug collection microsource discoverysystems usa lopac1280 sigmaaldrich tocriscreen mini tocris bioscience uk andkinase inhibitor libraries enzo life sciences merck kgaa selleck chemicals usahit validation with pbmcscryopreserved human pbmcs were purchased from cellular technology limited usa thecells were thawed in accordance with the manufacturer™s instructions and seeded at ×cellswell onto 384well cell culture plates in rpmi1640 medium containing fbs andcompounds four hours later the cells were stimulated with ngml lps for hours and mm atp sigmaaldrich for an additional hour the relative cytokine levels were measuredusing the htrf kit as described abovestatistical analysesstatistical analyses were performed using the excel and graphpad prism software programsgraphpad software usa statistical significance was evaluated using the paired ttestp was considered statistically significant the z™factor and sb ratio were calculated asfollowsz0 ¼ 00 ð3shc þ 3slcþðmhc 00 mlcþsb ¼ mhcmlcwhere σ is the standard deviation sd μ is the mean hc is the high control and lc is the lowcontrolsupporting informations1 fig htrf ratios of the assay controls each of the plates contained high stimulated and low not stimulated controls treated with dmso values falling outside of themean ± 3sd were excluded as outliers red crosses except when z™factors and sb ratios werecalculatedtifs2 fig scatter plot of the compound screening percent inhibitions for il1 xaxis and il yaxis within a range of to are shown seven hit compounds are markedtifs1 table karyotype analysis of ipsmlspdfs2 table a total list of compounds with the assay dataxlsxs1 raw imagepdfacknowledgmentswe thank drs takayuki tanaka mitsujiro osawa and yohei nishi cira kyoto universitykyoto japan for fruitful discussions and technical assistance dr peter karagiannis cirakyoto university kyoto japan for proofreading the paper ms harumi watanabe cira one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningkyoto university kyoto japan for providing administrative assistance and dr akitsu hottacira kyoto university kyoto japan for providing 293t cellsauthor contributionsconceptualization ryosuke seki megumu k saitodata curation akira ohta megumu k saitofunding acquisition megumu k saitoinvestigation ryosuke seki akira niwa yoshinori sugiminemethodology akira ohtaresources akira ohtasupervision akira niwa haruna naito tatsutoshi nakahata megumu k saitowriting “ original draft ryosuke sekiwriting “ review editing megumu k saitoreferences mestas j hughes cc of mice and not men differences between mouse and human immunology jimmunol “ epub 104049jimmunol17252731 pmid holsapple mp west lj landreth ks species comparison of anatomical and functional immune system development birth defects res b dev reprod toxicol “ epub 101002bdrb10035 pmid geissmann f manz mg jung s sieweke mh merad m ley k development of monocytes macrophages and dendritic cells science “ 101126science pmid takahashi k tanabe k ohnuki m narita m ichisaka t tomoda k induction of pluripotent stemcells from adult human fibroblasts by defined factors cell “ 101016jcell200711019 pmid takahashi k yamanaka s induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors cell “ 101016jcell200607024pmid lo cicero a jaskowiak al egesipe al tournois j brinon b pitrez pr a high throughput phenotypic screening reveals compounds that counteract premature osteogenic differentiation of hgpsipsderived mesenchymal stem cells sci rep epub 101038srep34798 pmid pubmed central pmcid pmc5064407thorne n malik n shah s zhao j class b aguisanda f highthroughput phenotypic screening of human astrocytes to identify compounds that protect against oxidative stress stem cellstransl med “ epub 105966sctm20150170 pmid pubmed central pmcid pmc4835244 darville h poulet a rodetamsellem f chatrousse l pernelle j boissart c human pluripotentstem cellderived cortical neurons for high throughput medication screening in autism a proof ofconcept study in shank3 haploinsufficiency syndrome ebiomedicine “ epub 101016jebiom201605032 p

cancer7556

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: curative therapeutic options for a number of immunological disorders remain to be established and approaches for identifying drug candidates are relatively limited furthermorephenotypic screening methods using induced pluripotent stem cell ipscderived immunecells or hematopoietic cells need improvement in the present study using immortalizedmonocytic cell lines derived from ipscs we developed a highthroughput screening htssystem to detect compounds that inhibit il1 secretion and nlrp3 inflammasome activation from activated macrophages the ipscs were generated from a patient with neonatalonset multisystem inflammatory disease nomid as a model of a constitutively activatednlrp3 inflammasome hts of compounds including fdaapproved drugs and compounds with known bioactivity identified compounds as predominantly il1 inhibitorssince these compounds are known inflammasome inhibitors or derivatives of these resultsprove the validity of our hts system which can be a versatile platform for identifying drugcandidates for immunological disorders associated with monocytic lineage cellsintroductionone of the main cell types affected by immunological disorders are white blood cells such aslymphocytes monocytes and neutrophils although our understanding of the cellular pathophysiology of immunological disorders has greatly benefited from in vitro studies usingpatientderived primary hematopoietic cells or in vivo animal models these approaches haveseveral limitations patientderived hematopoietic cells cannot be obtained in sufficient quantities and their in vitro phenotypes can be affected by the in vivo conditions of the patient suchas the cytokine milieu or the administration of therapeutic agents while animal models haveprovided important insights into these disorders species differences in the immunologicala1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation seki r ohta a niwa a sugimine y naitoh nakahata t induced pluripotentstem cellderived monocytic cell lines from anomid patient serve as a screening platform formodulating nlrp3 inflammasome activity one e0237030 101371 pone0237030editor xiaoping bao purdue university unitedstatesreceived march accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237030copyright seki this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfunding this work was supported by the grant forthe core center for ips cell research of researchcenter network for realization of regenerativemedicine from the japan agency for medicalresearch and development amed [tn and mks] the program for intractable diseases researchutilizing diseasespecific ips cells of amed and [tn and mks]practical research project for allergic diseasesand immunology research on allergic diseasesand immunology of amed [mks]practical research project for rareintractablediseases of amed [mks] and thedevelopment causes discrepancies in the function and phenotype of the immune cells [“]overall highthroughput screening hts of therapeutic compounds using patientderivedcells or animal models is usually not feasiblethe establishment of disease or patientspecific induced pluripotent stem cells ipscs has led to the development of a new field of disease modeling owing to their pluripotencyand capacity for selfrenewal ipscs can function as an unlimited source of patientderivedsomatic cells and progenitor cells ipscs have also been used as a source of phenotypebasedhts [“] however several roadblocks remain for ipscbased hts as follows obtaining alarge number of differentiated progenies from pscs is cost and laborintensive and theyield and function of the differentiated cells often vary among clones and experimentalbatchesjapan society for the promotion of science jspswe have established ipscs from patients with autoinflammatory syndromes including neokakenhi grant number [mks]nippon shinyaku co ltd provided support inthe form of salaries to the authors [rs and hn]the specific roles of rs and hn are articulated inthe ˜author contributions™ section the funders hadno role in study design data collection andanalysis decision to publish or preparation of themanuscriptcompeting interests rs and hn are employeesof nippon shinyaku co ltd this employmentdoes not alter our adherence to one policieson sharing data and materialsnatalonset multisystem inflammatory disease nomid also known as chronic infantile neurological cutaneous and articular [cinca] syndrome nakajonishimura syndrome and blau syndrome for disease modeling in these studies ipscderived myeloid cells wereimmortalized by transducing lentiviral vectors that encoded myc bmi1 and mdm2 anddisease phenotypes were recapitulated in vitro thus ipscderived immortalized myeloid celllines ipsmls can be expanded from one experimental batch with reduced financial and laborcosts they also can be stored and differentiated into terminally differentiated progenies therefore ipsmls can overcome the roadblocks associated with ipscbased hts nomid is the most severe form of cryopyrinassociated periodic syndrome caps anautoinflammatory disease caused by heterozygous mutations in the nlrp3 gene [ ]nacht lrr and pyd domainscontaining protein nlrp3 is expressed mainly in myelomonocytic lineage cells and acts as a sensor of cellular stress induced by various pathogens andsterile stimuli in normal macrophages a œpriming stimulus such as lipopolysaccharidelps induces the expression of nlrp3 and prointerleukin il1 an inactive form of theproinflammatory cytokine il1 then an œactivating stimulus such as adenosine triphosphate atp enhances the assembly of a protein complex known as nlrp3 inflammasomethis inflammasome contains the protease caspase1 which processes proil1 to the matureform on the other hand lps stimulation of monocytic cells obtained from untreated capspatients induces robust il1 secretion without secondary activating signals due to autoactivation of nlrp3 inflammasome indeed antiil1 therapy for caps patients has beenproven effective [ ] however antiil1 therapy has several weak points the efficacy ofantiil1 therapy is often inadequate for patients with severe phenotypes il1 maturation is mediated not only by nlrp3 inflammasome but also other inflammasomes and proteases [ ] thus a complete blockade of il1 may result in excessive immunosuppressionmoreover the cost and lifelong injection of biologics worsen the patients™ quality of life therefore other therapeutic approaches such as the direct inhibition of nlrp3 inflammasomeactivity are under considerationnlrp3 inflammasome is an attractive drug target because nlrp3 inflammasome activation is associated with the pathogenesis of various chronic inflammatory conditions recently several selective nlrp3 inhibitors entered the clinical phase their chemicalstructures are undisclosed but presumed to be sulfonylureas or their derivatives mcc950 asulfonylureabased potent selective inhibitor of nlrp3 inflammasome activation wasalso recently identified as a direct nlrp3 inhibitor by binding to the walker b atphydrolysismotif of the nacht domain [ ] given that capsrelated mutations frequently occursin the nacht domain it is not surprising that most capsrelated nlrp3 mutantsescape efficient mcc950 inhibition therefore novel nlrp3 inflammasome inhibitorseffective for diseases including caps are sought one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningin the present study we developed an hts system to identify compounds that regulate theactivity of nlrp3 inflammasome using ipscs generated from a nomid patient we established ipsmls which we used as a prototype of nlrp3activated immune cells by immortalizing ipscderived monocytic progenitor cells we conducted hts of approximately compounds and validated their inhibitory effect on the secretion of il1resultsfunctional monocytic cells on feeder and serumfree monolayer culturewe previously reported that macrophages derived from ipscs carrying diseaseassociatedmutations in the nlrp3 gene showed excessive secretion of il1 without secondary signals these macrophages were considered functional based on compatible appearances withprimary macrophages under an electron microscope the secretion of proinflammatory cytokines and the phagocytosis of listeria monocytogenes macrophages in that study wereobtained via a differentiation protocol using op9 feeder layers recently we have updated ourmonocytemacrophage differentiation system to a defined condition without feeder cells andserums which can also produce functional macrophages we therefore examined whethermonocytes obtained with the feeder and serumfree monolayer differentiation system exhibited a similar in vitro phenotypefor this a nlrp3mutated ipsc clone derived from a nomid patient with a somaticnlrp3y570c mutation was differentiated into monocyticlineage cells since thenlrp3 mutation was a somatic mosaicism the nlrp3nonmutated wildtype clonederived from the same donor was used as an isogenic control ipsc line the differentiatedcells showed a mononuclear and slightly foamy appearance consistent with the appearance ofin vitro differentiated monocytes fig 1a and expressed the hematopoietic cell marker cd45the myeloid cell marker cd11b and the monocytic cell marker cd14 fig 1bwe collected floating monocytic cells from the culture supernatant and evaluated theircytokine production we used lps as a priming signal and silica as a secondary inflammasomeactivating signal as expected the wildtype ipscderived monocytes required twosequential signals to secrete il1 fig 1c while the monocytes carrying the nlrp3 mutationshowed excessive il1 secretion without a secondary signal fig 1d both clones showedrobust secretion of il6 confirming the appropriate downstream signal transduction of lpsstimulation fig 1c and 1d overall the monocytic cells differentiated under feeder andserumfree condition showed the abnormal il1 secretion associated with the in vitro phenotype of nomidestablishment of ipsmls from monocytic progenitor cellswe next established an ipsml line from the ipscderived monocytic cells for this we recovered monocytic lineage cells and introduced lentiviral vectors encoding myc bmi1 andmdm2 after “ days culture in the presence of macrophage colonystimulating factormcsf the cells started to continuously proliferate in an exponential manner fig 2a theipsmls showed a small mononuclear appearance consistent with the appearance of monocyticlineage cells fig 2b the integration of transgenes into the genome of ipsmls was confirmed fig 2c the ipsmls expressed cd45 cd11b and cd14 similar to the originalipscderived monocytic cells fig 2d karyotype analysis of the ipsml demonstrated thatmost of the cells maintain normal karyotype except for some normal variations s1 tablebecause monocytes are heterogeneous progenitor cells that can differentiate into macrophagesand dendritic cells [ ] they might be composed of a heterogeneous population potentiallymaking their cytokine production insufficient we therefore compared the cytokine profiles of one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig functional monocytic cells on feeder and serumfree monolayer culture a a representative maygiemsa staining image of monocytic cells derived fromipscs b a flow cytometric analysis of monocytic cells the staining profiles of specific antibodies thick lines and isotypematched controls gray areas are showncd il1 and il6 secretion from monocytic cells with wildtype c and mutant d nlrp3 cells were stimulated with lps ngml for hours and silica μgml for an additional hour bars show the mean ± sem of four experiments � p paired ttest101371 pone0237030g001ipsmls and terminallydifferentiated ipsmlderived macrophages the ipsmls carrying anlrp3 mutation nomidmls were differentiated into macrophages mlmps fig 2esince mlmps produced an increased amount of il1 and il6 compared with nomidmlsfig 2f and 2g and showed less variability than ipscderived monocytes fig 2h wedecided to use mlmps to construct our htsestablishment of an hts platform using mlmpsto establish our hts system we differentiated nomidmls into mlmps and disseminatedthem onto 384well plates we added the compounds at the same time as when we disseminated the cells into the culture wells then the mlmps were cultured with appropriate concentrations of compounds for hours in order for the cells to firmly adhere to the plate surfaceand they were stimulated with lps for another hours fig 3a after incubation the supernatant was collected and the relative concentrations of cytokines were measured using ahomogeneous time resolved fluorescence htrf assay in this protocol a caspase1 inhibitor one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig establishment of ipsmls from monocytic cells a cumulative growth curve of ipsmls the number of cells was counted and cumulated in every passageculture the day of lentiviral transduction was regarded as day b a representative maygiemsa staining image of ipsmls c a reverse transcriptionpcr analysisof ipsmls transgenespecific primer pairs were used my myc b bmi1 md mdm2 as a positive control lentiviral expression vectors were used d a flowcytometric analysis of ipsmls the staining profiles of specific antibodies thick lines and isotypematched controls gray areas are shown e a phase contrastimage of mlmps f g il1 f and il6 g secretion from nomidmls and mlmps cells were stimulated with lps ngml for hours the bars show themean ± sem of four experiments �� p paired ttest h the coefficient of variation cv of lpsstimulated ipscderived monocytes fig 1c and 1dnomidmls and mlmps f g101371 pone0237030g002acyvadcho specifically inhibited the secretion of il1 in a dosedependent mannerfig 3b in contrast the proteasome inhibitor mg132 which inhibits the nfκb pathway byblocking the degradation of iκb decreased the production of both il1 and il6 in a one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig establishment of an hts platform using mlmps a schematic illustration of the compound screening b dosedependent inhibition of il1 closedcircles and il6 open circles secretion by acyvadcho mg132 and mcc950 bars show the mean ± sem of five experiments c the quality of the assaysystem was evaluated signaltobackground ratios and z™factors of plates for il1 closed and il6 open are shown d criteria and number of hitcompounds [il1] and [il6] indicate the percent inhibition of il1 and il6 secretion respectively e doseresponse curves of the hit compounds bars showthe mean ± sd of four wells101371 pone0237030g003dosedependent manner fig 3b these data proved the specificity of the hts system wealso evaluated the inhibitory effect of mcc950 mcc950 inhibited the secretion of il1 withan approximate ic50 at μm without inhibiting the secretion of il6 fig 3b overall theipsmlbased cytokine assay in combination with the htrf system successfully detected the one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeninginhibitory effect of previously reported compounds proving the application of our system tohtshts using annotated compoundsto identify compounds that inhibit the activation of mutant nlrp3 we next performed htsusing compounds including fdaapproved drugs and compounds with known bioactivity to evaluate the quality of the assay system the z™factor and signalbackground sb ratio of each plate were calculated using the data from dmso controls with or without stimuli s1 fig regarding il1 the z™factor and sb ratio were consistently high for each384well plate z™factor ± and sb ratio ± mean±se fig 3c since a z™factor over indicates an excellent assay these data proved the appropriateness of our hts system the z™factor and sb ratio for il6 were slightly lower than those for il1 z™factor± and sb ratio ±in the first screening we assessed the inhibitory effects of all compounds influencing cytokine secretion at μm s2 table and s2 fig we first selected the compounds thatinduced a more than reduction in il1 secretion fig 3d of these the compoundsthat also nonspecifically inhibited or enhanced il6 secretion were excluded we adopted amore relaxed criterion for il6 modulators because the z™factors of the il6 assay were lowerthan those of the il1 assay we also excluded duplicate compounds and known steroidswith broad antiinflammatory effects for the remaining compounds we evaluated thereproducibility of the inhibitory effects at different dose twelve compounds showed ic50values at less than μm and predominant il1 inhibition at least at two different dosesfinally seven compounds consistently showed predominant il1 inhibition fig 3e allseven compounds have previously been reported to inhibit nlrp3 inflammasome [“]indicating their inhibitory effects on both wildtype and mutant cellsvalidation of hit compounds with primary human cellswe also wondered if the effect of these compounds could be recapitulated in primary humancells we used primary peripheral blood mononuculear cells pbmcs obtained from healthyvolunteers for the validation to obtain prompt il1 secretion from nonmutant cells westimulated the pbmcs with lps and atp compounds modulating the activity of hsp9017aag and herbimycin a showed a selective inhibitory effect on il1 secretion comparable to the effects seen in mlmps fig on the other hand the remaining compoundsaside from the pancaspase inhibitor zvadfmk inhibited both il1 and il6 fig indicating that they had offtarget effects or their il1 inhibitory effects were nonspecificdiscussionwe established a diseaseassociated phenotypic hts system for nomid using ipsmls ourhts platform showed a stable z™factor and sb ratio for the amount of il1 to avoid falsepositives such as nonspecific inhibitors and cytotoxic compounds we also measured theamount of il6 an inflammasomeindependent cytokine we identified compounds as predominant inhibitors of il1 secretion from among candidates the compounds identified here have already been reported as inhibitors of nlrp3 inflammasome proving thevalidity of our strategy screening a larger number of compounds may help identify novel andmore specific compoundswe could efficiently immortalize monocytic progenitor cells derived from human ipscsmoreover these ipsmls could differentiate into terminally differentiated macrophagesmlmps as previously reported [ ] mlmps showed enhanced cytokine secretion and one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig hit validation with pbmcs dosedependent inhibition of il1 closed circles and il6 open circles secretion from pbmcs of a healthy donor cellswere stimulated with lps ngml for hours and atp mm for an additional hour bars show the mean ± sd of four wells101371 pone0237030g004therefore were deemed suitable for hts in this manner the financial and labor costs of htscan be dramatically decreased our strategy can be applied to other immunological disordersin which monocytemacrophagelineage cells play critical pathological roles in addition theimmortalization of ipscderived hematopoietic cells for hts can be expanded to otherhematopoietic disorders one concern associated with the immortalization of monocytic cellsis that the immortalization can affect the characteristics of the cells especially regarding thecell cycle and death which may affect the immunological functions to avoid any potentialchanges in the cellular phenotype we first expanded the ipsmls generated a larger numberof frozen stocks and then used the same passage number of the stocks for a series of htssessionsmodulating the function of the inflammasome by small compounds is a promising frontierfor controlling diseases associated with inflammasomemediated chronic inflammation somecompounds such as mcc950 have already been shown to be markedly effective without anysignificant side effects in animal models nevertheless identifying more candidates to modulate nlrp3 inflammasome is desirable as mcc950 inefficiently inhibits capsrelated nlrp3mutants interestingly we identified several compounds effective in mutant cells that were previously reported to inhibit the activation of nlrp3 inflammasome indicating that the activation of mutant nlrp3 shares activating signals to some degree with wildtype nlrp3however several compounds that exerted predominantly il1 inhibitory effects on mutantcells were nonselective on pbmcs which could be explained by offtarget effects or false positives offtarget effects including cytotoxicity could be the result of a higher sensitivity bypbmcs to the true positives the clear reason is difficult to elucidate however because of theheterogeneity of the pbmc population on the other hand ipsmls consist of relativelyhomogenous cell populations and may therefore be more useful than primary cells in in vitro one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningexperiments as for nonspecific inhibitors and false positives in hts the upregulation of certain signaling pathways irrelevant to the inflammasome in ipsmls might be one cause cytotoxic compounds may also suppress cytokine secretion in a nonspecific manneradditionally our hts system relies on the amount of secreted cytokines which can varybetween cells and only indirectly describes the inflammasome activity thus a combination ofmeasuring the cytotoxicity and direct detection of the inflammasome activity should improvethe accuracy of our hts systemto conclude although phenotypic screenings have been performed with various cell typesdifferentiated from human pscs the number of screenings with immune cells is still limitedour hts system provides a large number of functional monocytic lineage cells and a versatileplatform for compound screening to modify diseaseassociated phenotypes therefore it canbe used to screen candidate compounds for the treatment of congenital immunological disorders associated with monocytic lineage cellsmaterials and methodsstudy approvalthis study was approved by the ethics committee of kyoto university r0091g0259 andwritten informed consent was obtained from the patient™s guardians in accordance with thedeclaration of helsinkimaintenance and differentiation of human ipscswe previously established ipscs from a nomid patient cira188ai with nlrp3 somaticmosaicism y570c undifferentiated ipscs were maintained on mitotically inactivatedsnl feeder cells with primate es cell medium reprocell japan supplemented with ngml bfgf wako pure chemicals industries japan feeder and serumfree monocytic celldifferentiation was performed in accordance with previously described protocols with somemodifications [ ]lentivirus productionlentiviral constructs encoding myc bmi1 and mdm2 in the csiiefrfa vector and twoplasmids for lentiviral vector packaging pcmvvsvgrsvrev and pcaghivgp werekindly provided by dr satoru senju kumamoto university kumamoto japan and hiroyukimiyoshi riken bioresource center tsukuba japan plasmids were transfected into 293tcells crl3216 atcc usa by lipofection lipofectamine ltx thermo fisher scientificusa and days later viral ps in the culture supernatants were concentrated by centrifugation at rpm for hours at ˚cgeneration of ipsmlsipsmls were generated as previously described with some modifications [ ] briefly onday of the monocytic cell differentiation from ipscs floating cells were collected andinfected with lentiviruses the cells were cultured in stempro34 serumfree medium thermofisher scientific containing mm lglutamine in the presence of gmcsf ngml andmcsf ngml rd systems usa after approximately days proliferating ipsmlsappeared for macrophage differentiation ipsmls within passages after thawing were cultured in rpmi1640 medium sigmaaldrich usa containing fetal bovine serum equitechbio usa and mcsf ngml for days with a medium change on day adherent one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningcells were collected by treatment with accumax innovative cell technologies usa and usedfor the subsequent experimentsmaygiemsa stainingcells were seeded onto glass slides by cytospin thermo fisher scientific and stainedwith maygrunwald and giemsa staining solution merck kgaa germany in accordancewith the manufacturer™s instructions the slides were examined using a biorevo bz9000keyence japan a planapo × objective nikon japan and the bzii viewer software program keyence were used for the image acquisitionflow cytometrycells were treated with fcr blocking reagent miltenyi biotec germany and stained with primary antibodies cd45fitc becton dickinson and company usa cd11bpe andcd14apc beckman coulter usa at dilutions of for negative controls primary antibodies were replaced with mouse igg1 bd dapi sigmaaldrich was used to exclude deadcells the flow cytometric analysis data were collected using a macsquant analyzer miltenyibiotec and analyzed using the flowjo software program treestar usareverse transcriptionpolymerase chain reaction pcrrna samples were prepared using an rneasy mini kit qiagen germany total rna ng was reverse transcribed into cdna using a primescript rt master mix kit takarajapan pcr was performed on a veriti thermal cycler thermo fisher scientific withtakara ex taq hs polymerase using approximately ng cdna the forward primers targeting the coding region of the genes were ™gatcagcaacaaccgaaaat3™ myc™ccattgaattctttgaccagaa3™ bmi1 and ™gctgaagagggctttgatg3™mdm2 the reverse primer targeting wpre was ™gttgcgtcagcaaacacagt3™measurement of cytokinescells were seeded at × cellswell onto a 96well cell culture plate in rpmi1640 mediumcontaining fbs the cells were stimulated with ngml lps from e coli k12 invivogen usa for hours and μgml silica us silica usa for an additional hour aftercentrifugation the supernatants were collected the concentrations of cytokines in the culturesupernatants were analyzed using a flowcytomix kit ebioscience usa and a macsquantanalyzer in accordance with the manufacturer™s instructioncompound screeningcells were seeded at × cellswell onto 384well cell culture plates in rpmi1640 mediumcontaining fbs and compounds four hours later the cells were stimulated with ngmllps for hours after centrifugation the supernatants were transferred to 384well smallvolume white plates using a biomek nxp beckman coulter the relative cytokine levels weremeasured using an htrf kit cisbio bioassays france and a powerscan4 microplatereader ds pharma biomedical japan with excitation at nm and emission at and nm the htrf signals were calculated as the htrf ratio ×em nmem nmand then the percent inhibition was calculated using dmso controls cell seeding and reagentdispensation were performed with a multidrop combi thermo fisher scientific thereagents purchased are as follows acyvadcho mg132 merck kgaa mcc950sigmaaldrich fdaapproved drug library iccb known bioactives library enzo life one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningsciences usa usdrug collection international drug collection microsource discoverysystems usa lopac1280 sigmaaldrich tocriscreen mini tocris bioscience uk andkinase inhibitor libraries enzo life sciences merck kgaa selleck chemicals usahit validation with pbmcscryopreserved human pbmcs were purchased from cellular technology limited usa thecells were thawed in accordance with the manufacturer™s instructions and seeded at ×cellswell onto 384well cell culture plates in rpmi1640 medium containing fbs andcompounds four hours later the cells were stimulated with ngml lps for hours and mm atp sigmaaldrich for an additional hour the relative cytokine levels were measuredusing the htrf kit as described abovestatistical analysesstatistical analyses were performed using the excel and graphpad prism software programsgraphpad software usa statistical significance was evaluated using the paired ttestp was considered statistically significant the z™factor and sb ratio were calculated asfollowsz0 ¼ 00 ð3shc þ 3slcþðmhc 00 mlcþsb ¼ mhcmlcwhere σ is the standard deviation sd μ is the mean hc is the high control and lc is the lowcontrolsupporting informations1 fig htrf ratios of the assay controls each of the plates contained high stimulated and low not stimulated controls treated with dmso values falling outside of themean ± 3sd were excluded as outliers red crosses except when z™factors and sb ratios werecalculatedtifs2 fig scatter plot of the compound screening percent inhibitions for il1 xaxis and il yaxis within a range of to are shown seven hit compounds are markedtifs1 table karyotype analysis of ipsmlspdfs2 table a total list of compounds with the assay dataxlsxs1 raw imagepdfacknowledgmentswe thank drs takayuki tanaka mitsujiro osawa and yohei nishi cira kyoto universitykyoto japan for fruitful discussions and technical assistance dr peter karagiannis cirakyoto university kyoto japan for proofreading the paper ms harumi watanabe cira one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningkyoto university kyoto japan for providing administrative assistance and dr akitsu hottacira kyoto university kyoto japan for providing 293t cellsauthor contributionsconceptualization ryosuke seki megumu k saitodata curation akira ohta megumu k saitofunding acquisition megumu k saitoinvestigation ryosuke seki akira niwa yoshinori sugiminemethodology akira ohtaresources akira ohtasupervision akira niwa haruna naito tatsutoshi nakahata megumu k saitowriting “ original draft ryosuke sekiwriting “ review editing megumu k saitoreferences mestas j hughes cc of mice and not men differences between mouse and human immunology jimmunol “ epub 104049jimmunol17252731 pmid holsapple mp west lj landreth ks species comparison of anatomical and functional immune system development birth defects res b dev reprod toxicol “ epub 101002bdrb10035 pmid geissmann f manz mg jung s sieweke mh merad m ley k development of monocytes macrophages and dendritic cells science “ 101126science pmid takahashi k tanabe k ohnuki m narita m ichisaka t tomoda k induction of pluripotent stemcells from adult human fibroblasts by defined factors cell “ 101016jcell200711019 pmid takahashi k yamanaka s induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors cell “ 101016jcell200607024pmid lo cicero a jaskowiak al egesipe al tournois j brinon b pitrez pr a high throughput phenotypic screening reveals compounds that counteract premature osteogenic differentiation of hgpsipsderived mesenchymal stem cells sci rep epub 101038srep34798 pmid pubmed central pmcid pmc5064407thorne n malik n shah s zhao j class b aguisanda f highthroughput phenotypic screening of human astrocytes to identify compounds that protect against oxidative stress stem cellstransl med “ epub 105966sctm20150170 pmid pubmed central pmcid pmc4835244 darville h poulet a rodetamsellem f chatrousse l pernelle j boissart c human pluripotentstem cellderived cortical neurons for high throughput medication screening in autism a proof ofconcept study in shank3 haploinsufficiency syndrome ebiomedicine “ epub 101016jebiom201605032 p Answer:

Colon_Cancer

preoperative heart rate variability a prognosticindicator for overall survival and cancer recurrencein patients with primary colorectal cancer one e0237244 101371 pone0237244editor louise emilsson university of oslonorwayreceived february accepted july published august copyright strous this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement data cannot beshared publicly because of ethical concernspatients were included on a no objection base toconduct retrospective data studies and publishfindings but were not asked for permission topublish full encrypted data data are available fromthe viecuri institutional data access contact viawetenschapsbureauviecurinl for researcherswho meet the criteria for access to confidentialdata heart rate variability hrv represents efferent vagus nerve activity which is suggested tobe inversely related to fundamental mechanisms of tumorigenesis and to be a predictor ofprognosis in various types of cancer hrv is also believed to predict the occurrence andseverity of postoperative complications we aimed to determine the role of preoperativehrv as a prognostic factor in overall and cancer free survival in patients with colorectalcancermethodsretrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between january and december who underwent curative surgical treatment hrv was measured as timedomain parameters sdnn standarddeviation of nnintervals and rmssd root mean square of successive differencesbased on preoperative second ecgs groups were created by baseline hrv low hrvsdnn 20ms or rmssd 19ms and normal hrv sdnn �20ms or rmssd �19msprimary endpoints were overall and cancer free survivalresultsa total of patients were included in this study hrv was not significantly associated withoverall survival sdnn 20ms vs sdnn �20ms244 vs adjusted hr “ p rmssd 19ms vs rmssd �19ms270 vs adjustedhr “ p or cancer recurrence sdnn 20ms vs�20ms201 vs adjusted hr “ p rmssd 19ms vs�19ms vs adjusted hr “ p there was no one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfunding the authors received no specific fundingfor this workcompeting interests the authors have declaredthat no competing interests existsignificant association between hrv and cealevel at one year followup or between hrvand occurrence of a postoperative complication or the severity of postoperativecomplicationssheart rate variability was not associated with overall or cancer free survival in patients withprimary colorectal cancer who underwent curative surgical treatment these results do notalign with results found in studies including only patients with advanced cancer which suggests that there is only an association in the other direction cancer causing low hrvintroductionin there were over million newly diagnosed colorectal cancer patients worldwide andover in the netherlands alone it is the fourth most common cause of death worldwide to improve survival it is of importance to get a better insight into modifiable prognosticfactors emerging evidence suggests that vagal nerve activity indexed by heart rate variabilityhrv could be one of these prognostic factors [“]hrv is the physiological phenomenon of the fluctuation in time intervals between adjacentheartbeats and represents efferent vagus nerve activity to the heart [“] it has been suggestedthat efferent vagal activity is inversely related with fundamental mechanisms of tumorigenesisas inflammation oxidative stress and excessive sympathetic activity these mechanisms arebelieved to be controlled by the vagus nerve via a bidirectional braintoimmune pathwaysthrough the release of neurotransmitters via the cholinergic antiinflammatory pathway a higher vagal tone may reflect a more flexible topdown regulation of the immunesystem and physiological activity moderated by the brain absence of vagus activity due tovagotomy has been shown to increase the risk of developing colorectal cancer in addition to influencing development of cancer vagus nerve activity seems to be a predictor of prognosis in various types of cancer recent studies show an association betweendecreased activity of the vagus nerve and worse survival in patients with cancer of the gastrointestinal tract liver pancreas lung prostate and breast among others also patientswith normal hrv seem to live longer in different sorts of metastatic cancer independent ofconfounders in patients with colorectal cancer a low hrv at baseline has shown to beassociated with higher cea levels at months after diagnosis which predicts a poorer prognosis in patients undergoing curative treatment for colorectal cancer hrv does not only seemto influence cancer prognosis a recent study showed that patients with lower hrv have moreintraoperative blood loss and more and more severe postoperative complications identifying patients with low hrv is easy and noninvasive when its predictive value forthe prognosis of cancer patients is of satisfactory significance vagus nerve activation prior toor during cancer treatment could theoretically be beneficial in improving prognosis alsoif we could predict the occurrence and severity of postoperative complications based on hrvimproving hrv before surgery could possibly accelerate postoperative recovery and indirectlyaffect patients™ prognosis recent studies focussing on improving hrv by improving physicalfitness by means of physical exercise show promising results in both older men and woman however the only previous study on colon cancer and hrv including patients receiving curative treatment included a small sample and did not examine whether hrv predicts one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancersurvival in these patients to clarify the predictive value of hrv in prognosis of patientswith colorectal cancer further exploration is needed current studies identifying hrv as aprognostic factor did not specifically focus on colorectal cancer have small study populationsdid not correct for confounders and mainly focused on metastatic disease [“]the aim of this study was to determine the role of preoperative hrv as a prognostic factorin overall and cancer free survival in patients with primary colorectal cancer who underwentcurative surgical treatmentmethodsdata collectiondata from the netherlands cancer registry ncr were used the ncr collects data on allnewly diagnosed cancer patients in the netherlands information on patient and tumour characteristics diagnosis and treatment is routinely collected from the medical records by trainedadministrators of the cancer registry anatomical site of the tumour is registered according tothe international classification of diseases oncology the tumournodemetastasis tnmclassification is used for stage notification of the primary tumour according to the editionvalid at time of cancer diagnosis quality of the data is high due to thorough training of theregistration team and consistency checks for the study population additional data were collected from the medical records of thepatients this encompassed information on hrv cealevels asa classification comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroid disease pulmonary disease vascular disease neurological disease and otheroccurrence and severity of postoperative complications and cancer recurrence groups ofcomorbidities were chosen based on matching features within these groups and their potentialinfluence on hrv or the endpoint being analysed severity of the postoperative complicationsaccording to the claviendindo classification was also documented medical records wereassessed between january and july and reevaluated for revision of this between the 20th and 25th of april this study was approved by the research committee and the board of directors of viecurimedical centre data was obtained under the law ˜scientific research and statistics in the interest of public health where asking for permission is not possible or appropriate for several reasons™ in the netherlands unless patients objected to use of their personal medical record forscientific research data was encrypted with an encryption key provided by the ncr encryption was shortly lifted to access the patients™ number for accessing hisher medical record following extraction data were encrypted againstudy populationthe study population included all consecutive patients diagnosed with primary colorectal adenocarcinoma between january and january at viecuri medical centre who underwent curative surgical treatment patients with metastatic disease at time of surgery orcarcinoma in situ were excluded as their treatment and prognosis differs from those receivingcurative treatment for colorectal cancer metastasis found within months after surgery wereconsidered present at time of surgery and therefore excluded other excluded patients werepatients with neuroendocrine tumours because of different tumour characteristics and prognosis patients with cardiac arrhythmias including atrial and ventricular extrasystole pacemakers patients taking betablockers as this enhances hrv indexes or patients withbradycardia heart rate bpm or tachycardia heart rate bpm as this precluded reliable calculation of hrv we did not exclude patients taking alphablockers calcium one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerinhibitors diuretics amiodarone aceinhibitors or arb™s as these types of medicationreduce central sympathetic functioning rather than peripheral and their influence on hrv istherefore less univocal and sometimes completely absent heart rate variabilityheart rate variability was analysed using a 12lead 10second ecg 150hz used for preoperative screening in case of multiple ecg™s per patient the most recent ecg before date of surgery was used for hrvanalysis in case of multiple ecg™s per patient on the same date theecg with the best quality was chosen meaning an ecg without motion artefacts in case ofmotion artefacts there was always an ecg without motion artefacts available recorded on thesame date time between two consecutive rpeaks was measured in lead ii with an accuracy of02ms using museecg hrv was presented using the timedomain hrv parameters sdnnstandard deviation of nnintervals and rmssd root mean square of successive differences in milliseconds calculated using the following calculations rsdnn ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðrri 00 rrmeanþ2pnn 00 rmssd ¼rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðrriþ1 00 rriþ2pn 00 n 00 ð1þð2þsdnn and rmssd obtained from 10s ecgs were found to correlate with results of ecgsof longer durations power spectral analysis hrv parameters as lf and hf can only beobtained in longer recording ecgs and were therefore not implementable in this study sdnn and rmssd were both analysed as continuous variables as well as binary variablesusing cutoffs of 20ms versus �20ms and 19ms versus �19ms respectively in case of ansdnn 20ms or rmssd 19ms hrv was classified as low and in case of sdnn �20ms orrmssd �19ms as normal these cutoff values were based on cutoff values used in otherstudies showing an association between lowhrv as sdnn 20ms and rmssd 19ms andcolorectal cancer as there is no standardised definition of low and normal hrv endpoints and definitionsthe primary endpoints of this study were overall and cancer free survival overall survival wasdefined as the time between the date of surgery to the date of death or last followup date inmonths cancer free survival was defined as the time in months from the date of surgery untilthe date of cancer recurrence defined as the first date of either radiologic or pathologic diagnosis of metastases or tumour recurrence of colorectal cancer patients dying without cancerrecurrence were censored on day of death secondary endpoints were elevated cealevel ugl at oneyear followup occurrence of postoperative complications within daysafter surgery and severity of postoperative complications according to the claviendindoclassificationstatistical analysisin this retrospective observational cohort study we utilized descriptive statistics to provide anoverview of control variables of the study population patient characteristics as age sex bmicomorbidities and asaclassification heart rate and tumour characteristics as tnmstagetumour localisation and tumour differentiation and their association with hrv and one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerprognosis normal distribution of the continuous variables heart rate age and bmi as well assdnn and rmssd were tested with a kolmogorovsmirnov test because of normal distribution heart rate age and bmi were compared between hrvgroups using unpaired ttest allother variables were categorical and were compared between hrvgroups using chisquarestatistics as groups were all of sufficient powerdifferences in overall survival and cancer free survival in months according to sdnn andrmssd were visualized by means of kaplanmeier curves and statistically tested using the logrank test multivariate coxregression analyses were conducted to calculate the prognosticassociation between hrv and overall and cancer free survival while adjusting for other prognostic variables multivariate logistic regression was used to assess the independent effect ofsdnn and rmssd on cealevels and the occurrence and severity of postoperative complications variables included for adjustment were chosen by means of forward stepwise selectionbased on clinical judgment differences at baseline eg differences on any predictor betweenpatients who later died or not and database availability and depended on the analysed endpoint those included patient demographics age sex bodymassindex comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroiddisease pulmonary disease vascular disease neurological disease other including crohn™sdisease hepatitis kidney failure disorders anaemia depression arthritis tumour characteristics localisation stage differentiation and the occurrence of postoperative complicationswhen the later was not an outcome differences in cealevel at baseline and one year checkup between and within groups of low hrv and normal hrv were assessed with a repeatedmeasures linear model and tested using the tukey test to test the implication of a longer timebetween ecg and treatment all analyses were repeated after excluding patients with an ecgolder than months a twotailed pvalue � was considered significant in all analysesdata were analysed using ibm spss statistics version ibm corp ny armonk usaresultsof colorectal cancer patients that underwent a surgical resection a total of patientswere included in this study reasons for exclusion are presented in fig median sdnn andrmssd were 204ms interquartile range iqr 115ms351ms and 175ms iqr 99ms299ms respectively table shows descriptive data of the included patients by hrv groupsbaseline heart rate and age were negatively associated with hrv the group of patients withlow hrv contains more patients with a history of cardiac disease regardless of the hrv defining parameter when defining low hrv by rmssd 19ms more patients in this group havehypertension as comorbidity this group also contains more patients with an asa classification greater than oneduring a median followup of months iqr “ all causedeath occurred in patients cancer recurrence occurred in patients during a median followup of months iqr “to rule out any distort in results caused by a delay between ecg and treatment all analyseswere repeated after exclusion of ecg™s older than months this did not lead to any new significant results therefore these results were not displayed in detail in this papersurvivalin low hrv groups slightly more patients died compared to normal hrv groups sdnn20ms versus �20ms versus respectively rmssd 19ms versus�19ms versus respectively these observed differences between hrvgroups in overall survival were not significant fig a sdnn p b rmssd one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig flowchart of the study101371 pone0237244g001p after adjustment for some potential confounders these associations remained notsignificant sdnn 20ms versus �20ms hr “ p and rmssd19ms versus �19ms hr “ p tables and age cardiac disease tumour stage and postoperative complications had a significant influence on overall survival age also differed at baseline and was identified as a confounder when defining low andnormal hrvgroups by sdnn cardiac disease was identified as confounder when conducting sensitivity analyses with sdnn and rmss as continuous variables results remained thesame there was no association between hrv and overall survival sdnn hr “ p and rmssd hr “ p in low hrv groups slightly more patients had recurrence of cancer compared to normalhrv groups sdnn 20ms versus �20ms versus respectivelyrmssd 19ms versus �19ms versus respectively these observed differences between hrv groups in cancer free survival were not significant fig a sdnnp b rmssd p as in overall survival after adjustment for some potentialconfounders these associations remained not significant sdnn 20ms versus �20mshr “ p and rmssd 19ms versus �19ms hr “ p tables and in sdnnbased groups baseline heart rate was identified asa confounding variable sensitivity analyses with sdnn and rmssd as continuous variables one 101371 pone0237244 august one 0ctable descriptive data of included patients according to prespecified hrv groupssdnn 20ms n sdnn �20ms n prmssd 19ms n rmssd �19ms n phrv and prognosis in colorectal cancerheart rate�age�age n year� yearsex nmalefemale [“] [“] [“] [“] [“] [“] [“] [“] mean bmi sd [“] [“] [“] [“]comorbidities ncardiac diseasehypertensiondiabetes mellitusthyroid diseasepulmonary diseasevascular diseaseneurological diseaseotherasa nasa1asa2asa34localisation tumour nright colonleft colonrectumtumour stage niiiiii differentiation grade nwellmoderate poorundifferentiated � non normaldistributed data presented as median with interquartile range101371 pone0237244t001did not alter these results there was no association between hrv and cancer free survivalsdnn hr “ p and rmssd hr “p cealevelcealevel at baseline and one year checkup was registered in patients and elevated in of these patients this was elevated at one year checkup in only patients low hrv was notsignificantly associated with elevated cealevels at one year checkup as shown in table sensitivity analyses with sdnn and rmssd as continuous variables did not alter these resultssdnn hr “ p and rmssd hr “ one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig kaplanmeier curves for overall survival in groups of low hrv and normal hrv presented as a sdnn andb rmssd101371 pone0237244g002 one 101371 pone0237244 august one 0ctable multivariate analyses of associations of sdnn and covariates with overall and cancer free survivalhrv and prognosis in colorectal cancersdnn20ms�20msheart rateagebmicardiac diseasenoyeshypertensionnoyesasaclassificationasa1asa2asa34localisationright colonleft colonrectumtumour stageiiiiiioverall survivalhr cipcancer free survivalhr ci “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ “ “ reference “ “ reference “ “postoperative complicationnoyes reference reference “ “101371 pone0237244t002p adjusting for covariates was not possible because of the small number of patientswith an elevated cealeveldifferences between cealevel at baseline and one year checkup were compared betweenand within hrvgroups results were displayed in fig there were no significant differencesin cealevel at baseline and one year checkup between the low hrv group and normal hrvgroup defined by sdnn and rmssd p and p respectively also there were nosignificant differences in cealevel at baseline and at one year checkup within the low hrvgroup and normal hrv group defined by sdnn and rmssd p and p respectivelypostoperative complicationsin patients one or more postoperative complications occurred within days after surgerythe occurrence of a postoperative complication was not significantly associated with lowhrv defined as sdnn 20ms or rmssd 19ms even after adjustment for some potentialconfounders table heart rate age cardiac disease and hypertension were identified asconfounding covariates when conducting sensitivity analyses with sdnn and rmss as continuous variables the association between occurrence of a postoperative complication with one 101371 pone0237244 august one 0ctable multivariate analyses of associations of rmssd and covariates with overall and cancer free survivalhrv and prognosis in colorectal cancerrmssd19ms�19msheart rateagebmicardiac diseasenoyeshypertensionnoyesasaclassificationasa1asa2asa34localisationright colonleft colonrectumtumour stageiiiiiioverall survivalhr cipcancer free survivalhr ci “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “postoperative complicationnoyes reference reference “ “101371 pone0237244t003baseline hrv remained not significant sdnn hr “ p andrmssd “ p the same applied when postoperative complicationswere graded according to the claviendindo classification and the complication that is gradedthe highest for each patient is compared to the absence of postoperative complicationstable discussionin this observational cohortstudy we determined the heart rate variability in preoperativeecgs of patients with primary colorectal cancer who underwent curative surgical treatment hrv refers to physiological variations in beattobeat intervals it was presented intimedomain parameters sdnn and rmssd hrv was not significantly associated with overall survival or cancer free survival independent of some risk factors also this study showedno significant differences in cealevels at one year checkup between patients with low hrvand patients with normal hrv patients with low hrv did not have more or more severepostoperative complications compared to patients with normal hrvtumorigenesis has three fundamental mechanisms inflammation promoting oxidativestress and stimulating tumour growth oxidative stress causing dnadamage and one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig kaplanmeier curves for cancer free survival in groups of low hrv and normal hrv presented as a sdnnand b rmssd101371 pone0237244g003 one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable univariate analyses of low hrv and risk of elevated cealevel at one year checkupcea μglor cipcea μglor ciindependent of baseline ceaindependent of baseline ceasdnn 20ms n “rmssd 19ms n “sdnn �20ms n normal baseline cea � μglsdnn 20ms n sdnn �20ms n elevated baseline cea μglsdnn 20ms n reference “ reference “rmssd �19ms n normal baseline cea � μglrmssd 19ms n rmssd �19ms n elevated baseline cea μglrmssd 19ms n reference “ reference “sdnn �20ms n referencermssd �19ms n referencep101371 pone0237244t004interfering with subsequent repair mechanisms and sympathetic neurotransmitters stimulating tumour metastasis and progression it has been suggested that afferent fibres of thevagus nerve inform the brain about peripheral inflammation this is followed by a braintoimmune response via the efferent route of the vagus nerve that modulates the function ofimmuneregulatory cells through the release of neurotransmitters via the cholinergic antiinflammatory pathway malfunctioning of this route may play part in the onset of cancer this theory has been supported by studies of patients with gastric ulcers who underwent avagotomy who then showed an increased risk of developing lung and colorectal cancer [“] the vagus nerve is also believed to modulate tumour progression an active vagus nervecan inhibit inflammation oxidative stress and the release of sympathetic neurotransmittersthat stimulate tumour metastasis and progression [“] absence of this inhibitory effect inturn results in metastasis and tumour progression as shown in a study of erin showingthat mice who underwent chemical vagotomy developed more metastasis of breastcancer cellsthan controls epidemiologically low hrv has been associated with worse overall survivalover time in patients with recurrent or metastatic cancer of various types even after correctionfor confounders [“] however the results of the present study do not support thesefindingsto our knowledge this is the first study including only patients with colorectal cancer whoare eligible for curative treatment by partial bowel resection and not those receiving palliativetreatment de couck studied the relationship between hrv and tumour burden in bothcurative and palliative patients with prostate cancer or nonsmall cell lung cancer independent of confounders the hypothesised inverse relationship of hrv and the tumour markerpsa at and months after diagnosis was only significant in patients with stage iv prostatecancer not stage ii and iii in colorectal cancer mouton found that low hrv definedas sdnn ms predicted significantly higher levels of the tumour marker cea at months after diagnosis again these results were only found in patients receiving palliativetreatment not curative only one previous study showed a significant inverse relationshipbetween hrv and mortality in cancer in general independent of stage this study of guo had a large study population of patients with various types of cancer low hrv wasdefined as sdnn 70ms and was significantly associated with shorter survival times thissuggests that hrv is a predictive indicator of survival time not only in palliative but also incurative patients however results were not controlled for cancer type which could affect bothhrv and survival and should therefore be interpreted with caution the fact that thepatients recruited in the present sample were only with less advanced cancer may partlyexplain the lack of prognostic role in hrv in this sample one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig bar chart for cealevel at baseline and one year checkup in groups of low hrv and normal hrv presented as asdnn and b rmssd101371 pone0237244g004 one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable low hrv and risk of occurrence of a postoperative complication within dayspostoperative complicationor ciunadjustedsdnn 20ms n “sdnn �20ms n referenceadjusted�sdnn 20ms n “sdnn �20ms n referenceunadjustedrmssd 19ms n “rmssd �19ms n referenceadjusted�rmssd 19ms n “rmssd �19ms n reference�adjusted for heart rate age cardiac disease and hypertension101371 pone0237244t005psome of these previous studies suggest a bidirectional relationship between vagus nerveactivity and cancer however based on current evidence on this subject we cannot supportthis hypothesis the positive association between low hrv and worse prognosis found inpatients with colorectal cancer receiving palliative treatment but not in patients receivingcurative treatment suggest that this relation is not bidirectional but that advanced cancer isassociated with low hrv midlatestage tumours are often accompanied by damage to autonomic nerves resulting in decreased hrv a study of de couck showed that cancerpatients in general have a significantly lower hrv than healthy people the same resultswere found in studies of bijoor where rmssd was found to be significantly lower inpatients with early and advanced stage cancer compared to a healthy control group when comparing patients with advanced stage cancer tnm iii and iv to patients with anearly stage of cancer tnm i and ii rmssd was found to be significantly lower in patientswith advanced stages of cancer thus though experimental studies in animals showthat vagal nerve functioning can causally slow tumorigenesis the human data suggests that themalignant tumour causes vagal nerve dysfunction and therewith decreased hrv besides the proposed influence of low hrv on survival of colorectal cancer patientsthrough development and increased progression of cancer reimer suggested that lowhrv could influence survival of those undergoing surgical treatment due to more and moresevere postoperative complications however the results found in this study were notconcurrent with those of reimer who included patients with asa undergoingelective surgery their analysis of hrv was through powerspectrum parameters based on longer ecgrecordings instead of the timedomain parameters based on 10s ecgs used in thisstudy but the difference in used parameters used in both studies is probably not the explanation for the differences in results between both studies since it has been demonstrated thatrmssd and sdnn based on ecg recordings of 10s are in substantial agreement with those of45min and a 10s ecg therefore suffices to determine time domain hrv parameters howeverreimer did not correct for possible confounders in their study patients with low hrvwere more likely to have diabetes a known risk factor for postoperative ileus and wound infections both found to be common postoperative complications in their low hrv group correcting for this comorbidity may change the significance of their findings a study of one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable low hrv and severity of postoperative complications according to claviendindo classificationunadjustedminor grade i and iior cipmajor grade iii iv vor cisdnn 20msn “n “sdnn �20msn referencen referenceadjusted�sdnn 20msn “sdnn �20msn referenceunadjustedrmssd 19msn “rmssd �19msn referenceadjusted�n “n referencen “n referencermssd 19msn “n “rmssd �19msn referencen referencep�adjusted for heart rate age categories vs � and hypertension101371 pone0237244t006scheffler did not show any significant preoperative differences in hrv between patientswith or without postoperative complications or between patients with postoperative complications of different severity levels thus also in relation to predicting postoperative complications results are not uniformthis study was subject to a number of limitations due to its retrospective character confounding may have occurred we attempted to correct for all possible confounders withindatabase availability with regression analyses also not all possible confounding factors werewithin database availability reasons for diagnostic procedure smoking alcohol abuse are presumed to have an effect on hrv and outcomes but were not documented in enough patientsto be taken into account when conducting multivariate analyses analysed ecgs were thoseperformed before or at time of preoperative screening in this cohort ecg™s were madewithin the year before surgery only ecg™s were older than five years at time of surgery theprecise effects of these differences in time from ecg to cancer treatment on hrv areunknown to test the implication o

cancer7557

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: preoperative heart rate variability a prognosticindicator for overall survival and cancer recurrencein patients with primary colorectal cancer one e0237244 101371 pone0237244editor louise emilsson university of oslonorwayreceived february accepted july published august copyright strous this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement data cannot beshared publicly because of ethical concernspatients were included on a no objection base toconduct retrospective data studies and publishfindings but were not asked for permission topublish full encrypted data data are available fromthe viecuri institutional data access contact viawetenschapsbureauviecurinl for researcherswho meet the criteria for access to confidentialdata heart rate variability hrv represents efferent vagus nerve activity which is suggested tobe inversely related to fundamental mechanisms of tumorigenesis and to be a predictor ofprognosis in various types of cancer hrv is also believed to predict the occurrence andseverity of postoperative complications we aimed to determine the role of preoperativehrv as a prognostic factor in overall and cancer free survival in patients with colorectalcancermethodsretrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between january and december who underwent curative surgical treatment hrv was measured as timedomain parameters sdnn standarddeviation of nnintervals and rmssd root mean square of successive differencesbased on preoperative second ecgs groups were created by baseline hrv low hrvsdnn 20ms or rmssd 19ms and normal hrv sdnn �20ms or rmssd �19msprimary endpoints were overall and cancer free survivalresultsa total of patients were included in this study hrv was not significantly associated withoverall survival sdnn 20ms vs sdnn �20ms244 vs adjusted hr “ p rmssd 19ms vs rmssd �19ms270 vs adjustedhr “ p or cancer recurrence sdnn 20ms vs�20ms201 vs adjusted hr “ p rmssd 19ms vs�19ms vs adjusted hr “ p there was no one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfunding the authors received no specific fundingfor this workcompeting interests the authors have declaredthat no competing interests existsignificant association between hrv and cealevel at one year followup or between hrvand occurrence of a postoperative complication or the severity of postoperativecomplicationssheart rate variability was not associated with overall or cancer free survival in patients withprimary colorectal cancer who underwent curative surgical treatment these results do notalign with results found in studies including only patients with advanced cancer which suggests that there is only an association in the other direction cancer causing low hrvintroductionin there were over million newly diagnosed colorectal cancer patients worldwide andover in the netherlands alone it is the fourth most common cause of death worldwide to improve survival it is of importance to get a better insight into modifiable prognosticfactors emerging evidence suggests that vagal nerve activity indexed by heart rate variabilityhrv could be one of these prognostic factors [“]hrv is the physiological phenomenon of the fluctuation in time intervals between adjacentheartbeats and represents efferent vagus nerve activity to the heart [“] it has been suggestedthat efferent vagal activity is inversely related with fundamental mechanisms of tumorigenesisas inflammation oxidative stress and excessive sympathetic activity these mechanisms arebelieved to be controlled by the vagus nerve via a bidirectional braintoimmune pathwaysthrough the release of neurotransmitters via the cholinergic antiinflammatory pathway a higher vagal tone may reflect a more flexible topdown regulation of the immunesystem and physiological activity moderated by the brain absence of vagus activity due tovagotomy has been shown to increase the risk of developing colorectal cancer in addition to influencing development of cancer vagus nerve activity seems to be a predictor of prognosis in various types of cancer recent studies show an association betweendecreased activity of the vagus nerve and worse survival in patients with cancer of the gastrointestinal tract liver pancreas lung prostate and breast among others also patientswith normal hrv seem to live longer in different sorts of metastatic cancer independent ofconfounders in patients with colorectal cancer a low hrv at baseline has shown to beassociated with higher cea levels at months after diagnosis which predicts a poorer prognosis in patients undergoing curative treatment for colorectal cancer hrv does not only seemto influence cancer prognosis a recent study showed that patients with lower hrv have moreintraoperative blood loss and more and more severe postoperative complications identifying patients with low hrv is easy and noninvasive when its predictive value forthe prognosis of cancer patients is of satisfactory significance vagus nerve activation prior toor during cancer treatment could theoretically be beneficial in improving prognosis alsoif we could predict the occurrence and severity of postoperative complications based on hrvimproving hrv before surgery could possibly accelerate postoperative recovery and indirectlyaffect patients™ prognosis recent studies focussing on improving hrv by improving physicalfitness by means of physical exercise show promising results in both older men and woman however the only previous study on colon cancer and hrv including patients receiving curative treatment included a small sample and did not examine whether hrv predicts one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancersurvival in these patients to clarify the predictive value of hrv in prognosis of patientswith colorectal cancer further exploration is needed current studies identifying hrv as aprognostic factor did not specifically focus on colorectal cancer have small study populationsdid not correct for confounders and mainly focused on metastatic disease [“]the aim of this study was to determine the role of preoperative hrv as a prognostic factorin overall and cancer free survival in patients with primary colorectal cancer who underwentcurative surgical treatmentmethodsdata collectiondata from the netherlands cancer registry ncr were used the ncr collects data on allnewly diagnosed cancer patients in the netherlands information on patient and tumour characteristics diagnosis and treatment is routinely collected from the medical records by trainedadministrators of the cancer registry anatomical site of the tumour is registered according tothe international classification of diseases oncology the tumournodemetastasis tnmclassification is used for stage notification of the primary tumour according to the editionvalid at time of cancer diagnosis quality of the data is high due to thorough training of theregistration team and consistency checks for the study population additional data were collected from the medical records of thepatients this encompassed information on hrv cealevels asa classification comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroid disease pulmonary disease vascular disease neurological disease and otheroccurrence and severity of postoperative complications and cancer recurrence groups ofcomorbidities were chosen based on matching features within these groups and their potentialinfluence on hrv or the endpoint being analysed severity of the postoperative complicationsaccording to the claviendindo classification was also documented medical records wereassessed between january and july and reevaluated for revision of this between the 20th and 25th of april this study was approved by the research committee and the board of directors of viecurimedical centre data was obtained under the law ˜scientific research and statistics in the interest of public health where asking for permission is not possible or appropriate for several reasons™ in the netherlands unless patients objected to use of their personal medical record forscientific research data was encrypted with an encryption key provided by the ncr encryption was shortly lifted to access the patients™ number for accessing hisher medical record following extraction data were encrypted againstudy populationthe study population included all consecutive patients diagnosed with primary colorectal adenocarcinoma between january and january at viecuri medical centre who underwent curative surgical treatment patients with metastatic disease at time of surgery orcarcinoma in situ were excluded as their treatment and prognosis differs from those receivingcurative treatment for colorectal cancer metastasis found within months after surgery wereconsidered present at time of surgery and therefore excluded other excluded patients werepatients with neuroendocrine tumours because of different tumour characteristics and prognosis patients with cardiac arrhythmias including atrial and ventricular extrasystole pacemakers patients taking betablockers as this enhances hrv indexes or patients withbradycardia heart rate bpm or tachycardia heart rate bpm as this precluded reliable calculation of hrv we did not exclude patients taking alphablockers calcium one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerinhibitors diuretics amiodarone aceinhibitors or arb™s as these types of medicationreduce central sympathetic functioning rather than peripheral and their influence on hrv istherefore less univocal and sometimes completely absent heart rate variabilityheart rate variability was analysed using a 12lead 10second ecg 150hz used for preoperative screening in case of multiple ecg™s per patient the most recent ecg before date of surgery was used for hrvanalysis in case of multiple ecg™s per patient on the same date theecg with the best quality was chosen meaning an ecg without motion artefacts in case ofmotion artefacts there was always an ecg without motion artefacts available recorded on thesame date time between two consecutive rpeaks was measured in lead ii with an accuracy of02ms using museecg hrv was presented using the timedomain hrv parameters sdnnstandard deviation of nnintervals and rmssd root mean square of successive differences in milliseconds calculated using the following calculations rsdnn ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðrri 00 rrmeanþ2pnn 00 rmssd ¼rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðrriþ1 00 rriþ2pn 00 n 00 ð1þð2þsdnn and rmssd obtained from 10s ecgs were found to correlate with results of ecgsof longer durations power spectral analysis hrv parameters as lf and hf can only beobtained in longer recording ecgs and were therefore not implementable in this study sdnn and rmssd were both analysed as continuous variables as well as binary variablesusing cutoffs of 20ms versus �20ms and 19ms versus �19ms respectively in case of ansdnn 20ms or rmssd 19ms hrv was classified as low and in case of sdnn �20ms orrmssd �19ms as normal these cutoff values were based on cutoff values used in otherstudies showing an association between lowhrv as sdnn 20ms and rmssd 19ms andcolorectal cancer as there is no standardised definition of low and normal hrv endpoints and definitionsthe primary endpoints of this study were overall and cancer free survival overall survival wasdefined as the time between the date of surgery to the date of death or last followup date inmonths cancer free survival was defined as the time in months from the date of surgery untilthe date of cancer recurrence defined as the first date of either radiologic or pathologic diagnosis of metastases or tumour recurrence of colorectal cancer patients dying without cancerrecurrence were censored on day of death secondary endpoints were elevated cealevel ugl at oneyear followup occurrence of postoperative complications within daysafter surgery and severity of postoperative complications according to the claviendindoclassificationstatistical analysisin this retrospective observational cohort study we utilized descriptive statistics to provide anoverview of control variables of the study population patient characteristics as age sex bmicomorbidities and asaclassification heart rate and tumour characteristics as tnmstagetumour localisation and tumour differentiation and their association with hrv and one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerprognosis normal distribution of the continuous variables heart rate age and bmi as well assdnn and rmssd were tested with a kolmogorovsmirnov test because of normal distribution heart rate age and bmi were compared between hrvgroups using unpaired ttest allother variables were categorical and were compared between hrvgroups using chisquarestatistics as groups were all of sufficient powerdifferences in overall survival and cancer free survival in months according to sdnn andrmssd were visualized by means of kaplanmeier curves and statistically tested using the logrank test multivariate coxregression analyses were conducted to calculate the prognosticassociation between hrv and overall and cancer free survival while adjusting for other prognostic variables multivariate logistic regression was used to assess the independent effect ofsdnn and rmssd on cealevels and the occurrence and severity of postoperative complications variables included for adjustment were chosen by means of forward stepwise selectionbased on clinical judgment differences at baseline eg differences on any predictor betweenpatients who later died or not and database availability and depended on the analysed endpoint those included patient demographics age sex bodymassindex comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroiddisease pulmonary disease vascular disease neurological disease other including crohn™sdisease hepatitis kidney failure disorders anaemia depression arthritis tumour characteristics localisation stage differentiation and the occurrence of postoperative complicationswhen the later was not an outcome differences in cealevel at baseline and one year checkup between and within groups of low hrv and normal hrv were assessed with a repeatedmeasures linear model and tested using the tukey test to test the implication of a longer timebetween ecg and treatment all analyses were repeated after excluding patients with an ecgolder than months a twotailed pvalue � was considered significant in all analysesdata were analysed using ibm spss statistics version ibm corp ny armonk usaresultsof colorectal cancer patients that underwent a surgical resection a total of patientswere included in this study reasons for exclusion are presented in fig median sdnn andrmssd were 204ms interquartile range iqr 115ms351ms and 175ms iqr 99ms299ms respectively table shows descriptive data of the included patients by hrv groupsbaseline heart rate and age were negatively associated with hrv the group of patients withlow hrv contains more patients with a history of cardiac disease regardless of the hrv defining parameter when defining low hrv by rmssd 19ms more patients in this group havehypertension as comorbidity this group also contains more patients with an asa classification greater than oneduring a median followup of months iqr “ all causedeath occurred in patients cancer recurrence occurred in patients during a median followup of months iqr “to rule out any distort in results caused by a delay between ecg and treatment all analyseswere repeated after exclusion of ecg™s older than months this did not lead to any new significant results therefore these results were not displayed in detail in this papersurvivalin low hrv groups slightly more patients died compared to normal hrv groups sdnn20ms versus �20ms versus respectively rmssd 19ms versus�19ms versus respectively these observed differences between hrvgroups in overall survival were not significant fig a sdnn p b rmssd one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig flowchart of the study101371 pone0237244g001p after adjustment for some potential confounders these associations remained notsignificant sdnn 20ms versus �20ms hr “ p and rmssd19ms versus �19ms hr “ p tables and age cardiac disease tumour stage and postoperative complications had a significant influence on overall survival age also differed at baseline and was identified as a confounder when defining low andnormal hrvgroups by sdnn cardiac disease was identified as confounder when conducting sensitivity analyses with sdnn and rmss as continuous variables results remained thesame there was no association between hrv and overall survival sdnn hr “ p and rmssd hr “ p in low hrv groups slightly more patients had recurrence of cancer compared to normalhrv groups sdnn 20ms versus �20ms versus respectivelyrmssd 19ms versus �19ms versus respectively these observed differences between hrv groups in cancer free survival were not significant fig a sdnnp b rmssd p as in overall survival after adjustment for some potentialconfounders these associations remained not significant sdnn 20ms versus �20mshr “ p and rmssd 19ms versus �19ms hr “ p tables and in sdnnbased groups baseline heart rate was identified asa confounding variable sensitivity analyses with sdnn and rmssd as continuous variables one 101371 pone0237244 august one 0ctable descriptive data of included patients according to prespecified hrv groupssdnn 20ms n sdnn �20ms n prmssd 19ms n rmssd �19ms n phrv and prognosis in colorectal cancerheart rate�age�age n year� yearsex nmalefemale [“] [“] [“] [“] [“] [“] [“] [“] mean bmi sd [“] [“] [“] [“]comorbidities ncardiac diseasehypertensiondiabetes mellitusthyroid diseasepulmonary diseasevascular diseaseneurological diseaseotherasa nasa1asa2asa34localisation tumour nright colonleft colonrectumtumour stage niiiiii differentiation grade nwellmoderate poorundifferentiated � non normaldistributed data presented as median with interquartile range101371 pone0237244t001did not alter these results there was no association between hrv and cancer free survivalsdnn hr “ p and rmssd hr “p cealevelcealevel at baseline and one year checkup was registered in patients and elevated in of these patients this was elevated at one year checkup in only patients low hrv was notsignificantly associated with elevated cealevels at one year checkup as shown in table sensitivity analyses with sdnn and rmssd as continuous variables did not alter these resultssdnn hr “ p and rmssd hr “ one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig kaplanmeier curves for overall survival in groups of low hrv and normal hrv presented as a sdnn andb rmssd101371 pone0237244g002 one 101371 pone0237244 august one 0ctable multivariate analyses of associations of sdnn and covariates with overall and cancer free survivalhrv and prognosis in colorectal cancersdnn20ms�20msheart rateagebmicardiac diseasenoyeshypertensionnoyesasaclassificationasa1asa2asa34localisationright colonleft colonrectumtumour stageiiiiiioverall survivalhr cipcancer free survivalhr ci “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ “ “ reference “ “ reference “ “postoperative complicationnoyes reference reference “ “101371 pone0237244t002p adjusting for covariates was not possible because of the small number of patientswith an elevated cealeveldifferences between cealevel at baseline and one year checkup were compared betweenand within hrvgroups results were displayed in fig there were no significant differencesin cealevel at baseline and one year checkup between the low hrv group and normal hrvgroup defined by sdnn and rmssd p and p respectively also there were nosignificant differences in cealevel at baseline and at one year checkup within the low hrvgroup and normal hrv group defined by sdnn and rmssd p and p respectivelypostoperative complicationsin patients one or more postoperative complications occurred within days after surgerythe occurrence of a postoperative complication was not significantly associated with lowhrv defined as sdnn 20ms or rmssd 19ms even after adjustment for some potentialconfounders table heart rate age cardiac disease and hypertension were identified asconfounding covariates when conducting sensitivity analyses with sdnn and rmss as continuous variables the association between occurrence of a postoperative complication with one 101371 pone0237244 august one 0ctable multivariate analyses of associations of rmssd and covariates with overall and cancer free survivalhrv and prognosis in colorectal cancerrmssd19ms�19msheart rateagebmicardiac diseasenoyeshypertensionnoyesasaclassificationasa1asa2asa34localisationright colonleft colonrectumtumour stageiiiiiioverall survivalhr cipcancer free survivalhr ci “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “postoperative complicationnoyes reference reference “ “101371 pone0237244t003baseline hrv remained not significant sdnn hr “ p andrmssd “ p the same applied when postoperative complicationswere graded according to the claviendindo classification and the complication that is gradedthe highest for each patient is compared to the absence of postoperative complicationstable discussionin this observational cohortstudy we determined the heart rate variability in preoperativeecgs of patients with primary colorectal cancer who underwent curative surgical treatment hrv refers to physiological variations in beattobeat intervals it was presented intimedomain parameters sdnn and rmssd hrv was not significantly associated with overall survival or cancer free survival independent of some risk factors also this study showedno significant differences in cealevels at one year checkup between patients with low hrvand patients with normal hrv patients with low hrv did not have more or more severepostoperative complications compared to patients with normal hrvtumorigenesis has three fundamental mechanisms inflammation promoting oxidativestress and stimulating tumour growth oxidative stress causing dnadamage and one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig kaplanmeier curves for cancer free survival in groups of low hrv and normal hrv presented as a sdnnand b rmssd101371 pone0237244g003 one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable univariate analyses of low hrv and risk of elevated cealevel at one year checkupcea μglor cipcea μglor ciindependent of baseline ceaindependent of baseline ceasdnn 20ms n “rmssd 19ms n “sdnn �20ms n normal baseline cea � μglsdnn 20ms n sdnn �20ms n elevated baseline cea μglsdnn 20ms n reference “ reference “rmssd �19ms n normal baseline cea � μglrmssd 19ms n rmssd �19ms n elevated baseline cea μglrmssd 19ms n reference “ reference “sdnn �20ms n referencermssd �19ms n referencep101371 pone0237244t004interfering with subsequent repair mechanisms and sympathetic neurotransmitters stimulating tumour metastasis and progression it has been suggested that afferent fibres of thevagus nerve inform the brain about peripheral inflammation this is followed by a braintoimmune response via the efferent route of the vagus nerve that modulates the function ofimmuneregulatory cells through the release of neurotransmitters via the cholinergic antiinflammatory pathway malfunctioning of this route may play part in the onset of cancer this theory has been supported by studies of patients with gastric ulcers who underwent avagotomy who then showed an increased risk of developing lung and colorectal cancer [“] the vagus nerve is also believed to modulate tumour progression an active vagus nervecan inhibit inflammation oxidative stress and the release of sympathetic neurotransmittersthat stimulate tumour metastasis and progression [“] absence of this inhibitory effect inturn results in metastasis and tumour progression as shown in a study of erin showingthat mice who underwent chemical vagotomy developed more metastasis of breastcancer cellsthan controls epidemiologically low hrv has been associated with worse overall survivalover time in patients with recurrent or metastatic cancer of various types even after correctionfor confounders [“] however the results of the present study do not support thesefindingsto our knowledge this is the first study including only patients with colorectal cancer whoare eligible for curative treatment by partial bowel resection and not those receiving palliativetreatment de couck studied the relationship between hrv and tumour burden in bothcurative and palliative patients with prostate cancer or nonsmall cell lung cancer independent of confounders the hypothesised inverse relationship of hrv and the tumour markerpsa at and months after diagnosis was only significant in patients with stage iv prostatecancer not stage ii and iii in colorectal cancer mouton found that low hrv definedas sdnn ms predicted significantly higher levels of the tumour marker cea at months after diagnosis again these results were only found in patients receiving palliativetreatment not curative only one previous study showed a significant inverse relationshipbetween hrv and mortality in cancer in general independent of stage this study of guo had a large study population of patients with various types of cancer low hrv wasdefined as sdnn 70ms and was significantly associated with shorter survival times thissuggests that hrv is a predictive indicator of survival time not only in palliative but also incurative patients however results were not controlled for cancer type which could affect bothhrv and survival and should therefore be interpreted with caution the fact that thepatients recruited in the present sample were only with less advanced cancer may partlyexplain the lack of prognostic role in hrv in this sample one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig bar chart for cealevel at baseline and one year checkup in groups of low hrv and normal hrv presented as asdnn and b rmssd101371 pone0237244g004 one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable low hrv and risk of occurrence of a postoperative complication within dayspostoperative complicationor ciunadjustedsdnn 20ms n “sdnn �20ms n referenceadjusted�sdnn 20ms n “sdnn �20ms n referenceunadjustedrmssd 19ms n “rmssd �19ms n referenceadjusted�rmssd 19ms n “rmssd �19ms n reference�adjusted for heart rate age cardiac disease and hypertension101371 pone0237244t005psome of these previous studies suggest a bidirectional relationship between vagus nerveactivity and cancer however based on current evidence on this subject we cannot supportthis hypothesis the positive association between low hrv and worse prognosis found inpatients with colorectal cancer receiving palliative treatment but not in patients receivingcurative treatment suggest that this relation is not bidirectional but that advanced cancer isassociated with low hrv midlatestage tumours are often accompanied by damage to autonomic nerves resulting in decreased hrv a study of de couck showed that cancerpatients in general have a significantly lower hrv than healthy people the same resultswere found in studies of bijoor where rmssd was found to be significantly lower inpatients with early and advanced stage cancer compared to a healthy control group when comparing patients with advanced stage cancer tnm iii and iv to patients with anearly stage of cancer tnm i and ii rmssd was found to be significantly lower in patientswith advanced stages of cancer thus though experimental studies in animals showthat vagal nerve functioning can causally slow tumorigenesis the human data suggests that themalignant tumour causes vagal nerve dysfunction and therewith decreased hrv besides the proposed influence of low hrv on survival of colorectal cancer patientsthrough development and increased progression of cancer reimer suggested that lowhrv could influence survival of those undergoing surgical treatment due to more and moresevere postoperative complications however the results found in this study were notconcurrent with those of reimer who included patients with asa undergoingelective surgery their analysis of hrv was through powerspectrum parameters based on longer ecgrecordings instead of the timedomain parameters based on 10s ecgs used in thisstudy but the difference in used parameters used in both studies is probably not the explanation for the differences in results between both studies since it has been demonstrated thatrmssd and sdnn based on ecg recordings of 10s are in substantial agreement with those of45min and a 10s ecg therefore suffices to determine time domain hrv parameters howeverreimer did not correct for possible confounders in their study patients with low hrvwere more likely to have diabetes a known risk factor for postoperative ileus and wound infections both found to be common postoperative complications in their low hrv group correcting for this comorbidity may change the significance of their findings a study of one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable low hrv and severity of postoperative complications according to claviendindo classificationunadjustedminor grade i and iior cipmajor grade iii iv vor cisdnn 20msn “n “sdnn �20msn referencen referenceadjusted�sdnn 20msn “sdnn �20msn referenceunadjustedrmssd 19msn “rmssd �19msn referenceadjusted�n “n referencen “n referencermssd 19msn “n “rmssd �19msn referencen referencep�adjusted for heart rate age categories vs � and hypertension101371 pone0237244t006scheffler did not show any significant preoperative differences in hrv between patientswith or without postoperative complications or between patients with postoperative complications of different severity levels thus also in relation to predicting postoperative complications results are not uniformthis study was subject to a number of limitations due to its retrospective character confounding may have occurred we attempted to correct for all possible confounders withindatabase availability with regression analyses also not all possible confounding factors werewithin database availability reasons for diagnostic procedure smoking alcohol abuse are presumed to have an effect on hrv and outcomes but were not documented in enough patientsto be taken into account when conducting multivariate analyses analysed ecgs were thoseperformed before or at time of preoperative screening in this cohort ecg™s were madewithin the year before surgery only ecg™s were older than five years at time of surgery theprecise effects of these differences in time from ecg to cancer treatment on hrv areunknown to test the implication o Answer:

Colon_Cancer

low levels of physical activity pa and high levels of physical inactivity pi are associatedwith higher mortality and cardiovascular diseases higher age is associated with a decreaseof pa only yearolds achieve the pa times recommended by who theaim of this study was to identify levels of and determinants for moderate pa overall pa andpi in a sample of individuals aged � yearsmethodswe analyzed baseline data from an interventionstudy aiming to increase pa and decreasepi by automatically generated feedback letters to objectively measured pa and pi recruitment was multimodal including recontacting participants of previous studies and advertisements in regional public buses and newspapers at baseline participants wore anaccelerometer over a period of consecutive days pa was categorized using cutpointssuggested by freedsoon in light moderate and vigorous physical intensity as well asphysical inactivity potential determinants selfefficacy education were measured byquestionnaires or in a physical examination bmi multiple linear regression models were fitted to identify determinants for pa and piresultsn persons mean age years sd female participated in the studythe weekly amount of overall pa for men was on average minutes sd forwomen on average minutes sd of the women and of the menachieved the who recommendation of minutes moderate paday at baseline the timeof pi during the observation time period of days was on average minutes in men anda1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation kleinke f penndorf p ulbricht s do¨rr mhoffmann w van den berg n levels of anddeterminants for physical activity and physicalinactivity in a group of healthy elderly people ingermany baseline results of the movingstudy one e0237495 101371 pone0237495editor thalia fernandez universidad nacionalautonoma de mexico mexicoreceived november accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237495copyright kleinke this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0237495 august one 0cfunding the study was funded by the federalministry of education and research bmbf as asite project of the german centre forcardiovascular research dzhk funding sign81z7400174 the funders have had no influenceon the conceptualization and conduct of the studyand will not have any role in the data analysis andpublication of the resultscompeting interests the authors have declaredthat no competing interests existlevels of and determinants for physical activity and physical inactivity minutes in women in males age was found to be a significant negative determinantfor overall pa p and for moderate pa p0001 higher education was positivelyassociated with higher levels of overall pa p and moderate pa p in menbmi was a significant negative determinant for overall pa both in men p andwomen p as well as for moderate pa for women p only in women not inmen selfefficacy was to be a significant positive determinant for overall pa p aswell as negatively associated with pi p discussionthe participants of our study showed high levels of pa this is likely due to selection bias inthis convenience sample however also levels of pi are very high and those correspondwith average levels in the german population the determinants for higher pa and lower pidiffered between males and females thus strategies for improving pa and decrease pi arelikely different with respect to sex and should take individual factors eg age bmi intoaccounttrial registration numberdrks00010410 date may introductionphysical activity pa is defined as œany bodily movement produced by skeletal muscles thatrequires energy expenditure there is strong evidence that regular pa is a very effectivenonpharmacological and noninvasive healthpromoting method [“] a papromoting lifestyle is associated with a reduced risk of mortality and is correlated with improved overallhealth status additionally high levels of pa significantly decreases overall mortality by“ and cvd mortality by “ recent reviews showed that regular pa is alsoassociated with a “ reduced risk of developing dementia in older adults [ ] and is considered as one of the proxies of the concept of cognitive reserve one of the two most significant modifiable risk factors for dementia is pi additionally pa in particular aerobicexercise is positively associated with a less agerelated reduction of anic brain matter international pa guidelines recommend for healthy individuals aged over years at least minutesweek of moderate pa or at least minutesweek of vigorous pa or an equivalent combination of weekly pa in fact there is no difference to recommendations for healthyyounger individuals aged “ years pa should be performed in uninterrupted time periodsbouts of a duration of at least minutes [ ]data from the united kingdom show that of men and of women aged “years reach the recommended level of pa a study from norway showed that of menand of women aged “ years meet the recommendations in the age group “years only of women and of men reach the whorecommendations in the usanational health and nutrition survey nhanes the proportion of people aged over years who achieved the recommended amount of pa was in germany only of men and of women aged “ years achieve the who recommendations regardingpa selfreported via questionnaire in the age group “ years this proportion declines to in men and to in women one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivityin europe in of the total population was years or older in germany of the population was years or older in this proportion will furtherincrease to until older age is associated with an higher risk for chronic diseasesmultimorbidity [“] prevalences of cvd including coronary heart disease chd andstroke will increaselow pa is one of the leading risk factors for global mortality globally of theadults are insufficiently physically active a reduction of the prevalence of insufficient pais a global target of the who [ ] physical inactivity pi defined œas any waking behaviour characterized by an energy expenditure � mets metabolic equivalent of task whilein a sitting or reclining posture sedentary behaviour research network tremblay causes million deaths per year worldwide and in was estimated to beresponsible for million dalys disabilityadjusted life years globally besides lowlevels of pa also pi is a crucial risk factor for mortality [ ] frequently interrupted piis associated with positive effects on health status and a reduced risk for premature death lack of pa and a high level of pi are associated with an array of noncommunicable diseasesncd and eg responsible for approximately “ of breast and colon cancers of diabetes cases and approximately of ischaemic heart disease prevalencesworld healthanization additionally it seems that physical inactivity is an important preventablefactor for alzheimer™s dementia older people spent on average to hours a day sedentary which correspondents with “ of their waking time depending on the exact definition the distrubution of pi acrosseuropean countries ranged from in schweden up to in portugal in germany of men and of women showed a sedentary lifestyle low energy expenditure of theleisuretime expenditure in activities requiring � metabolic equivalents met the amount of pa and pi depends on individual factors such as age bmi gender education social status and selfefficacy [“] additionally environmental and policy factors weather conditions and length of the day have an effect on the amount of pa of people[ ]overweight and obesity bmi30 kgm2 have a negative influence on the level of pa andpi in the elderly in a crosssectional study in which subjects were surveyed from member states of the european union it was found that people with a low bmi kgm2and normal bmi “ kgm2 have low prevalence in pi both genders in contrast peoplewith a bmi above kgm2 showed a more sedentary lifestyle [ ]higher education has a positive influence on pa and pi varo eu study showed thatpeople with primary level education were more sedentary than participants with higher levelsof education greater difference in women further factors that influence the amount of pa and pi are marital status income wellbeing psychosocial variables such as selfefficacy and social and cultural parameters [ ]a report from the who about the prevention of noncommunicablediseases ncd insoutheastern european countries showed that the promotion of pa and reduction of pi arekey aspects in public health efforts promoting physical activity through mass media was a primary goal for immediate action in addition the global strategy on diet physical activityon health and the european charter on counteracting obesity underline the relevance of pa to fight against obesityto develop effective prevention strategies adapted to the elderly detailed information onthe levels of pa and pi and their determinants are necessary in this analysis we assessed thelevels of pa and pi and identified positive and negative determinants for pa and pi in a sample of healthy people aged � years one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitymaterials and methodsthe data for this analysis were retrieved from the baseline assessment of an intervention studywith the goal to increase pa and reduce pi in elderly people with a lowthreshold interventionmoving“motivation oriented intervention study for the elderly in greifswald thisstudy is a twoarm randomized controlled trial consisting of assessment of eligibility baselineexamination randomization intervention only the participants in the intervention groupand followup examinations at and months after baseline the study region was western pomerania a rural area in the northeast of germanystudy participants in the intervention group received two individual feedback letters containing objectively measured pa and pi times based on data from the accelerometer devicecaptured at baseline and months followup measurement feedbackletters were automatically generated in r software version or later lucent technologies murray hill njusaa comprehensive description of the study protocol for the moving study is publishedelsewhere inclusion of the participantsthe study participants had to meet the following inclusion criteria¢ age � years¢ ability to be physically active in daily lifeexclusion criteria were¢ inability to walk independently eg permanent use of a wheelchair¢ simultaneous participation in other studies addressing pa or pi¢ not accessible by telephone or cell phone¢ already fulfilling the who recommendations for pa selfreported for people � yearsat baseline � minutes moderate pa per weekrecruitment was performed in in several ways¢ recontacting participants of a previously performed study ¢ recruitment at venues frequented by older people eg senior sport hours in the publicswimming pool rehearsals of senior choirs events at meeting places for elderly people¢ persons contacted the study centre after reading s about the project in regional newspapers and television advertisements in regional buses and flyers and posters that were distributed in gp practices hospitals and meeting centers for elderly peopleall participants were informed in detail about the study and had to give their writteninformed consentmeasuresall study participants received a baseline examination consisting of the assessment of bloodpressure somatometry data body weight waist and hip circumference as well as sociodemographics sex age education job and partnership status general health status the ssa scaleselfefficacy towards physical exercise was used to asses the participants™ level of selfefficacy one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitythe result of that scale is a sum in which higher values indicate higher selfefficacy towards pa after that the study participants received an accelerometer device actigraph gt3xbtpensacola fl usa which captures and records pa and pi continuously at a sampling frequencyof hz over a period of seven consecutive days starting at midnight after the baseline examination participants were instructed to wear the accelerometer device for seven days only during daytime on the right hip and to remove it only at bedtime and for waterbased activities egshowering swimming in addition all study participants were asked to document their physicalactivities in a semistandardized protocol for each day of the observation time besides the objective assessment of pa and pi the participants were instructed to answer paperbased questionnaires to assess selfreported pa and pi with regard to the observation period of the accelerometerto asses selfreported pa the international physical activity questionnaire short formipaqsf in german version was used the ipaq consists of seven items addressing intensity and duration of pa in daily life over the last days by selfreport in addition the german physical activity questionnaire for german paq was used to assess type andduration of pa in daily life by selfreport pi was assessed by the measure of older adults™sedentary time most questionnaire in the german version body mass index was calculated from measured body height and body weight kgm2 � kgm2 and kgm2 � kgm2after seven days the participants had to bring the accelerometer device and the fulfilledquestionnaires back to the examination centerdata analysisdata assessment and documentation were conducted based on ecrfs in an itsupported documentation system which is characterized by an independent operation synchronization andmonitoring the software is based on the concept of offline clients and each staff member ofthe dzhk had individual login data the paperbased questionnaires were documented using the software cardiff teleform1electric paper lu¨neburg germany all questionnaires and the daily physical activity protocol contained 1d barcodes to ensure anonymization of the study participantsthe actilife software versions to actigraph cop pensacola fl usawas used to download the pa data from the accelerometer the raw data were calculated into10second epochs and saved in raw format as gt3x files a valid measurement day wasdefined as a record of at least hours of total wearing time measurements of at least validdays were required to be included into data analysis to categorize pa intensity we used specific cut points based on freedson pa intensity was divided into sedentary “counts light “ counts moderate “ counts and vigorous “counts pa nonwearing time was defined as having � minutes of continual zero countsrange � minutes between and countsdescriptive statistics were used to describe the population with respect to the levels of paand pi to identify determinants of pa and pi multiple linear regression models were calculated dependent variables for the regression models were overall and moderate pa as wellas pi the effect of the independent variables age bmi education and selfefficacy was examined for all regression models all independent variables were checked for multicollinearityusing a correlation pearson to include education as a determinant in the multiple linearregression models we used dummy variables referring to the gaußmarkovtheorem we analyzed the residuals and requirements of multiple linear regression like distribution andhomoscedasticity one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitystatistical significance was assumed for pvalues data processing and statistical calculations were performed with ibm spss statistics or later “ by ibm corp armonk new york usa all statistical analysis were performed based on pseudonymizeddata all identifying data were separated from the project data to the earliest possible timepoint all appointments were ensured facetoface by the study staffethicsthe study was approved by the ethics committee of the university medicine greifswaldethic approval protocol number bb07116 and registered at the german clinical trials register id drks00010410resultsof screended study participants could be included in the analyses fig fig number of recruited participants and participants included in the analysis101371 pone0237495g001 one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable descriptive characteristics of the study sample n characteristicssex womenage yreducation yrbody mass index kgm2 years years years � � currently smoking yesnumber of participants currently having a partnership yeswearing timen number of subjects sd standard deviation101371 pone0237495t001nmean sd or n sd sd participants were included in the analysis thereof women and men the mean age was years sd at the baseline measurement of pa participants had a mean wearing time of the accelerometer of minutes sd per weekwhich corresponds to an average of hoursday table overall there were no significant difference between women and men according tooverall pa mean women minutes mean men minutes per week tdf p men in higher age groups showed lower levels of weekly light pa themean number of minutes of moderate pa increased with higher education in men and inwomen both men and women with higher bmi showed lower levels of moderate and vigorouspa table male participants spent of their waking time in pi female participants olderage was associated with an increasing time of pi in men the level of pi increased with age“ yr minutes minutes and with higher education yr minutes yr minutes in women the time of pi was largely independent of age “ yr minutes minutes table n of the men and n of the women achieved the international recommendations for moderate pa � minutes moderate pa the proportion of participantswho fulfilled the recommendations decreased with age table a multiple linear regression was calculated to predict overall pa based on independent variables in table in men lower age p a lower bmi p and higher educationp were found to be significant positive determinants for overall pa higher bmi is anegative determinant for overall pa in women p additionally selfefficacy wasfound to be a significant positive determinant for overall pa p the overall model fitwas r2 for men and r2 for women table for moderate pa higher age p0001 was found to be a significant negative determinantin men in women a higher bmi was a significant negative determinant p for maleparticipants lower education yr was a significant negative determinant p aswell as years of education p table in women better selfefficacy was found to be a significant positive determinant p for pi table one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable mean duration of weekly pa [min] without specific cut points by intensity of pa and overall pamean duration of weekly [min]mean duration of weeklylight pa ci[min] moderate pa cimean duration of weekly[min] vigorous pa mean duration of weekly [min]overall pa cicimalefemalemalefemalemalefemalemalefemaleage yr“ ™‚ ™ “ ™‚ ™ “ ™‚ ™ ™‚ ™ education yr ™‚ ™ ™‚ ™ ™‚ ™ bmi kgm2 ™‚ ™ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “� ” ™‚ ™ “ “ “ “� ™‚ ™ currently having apartnership yesyes ™‚ ™ no ™‚ ™ total ™‚ ™ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “m mean ci confidence interval min minutes101371 pone0237495t002discussionthe results of this analysis show that the levels of light moderate and overall pa in our sampleof older people are high of the male and of the female study participants fulfilledthe age specific whorecommendations for moderate pa for people aged over years� minutes moderate pa or � minutes vigorous pa especially female participants inthe age group years were above average physically active in people in the same age allwomen in this age group n reached the recommendations for moderate pathe study results show that age is a significant negative determinant for moderate and overall pa in men it can be concluded that men become more physically inactive with age this isin contrast to other studies in which women are generally less physically active than their malecounterparts [ ] this finding can also be explained by the fact that women in our sample in particular were physically active to an aboveaverage extenteducation was found to be a significant positive determinant regarding moderate pa inmen and women and overall pa in men these results are consistent with other studies people one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable mean duration of weekly pi [min] proportion of sedentary time of total wake time in minutesmean min of weekly pi cimean proportion of daily pi daytime in malefemalemalecifemaleage yr“ ™‚ ™ “ ™‚ ™ “ ™‚ ™ ™‚ ™ education yr ™‚ ™ ™‚ ™ ™‚ ™ bmi kgm2 ™‚ ™ � ™‚ ™ � ™‚ ™ currently having a partnership yesyes ™‚ ™ no ™‚ ™ total ™‚ ™ ci confidence interval min minutes101371 pone0237495t003 “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “716s “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “with higher education tend to be more physically active and generally pay more attention totheir personal health [ ]bmi was found to be a significant determinant for moderate pa in women additionallyhigher bmi in men and women was statistically significantly associated with a decreasing levelof overall pa self efficacy was a significant negative determinant for pi only in women thusfemales with higher self efficacy showed lower levels of pi there was no systematic differencebetween the gender self efficacy was the only statistically significant predictor for pi thusmotivation can be seen as an important factor to reduce piour study participants spent most of their waking time in pi men spent and female per day in pi which correspondends to results from other studies examining the sameage group [ ] in industrialized countries high pi values are therefore associated with an enormous burden on health systems table number and proportion of study participants who fulfilled the who recommendations for moderate pa separately for men and womenn of age group of all menn of age group of all womenmalefemaleage yr“ n “ n “ n n total n of of of of of of of of of of n number of subjects fulfillment of who recommendations for moderate pa for people aged and older � minutes moderate pa or � minutes vigorous pa oran equivalent combination per week101371 pone0237495t004 one 101371 pone0237495 august one 0ctable multiple linear regression model to identify determinants for overall pa separately for men and womenlevels of and determinants for physical activity and physical inactivitypredictorsinterceptagebmieducationa yr yrmale n adj r2 β ci “ “ “ “ “ yr œabitur qualification for the university “otherselfefficacy “ “β regression coefficient ci confidence interval��p001�p005a reference high school degree101371 pone0237495t005p������female n adj r2 β ci “ “ “ “ “ “ “ “p����the participants showed a very good adherence in wearing the accelerometer device themean wearing time per day was hours only of participants had to be excluded fromthe analysis because of insufficient wearing time days and 10h per day thus the intervention in our study can be seen as feasible and practicable for this age groupthe public health relevance of pa and pi is high due to the demographic changes maintaining high levels of pa over older ages will become even more important regular pa is positively associated with several physical health outcomes besides that high levels of pa havealso the potential to maintain cognitive health over the long term into higher ages [ ] ingeneral high levels of pa can reduce incidence of dementia and cognitive restrictions significantly norton has shown that changeable risk factors such as pa and pi are responsible for around a third of alzheimer™s disease worldwide in europe usa and uk physicalinactivity was the strongest risk factor table multiple linear regression model to identify determinants for moderate pa separately for men and womenpredictorsinterceptagebmieducationa yr yrmale n adj r2 β ci “ “ “ “ “ yr œabitur qualification for the university “otherselfefficacy “ “β regression coefficient ci confidence interval��p001�p005a reference high school degree101371 pone0237495t006p�������female n adj r2 β ci “ “ “ “ “ “ “ “p���� one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable multiple linear regression model to identify determinants for pi separately for men and womenpredictorsinterceptagebmieducationa yr yrmale n adj r2 β ci “ “ “ “ “ yr œabitur qualification for the university “otherselfefficacy “ “β regression coefficient ci confidence interval��p001�p005a reference high school degree101371 pone0237495t007p��female n adj r2 β ci “ “ “ “ “ “ “ “p����effective and practicable strategies to increase pa and decrease pi are needed especially forthe elderly a report from who for the european region mentions specific suggestions toincrease peoples physical activity eg by promotion of green spaces or cycle paths etc therefore to design scale and implement effective noncommunicable disease preventionprogrammes accurate and valid data on physical activity levels and on sedentary behavior areneeded as well as valid knowledge about significant determinants of both pa and piregular pa is a highly effective healthpromoting method with strong evidence [ ] international recommendations for pa are existing for all age groups [ ] but it stillremains under debate how people should accumulate their recommended time of pa over theweek in recent years the number of interventions targeting pi has increased however there isstill a lack of recommendations for pi in addition it is also not conclusively establishedwhether pi is an independent risk factor for chronic diseases in summary recent literature points out that public health activities should emphasise increasing pa at any intensityespecially in the elderly limitationsthis analysis is based on a convenience sample of participants we used a variety of samplingmethods including the possibility of selfrecruitment some of which have likely increased theproportion of participants with above average pa compared to pa at the population level weobserved high levels of pa particularly among the older age groups and females which indicate some selection biasin general the use of accelerometer devices for objective assessment of pa allows a validand reliable record of pa intensity frequency and duration but data from the accelerometerdevice can potentially differ from the real levels of pa and pi especially in the elderly becauseseveral activities are carried out in standing eg gardening or sedentary positions eg gymnastic on stools which as a consequence can not be assessed reliablyin general the explanatory value for both pa models are acceptable the explanatory valuefor our pi model is low thus research should focus on further environmental and interpersonal factors eg the walkability of neigbourhoods and attractive activities for seniors one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivityconclusionthe number of guidelines and recommendations in pa and pi increases continuously however there are still certain aspects especially of pi which can not described in an accurate wayto our knowledge we still know only little about the independent negative health effects ofsedentary behavior in general there is international consensus regarding recommendationsfor pa but recommendations about pi are still under debatebecause of demographic change and the associated increase of proportion of older peoplethe need of global prevention strategies is high simultaneously knowledge to improve ourunderstanding of pa and pi in the elderly prevalence of dementia and chronic diseases egcvd will probably increase in the next decades therefore the relevance of modifiable riskfactors as preventive measures will rise our analyses confirm that especially individual factors eg age sex has the largest impacton pa the results for pi are less clear thus pi may have other predictors another importantfinding of this study is that pa and pi can be seen as mostly independent factors of activityparticipants with a high level of pa showed also high levels of pi further research should paymore attention to effective predictors for the reduction of pi and should focus more on environmental and interpersonal factors especially in pisupporting informations1 file datasetxlsxauthor contributionsconceptualization fabian kleinke sabina ulbricht marcus do¨rr wolfgang hoffmannneeltje van den bergdata curation neeltje van den bergformal analysis fabian kleinkemethodology wolfgang hoffmann neeltje van den bergproject administration fabian kleinkesoftware peter penndorfsupervision neeltje van den bergwriting “ original draft fabian kleinkewriting “ review editing sabina ulbricht marcus do¨rr wolfgang hoffmann neeltje vanden bergreferences australien institute of health and welfare insufficient physical activity [internet] available fromwwwaihwgovaugetmedia44533aa45704447d8d2a4d68d9fa2416insufficient physicalactivitypdfaspxinline true miles l physical activity and health br nutr found nutr bull “ allender s foster c boxer a occupational and nonoccupational physical activity and the social determinants of physical activity results from the health survey for england j phys act health jan “ 101123jpah51104 pmid lewis ba napolitano ma buman mp williams dm nigg cr future directions in physical activityintervention research expanding our focus to sedentary behaviors technology and dissemination j one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitybehav med [internet] feb “ available from 101007s10865016 pmid lollgen h lollgen d [risk reduction in cardiovascular diseases by physical activity] internist berl jan “ ahlskog je geda ye graffradford nr petersen rc physical exercise as a preventive or diseasemodifying treatment of dementia and brain aging mayo clin proc sep “ 104065mcp20110252 pmid blondell sj hammersleymather r veerman jl does physical activity prevent cognitive decline anddementia a systematic review and metaanalysis of longitudinal studies bmc public health may 101186147124581451

cancer7558

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: low levels of physical activity pa and high levels of physical inactivity pi are associatedwith higher mortality and cardiovascular diseases higher age is associated with a decreaseof pa only yearolds achieve the pa times recommended by who theaim of this study was to identify levels of and determinants for moderate pa overall pa andpi in a sample of individuals aged � yearsmethodswe analyzed baseline data from an interventionstudy aiming to increase pa and decreasepi by automatically generated feedback letters to objectively measured pa and pi recruitment was multimodal including recontacting participants of previous studies and advertisements in regional public buses and newspapers at baseline participants wore anaccelerometer over a period of consecutive days pa was categorized using cutpointssuggested by freedsoon in light moderate and vigorous physical intensity as well asphysical inactivity potential determinants selfefficacy education were measured byquestionnaires or in a physical examination bmi multiple linear regression models were fitted to identify determinants for pa and piresultsn persons mean age years sd female participated in the studythe weekly amount of overall pa for men was on average minutes sd forwomen on average minutes sd of the women and of the menachieved the who recommendation of minutes moderate paday at baseline the timeof pi during the observation time period of days was on average minutes in men anda1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation kleinke f penndorf p ulbricht s do¨rr mhoffmann w van den berg n levels of anddeterminants for physical activity and physicalinactivity in a group of healthy elderly people ingermany baseline results of the movingstudy one e0237495 101371 pone0237495editor thalia fernandez universidad nacionalautonoma de mexico mexicoreceived november accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237495copyright kleinke this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0237495 august one 0cfunding the study was funded by the federalministry of education and research bmbf as asite project of the german centre forcardiovascular research dzhk funding sign81z7400174 the funders have had no influenceon the conceptualization and conduct of the studyand will not have any role in the data analysis andpublication of the resultscompeting interests the authors have declaredthat no competing interests existlevels of and determinants for physical activity and physical inactivity minutes in women in males age was found to be a significant negative determinantfor overall pa p and for moderate pa p0001 higher education was positivelyassociated with higher levels of overall pa p and moderate pa p in menbmi was a significant negative determinant for overall pa both in men p andwomen p as well as for moderate pa for women p only in women not inmen selfefficacy was to be a significant positive determinant for overall pa p aswell as negatively associated with pi p discussionthe participants of our study showed high levels of pa this is likely due to selection bias inthis convenience sample however also levels of pi are very high and those correspondwith average levels in the german population the determinants for higher pa and lower pidiffered between males and females thus strategies for improving pa and decrease pi arelikely different with respect to sex and should take individual factors eg age bmi intoaccounttrial registration numberdrks00010410 date may introductionphysical activity pa is defined as œany bodily movement produced by skeletal muscles thatrequires energy expenditure there is strong evidence that regular pa is a very effectivenonpharmacological and noninvasive healthpromoting method [“] a papromoting lifestyle is associated with a reduced risk of mortality and is correlated with improved overallhealth status additionally high levels of pa significantly decreases overall mortality by“ and cvd mortality by “ recent reviews showed that regular pa is alsoassociated with a “ reduced risk of developing dementia in older adults [ ] and is considered as one of the proxies of the concept of cognitive reserve one of the two most significant modifiable risk factors for dementia is pi additionally pa in particular aerobicexercise is positively associated with a less agerelated reduction of anic brain matter international pa guidelines recommend for healthy individuals aged over years at least minutesweek of moderate pa or at least minutesweek of vigorous pa or an equivalent combination of weekly pa in fact there is no difference to recommendations for healthyyounger individuals aged “ years pa should be performed in uninterrupted time periodsbouts of a duration of at least minutes [ ]data from the united kingdom show that of men and of women aged “years reach the recommended level of pa a study from norway showed that of menand of women aged “ years meet the recommendations in the age group “years only of women and of men reach the whorecommendations in the usanational health and nutrition survey nhanes the proportion of people aged over years who achieved the recommended amount of pa was in germany only of men and of women aged “ years achieve the who recommendations regardingpa selfreported via questionnaire in the age group “ years this proportion declines to in men and to in women one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivityin europe in of the total population was years or older in germany of the population was years or older in this proportion will furtherincrease to until older age is associated with an higher risk for chronic diseasesmultimorbidity [“] prevalences of cvd including coronary heart disease chd andstroke will increaselow pa is one of the leading risk factors for global mortality globally of theadults are insufficiently physically active a reduction of the prevalence of insufficient pais a global target of the who [ ] physical inactivity pi defined œas any waking behaviour characterized by an energy expenditure � mets metabolic equivalent of task whilein a sitting or reclining posture sedentary behaviour research network tremblay causes million deaths per year worldwide and in was estimated to beresponsible for million dalys disabilityadjusted life years globally besides lowlevels of pa also pi is a crucial risk factor for mortality [ ] frequently interrupted piis associated with positive effects on health status and a reduced risk for premature death lack of pa and a high level of pi are associated with an array of noncommunicable diseasesncd and eg responsible for approximately “ of breast and colon cancers of diabetes cases and approximately of ischaemic heart disease prevalencesworld healthanization additionally it seems that physical inactivity is an important preventablefactor for alzheimer™s dementia older people spent on average to hours a day sedentary which correspondents with “ of their waking time depending on the exact definition the distrubution of pi acrosseuropean countries ranged from in schweden up to in portugal in germany of men and of women showed a sedentary lifestyle low energy expenditure of theleisuretime expenditure in activities requiring � metabolic equivalents met the amount of pa and pi depends on individual factors such as age bmi gender education social status and selfefficacy [“] additionally environmental and policy factors weather conditions and length of the day have an effect on the amount of pa of people[ ]overweight and obesity bmi30 kgm2 have a negative influence on the level of pa andpi in the elderly in a crosssectional study in which subjects were surveyed from member states of the european union it was found that people with a low bmi kgm2and normal bmi “ kgm2 have low prevalence in pi both genders in contrast peoplewith a bmi above kgm2 showed a more sedentary lifestyle [ ]higher education has a positive influence on pa and pi varo eu study showed thatpeople with primary level education were more sedentary than participants with higher levelsof education greater difference in women further factors that influence the amount of pa and pi are marital status income wellbeing psychosocial variables such as selfefficacy and social and cultural parameters [ ]a report from the who about the prevention of noncommunicablediseases ncd insoutheastern european countries showed that the promotion of pa and reduction of pi arekey aspects in public health efforts promoting physical activity through mass media was a primary goal for immediate action in addition the global strategy on diet physical activityon health and the european charter on counteracting obesity underline the relevance of pa to fight against obesityto develop effective prevention strategies adapted to the elderly detailed information onthe levels of pa and pi and their determinants are necessary in this analysis we assessed thelevels of pa and pi and identified positive and negative determinants for pa and pi in a sample of healthy people aged � years one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitymaterials and methodsthe data for this analysis were retrieved from the baseline assessment of an intervention studywith the goal to increase pa and reduce pi in elderly people with a lowthreshold interventionmoving“motivation oriented intervention study for the elderly in greifswald thisstudy is a twoarm randomized controlled trial consisting of assessment of eligibility baselineexamination randomization intervention only the participants in the intervention groupand followup examinations at and months after baseline the study region was western pomerania a rural area in the northeast of germanystudy participants in the intervention group received two individual feedback letters containing objectively measured pa and pi times based on data from the accelerometer devicecaptured at baseline and months followup measurement feedbackletters were automatically generated in r software version or later lucent technologies murray hill njusaa comprehensive description of the study protocol for the moving study is publishedelsewhere inclusion of the participantsthe study participants had to meet the following inclusion criteria¢ age � years¢ ability to be physically active in daily lifeexclusion criteria were¢ inability to walk independently eg permanent use of a wheelchair¢ simultaneous participation in other studies addressing pa or pi¢ not accessible by telephone or cell phone¢ already fulfilling the who recommendations for pa selfreported for people � yearsat baseline � minutes moderate pa per weekrecruitment was performed in in several ways¢ recontacting participants of a previously performed study ¢ recruitment at venues frequented by older people eg senior sport hours in the publicswimming pool rehearsals of senior choirs events at meeting places for elderly people¢ persons contacted the study centre after reading s about the project in regional newspapers and television advertisements in regional buses and flyers and posters that were distributed in gp practices hospitals and meeting centers for elderly peopleall participants were informed in detail about the study and had to give their writteninformed consentmeasuresall study participants received a baseline examination consisting of the assessment of bloodpressure somatometry data body weight waist and hip circumference as well as sociodemographics sex age education job and partnership status general health status the ssa scaleselfefficacy towards physical exercise was used to asses the participants™ level of selfefficacy one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitythe result of that scale is a sum in which higher values indicate higher selfefficacy towards pa after that the study participants received an accelerometer device actigraph gt3xbtpensacola fl usa which captures and records pa and pi continuously at a sampling frequencyof hz over a period of seven consecutive days starting at midnight after the baseline examination participants were instructed to wear the accelerometer device for seven days only during daytime on the right hip and to remove it only at bedtime and for waterbased activities egshowering swimming in addition all study participants were asked to document their physicalactivities in a semistandardized protocol for each day of the observation time besides the objective assessment of pa and pi the participants were instructed to answer paperbased questionnaires to assess selfreported pa and pi with regard to the observation period of the accelerometerto asses selfreported pa the international physical activity questionnaire short formipaqsf in german version was used the ipaq consists of seven items addressing intensity and duration of pa in daily life over the last days by selfreport in addition the german physical activity questionnaire for german paq was used to assess type andduration of pa in daily life by selfreport pi was assessed by the measure of older adults™sedentary time most questionnaire in the german version body mass index was calculated from measured body height and body weight kgm2 � kgm2 and kgm2 � kgm2after seven days the participants had to bring the accelerometer device and the fulfilledquestionnaires back to the examination centerdata analysisdata assessment and documentation were conducted based on ecrfs in an itsupported documentation system which is characterized by an independent operation synchronization andmonitoring the software is based on the concept of offline clients and each staff member ofthe dzhk had individual login data the paperbased questionnaires were documented using the software cardiff teleform1electric paper lu¨neburg germany all questionnaires and the daily physical activity protocol contained 1d barcodes to ensure anonymization of the study participantsthe actilife software versions to actigraph cop pensacola fl usawas used to download the pa data from the accelerometer the raw data were calculated into10second epochs and saved in raw format as gt3x files a valid measurement day wasdefined as a record of at least hours of total wearing time measurements of at least validdays were required to be included into data analysis to categorize pa intensity we used specific cut points based on freedson pa intensity was divided into sedentary “counts light “ counts moderate “ counts and vigorous “counts pa nonwearing time was defined as having � minutes of continual zero countsrange � minutes between and countsdescriptive statistics were used to describe the population with respect to the levels of paand pi to identify determinants of pa and pi multiple linear regression models were calculated dependent variables for the regression models were overall and moderate pa as wellas pi the effect of the independent variables age bmi education and selfefficacy was examined for all regression models all independent variables were checked for multicollinearityusing a correlation pearson to include education as a determinant in the multiple linearregression models we used dummy variables referring to the gaußmarkovtheorem we analyzed the residuals and requirements of multiple linear regression like distribution andhomoscedasticity one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitystatistical significance was assumed for pvalues data processing and statistical calculations were performed with ibm spss statistics or later “ by ibm corp armonk new york usa all statistical analysis were performed based on pseudonymizeddata all identifying data were separated from the project data to the earliest possible timepoint all appointments were ensured facetoface by the study staffethicsthe study was approved by the ethics committee of the university medicine greifswaldethic approval protocol number bb07116 and registered at the german clinical trials register id drks00010410resultsof screended study participants could be included in the analyses fig fig number of recruited participants and participants included in the analysis101371 pone0237495g001 one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable descriptive characteristics of the study sample n characteristicssex womenage yreducation yrbody mass index kgm2 years years years � � currently smoking yesnumber of participants currently having a partnership yeswearing timen number of subjects sd standard deviation101371 pone0237495t001nmean sd or n sd sd participants were included in the analysis thereof women and men the mean age was years sd at the baseline measurement of pa participants had a mean wearing time of the accelerometer of minutes sd per weekwhich corresponds to an average of hoursday table overall there were no significant difference between women and men according tooverall pa mean women minutes mean men minutes per week tdf p men in higher age groups showed lower levels of weekly light pa themean number of minutes of moderate pa increased with higher education in men and inwomen both men and women with higher bmi showed lower levels of moderate and vigorouspa table male participants spent of their waking time in pi female participants olderage was associated with an increasing time of pi in men the level of pi increased with age“ yr minutes minutes and with higher education yr minutes yr minutes in women the time of pi was largely independent of age “ yr minutes minutes table n of the men and n of the women achieved the international recommendations for moderate pa � minutes moderate pa the proportion of participantswho fulfilled the recommendations decreased with age table a multiple linear regression was calculated to predict overall pa based on independent variables in table in men lower age p a lower bmi p and higher educationp were found to be significant positive determinants for overall pa higher bmi is anegative determinant for overall pa in women p additionally selfefficacy wasfound to be a significant positive determinant for overall pa p the overall model fitwas r2 for men and r2 for women table for moderate pa higher age p0001 was found to be a significant negative determinantin men in women a higher bmi was a significant negative determinant p for maleparticipants lower education yr was a significant negative determinant p aswell as years of education p table in women better selfefficacy was found to be a significant positive determinant p for pi table one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable mean duration of weekly pa [min] without specific cut points by intensity of pa and overall pamean duration of weekly [min]mean duration of weeklylight pa ci[min] moderate pa cimean duration of weekly[min] vigorous pa mean duration of weekly [min]overall pa cicimalefemalemalefemalemalefemalemalefemaleage yr“ ™‚ ™ “ ™‚ ™ “ ™‚ ™ ™‚ ™ education yr ™‚ ™ ™‚ ™ ™‚ ™ bmi kgm2 ™‚ ™ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “� ” ™‚ ™ “ “ “ “� ™‚ ™ currently having apartnership yesyes ™‚ ™ no ™‚ ™ total ™‚ ™ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “m mean ci confidence interval min minutes101371 pone0237495t002discussionthe results of this analysis show that the levels of light moderate and overall pa in our sampleof older people are high of the male and of the female study participants fulfilledthe age specific whorecommendations for moderate pa for people aged over years� minutes moderate pa or � minutes vigorous pa especially female participants inthe age group years were above average physically active in people in the same age allwomen in this age group n reached the recommendations for moderate pathe study results show that age is a significant negative determinant for moderate and overall pa in men it can be concluded that men become more physically inactive with age this isin contrast to other studies in which women are generally less physically active than their malecounterparts [ ] this finding can also be explained by the fact that women in our sample in particular were physically active to an aboveaverage extenteducation was found to be a significant positive determinant regarding moderate pa inmen and women and overall pa in men these results are consistent with other studies people one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable mean duration of weekly pi [min] proportion of sedentary time of total wake time in minutesmean min of weekly pi cimean proportion of daily pi daytime in malefemalemalecifemaleage yr“ ™‚ ™ “ ™‚ ™ “ ™‚ ™ ™‚ ™ education yr ™‚ ™ ™‚ ™ ™‚ ™ bmi kgm2 ™‚ ™ � ™‚ ™ � ™‚ ™ currently having a partnership yesyes ™‚ ™ no ™‚ ™ total ™‚ ™ ci confidence interval min minutes101371 pone0237495t003 “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “716s “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “with higher education tend to be more physically active and generally pay more attention totheir personal health [ ]bmi was found to be a significant determinant for moderate pa in women additionallyhigher bmi in men and women was statistically significantly associated with a decreasing levelof overall pa self efficacy was a significant negative determinant for pi only in women thusfemales with higher self efficacy showed lower levels of pi there was no systematic differencebetween the gender self efficacy was the only statistically significant predictor for pi thusmotivation can be seen as an important factor to reduce piour study participants spent most of their waking time in pi men spent and female per day in pi which correspondends to results from other studies examining the sameage group [ ] in industrialized countries high pi values are therefore associated with an enormous burden on health systems table number and proportion of study participants who fulfilled the who recommendations for moderate pa separately for men and womenn of age group of all menn of age group of all womenmalefemaleage yr“ n “ n “ n n total n of of of of of of of of of of n number of subjects fulfillment of who recommendations for moderate pa for people aged and older � minutes moderate pa or � minutes vigorous pa oran equivalent combination per week101371 pone0237495t004 one 101371 pone0237495 august one 0ctable multiple linear regression model to identify determinants for overall pa separately for men and womenlevels of and determinants for physical activity and physical inactivitypredictorsinterceptagebmieducationa yr yrmale n adj r2 β ci “ “ “ “ “ yr œabitur qualification for the university “otherselfefficacy “ “β regression coefficient ci confidence interval��p001�p005a reference high school degree101371 pone0237495t005p������female n adj r2 β ci “ “ “ “ “ “ “ “p����the participants showed a very good adherence in wearing the accelerometer device themean wearing time per day was hours only of participants had to be excluded fromthe analysis because of insufficient wearing time days and 10h per day thus the intervention in our study can be seen as feasible and practicable for this age groupthe public health relevance of pa and pi is high due to the demographic changes maintaining high levels of pa over older ages will become even more important regular pa is positively associated with several physical health outcomes besides that high levels of pa havealso the potential to maintain cognitive health over the long term into higher ages [ ] ingeneral high levels of pa can reduce incidence of dementia and cognitive restrictions significantly norton has shown that changeable risk factors such as pa and pi are responsible for around a third of alzheimer™s disease worldwide in europe usa and uk physicalinactivity was the strongest risk factor table multiple linear regression model to identify determinants for moderate pa separately for men and womenpredictorsinterceptagebmieducationa yr yrmale n adj r2 β ci “ “ “ “ “ yr œabitur qualification for the university “otherselfefficacy “ “β regression coefficient ci confidence interval��p001�p005a reference high school degree101371 pone0237495t006p�������female n adj r2 β ci “ “ “ “ “ “ “ “p���� one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable multiple linear regression model to identify determinants for pi separately for men and womenpredictorsinterceptagebmieducationa yr yrmale n adj r2 β ci “ “ “ “ “ yr œabitur qualification for the university “otherselfefficacy “ “β regression coefficient ci confidence interval��p001�p005a reference high school degree101371 pone0237495t007p��female n adj r2 β ci “ “ “ “ “ “ “ “p����effective and practicable strategies to increase pa and decrease pi are needed especially forthe elderly a report from who for the european region mentions specific suggestions toincrease peoples physical activity eg by promotion of green spaces or cycle paths etc therefore to design scale and implement effective noncommunicable disease preventionprogrammes accurate and valid data on physical activity levels and on sedentary behavior areneeded as well as valid knowledge about significant determinants of both pa and piregular pa is a highly effective healthpromoting method with strong evidence [ ] international recommendations for pa are existing for all age groups [ ] but it stillremains under debate how people should accumulate their recommended time of pa over theweek in recent years the number of interventions targeting pi has increased however there isstill a lack of recommendations for pi in addition it is also not conclusively establishedwhether pi is an independent risk factor for chronic diseases in summary recent literature points out that public health activities should emphasise increasing pa at any intensityespecially in the elderly limitationsthis analysis is based on a convenience sample of participants we used a variety of samplingmethods including the possibility of selfrecruitment some of which have likely increased theproportion of participants with above average pa compared to pa at the population level weobserved high levels of pa particularly among the older age groups and females which indicate some selection biasin general the use of accelerometer devices for objective assessment of pa allows a validand reliable record of pa intensity frequency and duration but data from the accelerometerdevice can potentially differ from the real levels of pa and pi especially in the elderly becauseseveral activities are carried out in standing eg gardening or sedentary positions eg gymnastic on stools which as a consequence can not be assessed reliablyin general the explanatory value for both pa models are acceptable the explanatory valuefor our pi model is low thus research should focus on further environmental and interpersonal factors eg the walkability of neigbourhoods and attractive activities for seniors one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivityconclusionthe number of guidelines and recommendations in pa and pi increases continuously however there are still certain aspects especially of pi which can not described in an accurate wayto our knowledge we still know only little about the independent negative health effects ofsedentary behavior in general there is international consensus regarding recommendationsfor pa but recommendations about pi are still under debatebecause of demographic change and the associated increase of proportion of older peoplethe need of global prevention strategies is high simultaneously knowledge to improve ourunderstanding of pa and pi in the elderly prevalence of dementia and chronic diseases egcvd will probably increase in the next decades therefore the relevance of modifiable riskfactors as preventive measures will rise our analyses confirm that especially individual factors eg age sex has the largest impacton pa the results for pi are less clear thus pi may have other predictors another importantfinding of this study is that pa and pi can be seen as mostly independent factors of activityparticipants with a high level of pa showed also high levels of pi further research should paymore attention to effective predictors for the reduction of pi and should focus more on environmental and interpersonal factors especially in pisupporting informations1 file datasetxlsxauthor contributionsconceptualization fabian kleinke sabina ulbricht marcus do¨rr wolfgang hoffmannneeltje van den bergdata curation neeltje van den bergformal analysis fabian kleinkemethodology wolfgang hoffmann neeltje van den bergproject administration fabian kleinkesoftware peter penndorfsupervision neeltje van den bergwriting “ original draft fabian kleinkewriting “ review editing sabina ulbricht marcus do¨rr wolfgang hoffmann neeltje vanden bergreferences australien institute of health and welfare insufficient physical activity [internet] available fromwwwaihwgovaugetmedia44533aa45704447d8d2a4d68d9fa2416insufficient physicalactivitypdfaspxinline true miles l physical activity and health br nutr found nutr bull “ allender s foster c boxer a occupational and nonoccupational physical activity and the social determinants of physical activity results from the health survey for england j phys act health jan “ 101123jpah51104 pmid lewis ba napolitano ma buman mp williams dm nigg cr future directions in physical activityintervention research expanding our focus to sedentary behaviors technology and dissemination j one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitybehav med [internet] feb “ available from 101007s10865016 pmid lollgen h lollgen d [risk reduction in cardiovascular diseases by physical activity] internist berl jan “ ahlskog je geda ye graffradford nr petersen rc physical exercise as a preventive or diseasemodifying treatment of dementia and brain aging mayo clin proc sep “ 104065mcp20110252 pmid blondell sj hammersleymather r veerman jl does physical activity prevent cognitive decline anddementia a systematic review and metaanalysis of longitudinal studies bmc public health may 101186147124581451 Answer:

Colon_Cancer

open accesscitation iburg km mikkelsen l adair t lopez ad are cause of death data fit for purposeevidence from countries at different levels ofsocioeconomic development one e0237539 101371 pone0237539editor brecht devleesschauwer sciensanobelgiumreceived april accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237539copyright iburg this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement the data underlyingthe results presented in the study are available inthe who mortality database appswhointhealthinfostatisticsmortalitywhodpms with the and objectivemany countries have used the new anaconda analysis of causes of national death foraction tool to assess the quality of their cause of death data cod but no crosscountryanalysis has been done to verify how different or similar patterns of diagnostic errors anddata quality are in countries or how they are related to the local cultural or epidemiologicalenvironment or to levels of development our objective is to measure whether the usabilityof cod data and the patterns of unusable codes are related to a country™s level of socioeconomic developmentmethodswe have assessed the quality of national cod datasets from the who mortality database by assessing their completeness of cod reporting and the extent pattern and severityof garbage codes ie codes that provide little or no information about the true underlyingcod garbage codes were classified into four groups based on the severity of the error inthe code the vital statistics performance index for quality vspiq was used to measurethe overall quality of each country™s mortality surveillance systemfindingsthe proportion of ˜garbage codes™ varied from to across the countries countrieswith a high sdi generally had a lower proportion of high impact ie more severe garbagecodes than countries with low sdi while the magnitude and pattern of garbage codes differed among countries the specific codes commonly used did notsthere is an inverse relationship between a country™s sociodemographic development andthe overall quality of its cause of death data but with important exceptions in particularsome low sdi countries have vital statistics systems that are as reliable as more developedcountries however in lowincome countries where most people die at home the proportion one 101371 pone0237539 august one 0cfollowing selected variables country referenceyear icd10 code 5years age group sex andnumber of deaths data were also obtained fromthe world population prospect unpopulation division populationunwppdownloadstandardpopulation with the followingselected variables country reference year years age group sex and population numberfunding this study was funded under an awardfrom bloomberg philanthropies to the university ofmelbourne to support the data for health initiativewwwbloombergprogrampublichealthdatahealth grant number not applicablethe funders had no role in study design datacollection and analysis decision to publish orpreparation of the manuscriptcompeting interests the authors have declaredthat no competing interests existare cause of death data fit for purposeof unusable codes often exceeds implying that half of all causespecific mortality datacollected is of little or no use in guiding public policy moreover the cause of death patternidentified from the data is likely to seriously underrepresent the true extent of the leadingcauses of death in the population with very significant consequences for health priority setting garbage codes are prevalent at all ages contrary to expectations further researchinto effective strategies deployed in these countries to improve data quality can informefforts elsewhere to improve cod reporting systemsintroductiona key source of evidence for targeting health interventions to improve population health ishigh quality cause of death cod data that reliably document trends in mortality for differentdiseases and injuries however several studies have demonstrated that policy and practicein many countries are based on data that are far from accurate [“] in order to target effortsto improve the utility of cod data for policy it is important to first understand what the keydiagnostic errors area major problem with cod data is poor cause of death certification practices that result in˜garbage codes™ ie codes that provide little or no information about the true underlying causeof death garbage codes include what are often called ˜illdefined causes but encompass alarger universe of uninformative diagnoses the major consequence of garbage codes is thatthey obscure the true mortality pattern in a population for example if a death certificate onlystates septicemia as the cause of death there is no way of knowing whether this resulted forexample from an infected wound following an accident from a postoperative amputationdue to diabetes or from meningitis or pneumonia each of which would require different preventive strategies if the underlying cause that led to septicemia is not indicated on the deathcertificate public policy to prevent these deaths would be misinformed potentially leading toinefficient resource allocation to prevent themcod data provide the essential health intelligence for health policies across countries atvarious levels of socioeconomic development our premise is that a better understanding ofgarbage codes ie their levels and patterns in countries at different stages of socioeconomicdevelopment will help to target improvements in cod reporting systems in this study weinvestigate whether the usability of cod data and the patterns of garbage codes are related toa country™s socioeconomic development using the anaconda software tool [ s1 file]to assess the quality of national cod datasets several countries have used this tool to assesshow fit for purpose their data are [“] but there has not been any crosscountry analysis ofdata quality across a range of socioeconomic development levels and cod reporting systemsusing the common anaconda frameworkthe implication of our findings for public policy to improve population health is that if therelationship is found to be very weak or nonexistent then efforts to improve national civilregistration and vital statistics systems can expect to make significant progress towardsimproving the evidence base for policy without depending on further socioeconomicdevelopmentdata and methodswe carried out a crosssectional study using publicly available data from the who mortalitydatabase which contains cod data reported by its member states the countries one 101371 pone0237539 august one 0care cause of death data fit for purposewere selected on the basis that they used icd10 had provided data to who for a relatively recent year “ were located in all major regions of the world and differed in levels of socioeconomic development population data were taken from the un worldpopulation prospects with the youngest age group of “ years divided into and14years age groups using sprague™s interpolation we classified a country™s level of development using the socio demographic index sdiscore from the global burden of disease study gbd into three levels high middle and lowthe sdi is a summary measure of development expressed on a scale from to taking intoaccount the total fertility rate years of schooling and gross national income for the lowestsdi level the who mortality database only contained the few countries we selected for themiddle and high sdi levels we selected countries with recent data from different regions of theworld our study included low sdi countries middle sdi and high sdi countrieson the basis of the country specific icd10 codes used in gbd the most severecertification and coding errors that can mislead policy and public health planning were identified and categorized into four groups the fourtier garbage code typology used in anaconda is based on the premise that some garbage codes are worse than others depending onhow serious their impact is for guiding or misguiding policy debates and will thus likely impactdisease and injury control strategies differently ¢ level very high“codes with serious policy implications these are causes for which thetrue underlying cod could in fact belong to any of three broad cause group ie it is impossible to establish whether the true cause was a communicable disease a noncommunicabledisease or because of an injury a good example being ˜septicaemia™ reported as the underlying cause of death these are the most serious misdiagnoses among the universe of unusable codes since they could potentially grossly misinform understanding of the extent ofepidemiological transition in the population¢ level high“codes with substantial implications for policy these are causes for whichthe true underlying cod is likely to belong to one or two of the three broad cause groupsfor example ˜essential primary hypertension™¢ level medium“codes with important implications for policy these are causes forwhich the true underlying cod is likely to be within the same icd chapter for example˜unspecified cancer™ and thus are of some policy value¢ level low“codes with limited implications for policy these are diagnoses for whichthe true underlying cod is likely to be confined to a single disease or injury category egunspecified stroke would still be assigned as a stroke death and not to some other diseasecategory the implications of unusable causes classified at this level will therefore be muchless important for public policy but a more specific code would have increased their utilityfor specific public health analysesa full list of the composition of specific icd10 garbage codes for each of the four severitylevels is given in s2 filegiven the considerable differences in population age structure between countries at highand low levels of socioeconomic development we age standardised the pattern of garbagecodes the point of agestandardising was to investigate whether countries with a comparatively old age structure and hence relatively high average age at death might expect to have agreater fraction of garbage codes simply because of the higher likelihood of multiple comorbidity in the elderly we used the global age distribution of deaths from the latest gbd studyas the standard one 101371 pone0237539 august one 0care cause of death data fit for purposein addition to diagnostic accuracy the ability of any dataset to describe the true mortalitypattern in a population also depends on how complete it is both in terms of capturing alldeaths that occur and in assigning each a cod completeness of the cod reporting ie thepercentage of actual deaths in a population that are assigned a cod for each of the countries was calculated using the adairlopez empirical method incorporated into anaconda the empirical method models the relationship between the crude death rate cdr andits principal determinants namely the age structure of the population and the overall level ofmortality as reflected by the level of child mortality the predicted cdr based on these inputvariables for a population is then compared with the observed cdr to estimate death registration completeness given that the model was built largely from historical data where the levelsof adult mortality and child mortality are closely correlated the predictions of completenessfor populations where this assumption is not valid such as those severely affected by hivshould be interpreted cautiouslydatasets that are both incomplete and have a high proportion of garbage codes provide limited insight into the true health status of a population we combined the proportion of unrecorded deaths with the amount of garbage codes to provide a summary measure of the utilityof the data for policy this indicator is particularly important when investigating data qualityin countries with low completeness where the data available may only come from hospitalsand other health facilities where diagnostic facilities and physician availability is greaterpotentially overstating the policy utility of the dataa key output of any mortality surveillance system is a table showing the leading causes ofdeath for the population in countries where garbage codes are commonly assigned they frequently appear among the or leading causes and can seriously impact the overall utilityof the cod data this is particularly the case when they permeate the top leading causesand are œhigh impact providing little or no useful information for policythe anaconda software tool specifically developed for assessing quality of mortalityand cod data was used to investigate each dataset s1 file to identify the pattern and extentof garbage codes in the data their frequency among the leading causes the completeness ofthe dataset and to provide an overall summary index of the quality of the output of the mortality data system namely the vital statistics performance index for quality vspiq resultsthe proportion of garbage codes in the country datasets varied substantially by countryranging from to see fig while the relationship between sdi and amount of garbage codes in the data is broadly apparent the relatively low r2 arises from the presenceof outliers particularly uzbekistan kyrgyzstan nicaragua and colombia it is quite possiblethat specific certification and coding procedures have been introduced in these countries toavoid the use of garbage codes if these countries are omitted the strength of the inverse relationship is much more apparent further insights into the general characteristics of populationand mortality of the selected countries can be found in s2 filethe mean values for each sdi group indicate that on average countries of high sdi statushad a lower proportion of garbage codes in their data than middle and low sdi countries table interestingly there was large intercountry variation in theuse of garbage codes within each sdi level for example of the high sdi countries finlandhad the lowest amount of garbage codes in their data whereas in japan and france the level was five times higher affecting about one in three deaths for the middle sdigroup argentina thailand and tunisia had more than half of all cods coded to a garbagecode in thailand close to of these were high impact errors while for other countries in one 101371 pone0237539 august one 0care cause of death data fit for purposefig percentage of total garbage codes versus socio demographic index selected countries high sdi middle sdi low sdi101371 pone0237539g001this group notably uzbekistan turkey and colombia the use of garbage codes was much lessprominent surprisingly among the low sdi countries kyrgyzstan and nicaragua had comparatively low levels of garbage and respectively whereas in egypt at a similarsdi level twothirds of all deaths are coded to garbage codes and of these over were ofhigh impact table importantly the fraction of high impact garbage codes ranged from a low of finlandto egypt and for low impact codes from finland to brazil countries withhigh sociodemographic development had a lower proportion of high impact garbage codesmean compared with middle and low sdi countries table rankingcountries according to their percent of high impact garbage codes reveals a very substantialgap between the best and worst performing cod information systems and a surprising mixture of sdi levels fig kyrgyzstan has the secondbest performing system after finlandwith cod data in colombia of almost equal quality to the uk and that in nicaragua fallingbetween australia and canada uzbekistan turkey brazil and jordan all assign less causes tohigh impact garbage codes than both japan and francethe impact of population age structure on the overall level of garbage codes varied acrosscountries but was generally small contrary to what might have been expected table injapan and argentina the ageadjusted fraction of garbage codes was “ points lower than one 101371 pone0237539 august one 0ctable levels of garbage codes standardised by age and registration completeness for select countries ranked by their sociodemographic index sdiare cause of death data fit for purposecountryfinlandcanadaaustraliajapanfranceyearsdivalueunited kingdom high sdimeanturkeyargentinairanjordanthailandsouth africatunisiabrazilcolombiauzbekistanmiddle sdimeankyrgyzstanegyptnicaraguatajikistanlow sdi meantotal meansdi levelshigh impact gclow impact gca total gc ageadjb completeness of codc deaths of no policygc registration value sdi three levels collapsed from gbd five sdi levelshigh highmiddle high middle middlelow low low middlegarbage code gc levelshigh impact gc levels “low impact gc level total gc high lowdeath of no value for policyc 1b a�bthis equation calculates unavailable deaths for policy ie unregistered deaths plus deaths with a garbage code101371 pone0237539t001the unadjusted fraction while in france and south africa it increased by a similar amountcolumn a agestandardisation therefore had no impact on the mean level of garbage codesin each development categoryagestandardisation however masks the age pattern of garbage coding particularly its relative importance at younger adult ages where accurate and specific diagnoses are critical fuiding policies designed to prevent premature deaths the perception that garbage codes arelargely confined to deaths among the elderly due to the presence of comorbidities at oraround the time of death is not confirmed by the agespecific fractions of garbage codes shown one 101371 pone0237539 august one 0care cause of death data fit for purposefig percentage of high impact garbage codes in total causes of death selected countries c high sdi middlesdi low sdi101371 pone0237539g002in fig with exact fractions reported in s3 file garbage codes are prevalent at all ages andoften in similar proportions to what is observed for the age group indeed in some highlydeveloped countries eg finland canada uk the proportion of garbage codes is significantly higher at ages “ for both sexes than at older ages indeed in tunisia for males andfig garbage codes as a percentage of all deaths by age selected countries101371 pone0237539g003 one 101371 pone0237539 august one 0care cause of death data fit for purposein jordan and kyrgyzstan for both sexes this pattern is already evident from age in uzbekistan garbage coding is more common for deaths of children and adolescents than at ages and abovegiven that the utility of a country™s mortality information system is reduced not only by theamount of garbage codes but also by how complete it is in terms of capturing all deaths wemeasured their combined impact for two countries namely jordan and tajikistan the consolidated indicator of registration completeness and fraction of garbage codes was close to suggesting that useful information on only about one quarter of all deaths that occur injordan and tajikistan is available for policy purposes col c table overall the combinedindicator showed the proportion of deaths for which information was either missing or of littleor no value for guiding health policy was much higher in low and middle sdicountries compared to high sdi countriestable shows the strong influence of garbage codes on the leading cause distribution whenthese are ranked with high impact garbage codes marked in red and low impact in orangethe presence of high impact garbage codes among the leading causes of death will substantially distort the true picture of what are the common cod that most people die from amonghigh sdi countries only japan and france have high impact garbage codes for males amongthe ten leading causes of death canada australia and uk had only low impact causes in thiscategory and finland had neither for the middle and low sdi countries there were manymore high impact garbage codes listed among the leading cod with egypt having seventajikistan five and thailand and tunisia each with four colombia and nicaragua had nonefor females egypt had seven iran tunisia and tajikistan five with the remaining countrieshaving between one and threenotwithstanding some variation in patterns and volume of garbage coding across countriesthe most common garbage codes were remarkably similar in particular other illdefined andunspecified causes of death senility heart failure unspecified neoplasm septicemia respiratory failure unknown cause of death hypertension and unspecified diabetes were observedacross all sdi levels in addition low sdi countries tended to report atherosclerosis hepaticfailure intracerebral hemorrhage and unattended deaths all garbage codes as leading causesfig ranks countries according to a single consolidated summary measure of system performance namely the vital statistics performance index for quality vspiq eleven countries scored and above a level where they could be considered as having wellfunctioningsystems six of the remaining countries achieved scores that would classify them as havingmedium performing systems with lower scores mostly arising from the high proportion ofgarbage codes that bias their cod distributions of the countries only jordan tajikistanand tunisia were classified as having poorly functioning systemsdiscussionin general one would expect that in high sdi countries where all deaths are medically certified with good quality clinical and diagnostic services comparatively few deaths would beassigned unusable garbage codes and certainly much less than in countries where such services are less common our findings based on an analysis of cod datasets from across theworld produced evidence both for and against this hypothesis france and japan do not seemto have more reliable cod data to guide policy than turkey colombia kyrgyzstan and nicaragua in france in deaths is assigned a garbage code with of all deaths being certifiedas due to œother illdefined and unspecified causes of death r99 heart failure i509 orrespiratory arrest r092 similarly in japan œold age senility r54 is a commonly assignedcause of death accounting for one fifth of all garbage codes other research has found that one 101371 pone0237539 august one 0cdeificepsnuesaesidtraehnoitcrafnilarberecdeificepsnusaercnapainomuenpdeificepsnuevitcurtsboyranomuplhtiwesaesidrewoletucayrotaripsernoitcefnideificepsnulnoocyrtnuocknarselamwolleynisenotcapmiwoldernidekramsedocegabragtcapmihgihxesdnayrtnuocybhtaedfosesuacgnidaelpotelbatcimeahcsicinorhcfoealeuqescinorhcsisohrriccilohoclarevilfoebolreppurosuhcnorbs™remiehzlaesaesidgnuldeificepsnulaidracoymnoitcrafnideificepsnuetatsorptesnofomsalpoenetalhtiwesaesidesaesidtraehetucatnangilams™remiehzlacitorelcsorehtadnalnifgnignahybmrahdnanoitalugnartsfleslanoitnetniecalpdeificepsnunoitacoffusdeificepsnusaercnaptonekortssadeificepsegahrromeahnoitcrafnirocitorelcsorehtaesaesidtraehnoitcrafnilarberecfoealeuqesllecrevileudsitinomuenpdeificepsnunoitcrafnilarberecamonicractimovdnadoofotevitcurtsboyranomuplesaesiddeificepsnulaidracoymnoitcrafnideificepsnuetucaaitnemedfomsalpoentraehcimeahcsietatsorpdeificepsnuesaesidytilineseruliaftraehdeificepsnudeificepsnuhcamotslaidracoymnoitcrafnideificepsnurosuhcnorbdeificepsnugnuldeificepsnugnulainomuenpdeificepsnunapajcinorhcdeificepsnutnangilamcinorhcetucarosuhcnorbneilartsuaainomuenpohcnorbdeificepsnutonekortssadeificepsainomuenpdeificepsnuroegahrromeahnoitcrafniesaesidyranomuplrewoletucahtiwyrotaripsernoitcefnietatsorpdeificepsnuesaesidevitcurtsboesaesidtraehfomsalpoenaitnemedtraehcimeahcsilaidracoymnoitcrafnideificepsnudeificepsnugnulmodgnikcinorhccitorelcsorehtatnangilamdeificepsnucinorhcetucarosuhcnorbdetinudeificepsnusaercnapdeificepsnudeificepsnuesaesidesaesiddeificepsnudeificepsnutraehcimeahcsitserralaidracoymnoitcrafnideificepsnufomsalpoendeificepsnudnaetatsorpfosesuacytilatromdeificepsnugnuls™remiehzlalnooceruliaftraehcinorhcyrotaripseretucatnangilamdenifedllirehtorosuhcnorbecnarfcinorhcralucsavorbereccitorelcsorehtacinorhceruliaftraehrosuhcnorbetucayekrutesaesidyranomuplroegahrromeahdeificepsnunoitcrafnievitcurtsbocinorhctonekortssadeificepsfomsalpoentnangilametatsorpdeificepsnucitorelcsorehtaaitnemedesaesidtraehlaidracoymnoitcrafnideificepsnudeificepsnugnuletucarosuhcnorbadanacdeificepsnulnoocdeificepsnuesaesidyranomupletatsorpevitcurtsbofomsalpoencinorhctnangilamrewoletucahtiwyrotaripsernoitcefniaimeacitpesdeificepsnuroegahrromeahnoitcrafnitonekortssadeificepsesaesids™remiehzlatesnoetalhtiwdeificepsnuhcamotsainomuenpdeificepsnuesaesidyranomupldeificepsnuevitcurtsboesaesidesaesidtraehevitcurtsboyranomuplesaesiddeificepsnudeificepsnudeificepsnugnullaidracoymnoitcrafnideificepsnudnadenifeddeificepsnufosesuacytilatromdeificepsnugnuldeificepsnullirehtorosuhcnorberuliaftraehetucaainomuenpanitnegraare cause of death data fit for purposeesaesidtraehdnasuhcnorbfodenifedllifomsalpoenesaesidtraehcimeahcsignulfosnoitpircsedhcamotsesaesidsetebaidsutillemdeunitnoceruliaftraehdeificepsnuainomuenpcitorelcsorehtayramirplaitnesserosuhcnorblarberecdeificepsnudeificepsnuesaesidtraehnoisnetrepyhdeificepsnugnulnoitcrafnideificepsnusnoitacilpmocsetebaidsutillemtuohtiwdeificepsnudnaevisnetrepyhesaesidtraehevitsegnoceruliaftraehhtiwfosesuacytilatrommsalpoentnangilamdnasnoitacilpmoctnangilamtraehevisnetrepyhcinorhcdeificepsnutserracaidracdenifedllirehtolaidracoymnoitcrafnideificepsnunoitcrafnilarberecdeificepsnutropsnarttnediccadeificepsnulaidracoymnoitcrafnideificepsnulaidracoymnoitcrafnietucanarietucanadroj one 101371 pone0237539 august one 0cniderunjinosrepnoitcrafnilarberecytilinestserrayrotaripserdeificepsnudeificepsnutnetniidenmretednuesaesidtraehtnevedeificepsnucimeahcsicinorhclaidracoymnoitcrafnietucalarberecartniegahrromeaheruliaftraehdnaaeohrraidlarivrehtonamuhfositiretneortsagtonsesaesidycneicifedonummifomsalpoentnangilamdnasuhcnorbgnultonekortssadeificepsnigirosuoitcefnideifissalcsesaesidcitisarapdnasuoitcefninignitlusernoitcrafnidemuserperehwesleesaesid]ivh[surivroegahrromeahaimeacitpesrehtofomsalpoentnangilamcitapehartnistcudelibdnarevilytilinesainomuenpdeificepsnumsinagrooterusopxedeificepsnurotcafdeificepsnuainomuenpyrotaripsersetebaidsutillemdeificepsnudemrifnocmsinagrotonlsisoucrebutlyllacigooiretcablyllacigootsihrodeunitnocelbatllirehtodnaliahtdnadenifeddeificepsnufosesuacytilatromdnadenifeddeificepsnullirehtofosesuacytilatromhtuosacirfarotomdeificepsnudeificepsnuoteudtnediccaelcihevronoisulccociffartlarberecfosisonetsseiretrahtaeddednettanuyramirplaitnessedeificepsnunoisnetrepyhsnoitacilpmocsetebaidsutillemtuohtiwetatsorpdeificepsnudeificepsnuegahrromeahmraerifnoitcrafnirofosesuacytilatromfomsalpoentnangilamrosuhcnorbgnulybtluassadnarehtoecalptonekortsdenifedllirehtosadeificepsdeificepsnudnaainomuenpdeificepsnulaidracoymnoitcrafnideificepsnuetucalizarbsutillemsetebaidtnevesnoitacilpmocdeificepsnurehtohtiwidenmretednuegahrromeahdeificepstnetninoitcrafnirotonekortssadeificepslaidracoymnoitcrafnideificepsnudeificepsnugnuldnadenifeddeificepsnufosesuacytilatrometucarosuhcnorbllirehtoaisinutdeificepsnueruliafdeificepsnudnagnullanercinorhcrehtoybtluassarosuhcnorbevitcurtsbocinorhcegrahcsidmraerifdeificepsnuesaesidyranomuplfomsalpoentnangilametatsorpsisorelcsorehtaeruliaftraehecalpdeificepsnutonekortssadeificepsegahrromeahnoitcrafnirorewoletucahtiwyrotaripsernoitcefnitraehcimeahcsietucarehtosesaesidlarberecartniegahrromeahgnignahybmrahdnanoitalugnartsemohnoitacoffusfleslanoitnetnideificepsnutraehcimeahcsiralucsavoidracdeificepsnuesaesidosesaesiddebircsedevitcurtsboyranomuplcinorhcesaesidhcamotscinorhccitorelcsorehtadeificepsrehtoegrahcsiddnateertsyawhgihdeificepsnuhcamotsdnasisorbiffosisohrricrevildnadenifeddeificepsnullirehtofosesuacytilatromainomuenpdeificepsnuevitcurtsbodeificepsnudnayranomuplmraerifesaesidteertsegrahcsiddeificepsnuyawhgihdnalaidracoymnoitcrafnideificepsnucinorhcrehtoybtluassaetucaaibmoloclaidracoymnoitcrafniesaesidtraehtraehcimeahcsiesaesidetucasirotcepanignaevisnetrepyhcinorhcnatsikebzunoitcrafnirevilfosisohrricroegahrromeahdeificepsnunoitcrafnilaidracoymetucadeificepsnudnarehtosadeificepsesaesidtraehtonekortscitorelcsorehtanatszygrykare cause of death data fit for purposemsalpoentnangilamnilusninonetatsorpfosetebaidtnednepedlaidracoymnoitcrafnifosisohrriccilohoclalanerhtiwsutillemsnoitacilpmocrevilrevilfosisohrricerehwesletonegahrromeaherehwesletonfosisohrricdeificepsnudeifissalcdnarehtoderunjinosrepdeificepsnunielcihevrotomciffarttnediccaevitcurtsboyranomuplcinorhcesaesiddeificepsnudeifissalctonekortssadeificepsegahrromeahnoitcrafnirorevilainomuenpdeificepsnusisorelcsorehtaytilinesetucaeruliafyrotaripserlarberecartnieruliafcitapehdnasisorbiftserracaidracnoisnetrepyhlanercinorhcdeificepsnueruliafyramirplaitnesseeruliaftraehtpygelaidracoymnoitcrafnideificepsnuetucaaugaracintraehfosnoitpircsedesaesidesaesidfosesuacytilatromnoitcrafnisesaesidroegahrromeahnoisnetrepyhnoitcrafnidenifedllisutillemsetebaidtraehcimeahcsideificepsnudnalaidracoymtraehcimeahcsidnasnoitacilpmocdeificepsnucinorhcsisorelcsorehtadenifedllirehtoetucaetucarehtoytilinestonekortssadeificepsyramirplaitnessenatsikijatdeunitnocknarselamefyrtnuoc one 101371 pone0237539 august one 0clarberecfoealeuqess™remiehzlarehtolarbereccimeahcsicinorhcnoitcrafniesaesidnoitcrafnideificepsnuesaesidtraehdeificepsnudeificepsnuaitnemedlaidracoymnoitcrafnideificepsnuevitsegnoceruliaftraehhtiwdeificepsnuesaesidtraehesaesidesaesidtraehetucaevisnetrepyhs™remiehzlacitorelcsorehtas™remiehzlahtiwesaesidtesnoetaldnalnifdeunitnocelbatdeificepsnulnoocainomuenpdeificepsnudeificepsnutsaerbdeificepsnuhcamotsevitcurtsbocinorhcaitnemedralucsavesaesidyranomuplrewoletucahtiwnoitcefniyrotaripserdeificepsnudeificepsnurettulflairtadnasaercnapnoitallirbiflairtadeificepsnuevitcurtsboyranomuplcinorhcesaesiddeificepsnuevitcurtsboyranomuplcinorhcesaesiddeificepsnulaidracoymnoitcrafnideificepsnuetucaainomuenpdeificepsnus™remiehzlaesaesidtonekortssadeificepsdeificepsnuroegahrromeahnoitcrafnideificepsnuesaesidtraehlaidracoymnoitcrafnideificepsnudeificepsnugnulaitnemedtsaerbcitorelcsorehtaetucarosuhcnorbdeificepsnuadanacs™remiehzladeificepsnuesaesidnoitcrafnideificepsnularberecs™remiehzladeificepsnuesaesiddeificepsnusadeificepstraehcimeahcsignulegahrromeahnoitcrafnirodeificepsnuesaesiddeificepsnulaidracoymnoitcrafnideificepsnuaitnemedtsaerbtonekortscinorhcrosuhcnorbetucadeificepsnuneilartsuanoitcrafnitimovdeificepsnufoealeuqessitinomuenprosuhcnorberuliaftraehlarberecdnadoofoteudgnuldeificepsnuainomuenpdeificepsnuytilinesnapajlaidracoymnoitcrafnideificepsnuetucatonekortssadeificepsegahrromeahnoitcrafnirodeificepsnuesaesiddeificepsnudeificepsnutraehcimeahcsignulaitnemedmodgniktsaerbcinorhcrosuhcnorbdeificepsnudetinudeificepsnulnooclaidracoymetucatonekortstserrayrotaripsereruliaftraehrosuhcnorbdeificepsnudenifedllirehtotsaerbs™remiehzlaecnarfare cause of death data fit for purposetraehfosnoitpircseddenifedlliesaesiddnadenifeddeificepsnufosesuacytilatromesaesidnoisnetrepyhtraehcimeahcsiesaesidtraehsutillemsetebaiddnasnoitacilpmocytilinesllirehtoyramirplaitnessecinorhctserracaidracevisnetrepyhdeificepsnunoitcrafnilarbereclaidracoymnoitcrafnietucanarideificepsnutraehfonoitamroflamesaesidtraehdeificepsnulatinegnoccitorelcsorehtaeruliaftraehainomuenpdeificepsnudeificepsnutsaerbnoisnetrepyhyramirplaitnesselaidracoymsutillemsetebaidnoitcrafnideificepsnusnoitacilpmoctuohtiwetucadeificepsnuevisnetrepyhesaesidtraehevitsegnoceruliaftraehhtiwnoitcrafnideificepsnularberecnadrojdeunitnoclarberecartniegahrromeahgnuldnasuhcnorbcitapehartnistcudelibdnarevilmetsyssuovrenerehwesletonfosesaesiddeifissalcfomsalpoenfomsalpoeneruliafevitarenegedsetebaidsutillemtnangilamtnangilamlanercinorhcrehtodeificepsnuaimeacitpesrehtoainomuenpdeificepsnumsinagroytilinesllirehtodnaliahtdnadenifeddeificepsnufosesuacytilatromdeificepsnulnooceruliafyrotaripserrosuhcnorbdeificepsnugnultonekortssadeificepsaimeacitpesdeificepsnudeificepsnuroegahrromeahnoitcrafnidnadenifeddeificepsn

cancer7559

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: open accesscitation iburg km mikkelsen l adair t lopez ad are cause of death data fit for purposeevidence from countries at different levels ofsocioeconomic development one e0237539 101371 pone0237539editor brecht devleesschauwer sciensanobelgiumreceived april accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237539copyright iburg this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement the data underlyingthe results presented in the study are available inthe who mortality database appswhointhealthinfostatisticsmortalitywhodpms with the and objectivemany countries have used the new anaconda analysis of causes of national death foraction tool to assess the quality of their cause of death data cod but no crosscountryanalysis has been done to verify how different or similar patterns of diagnostic errors anddata quality are in countries or how they are related to the local cultural or epidemiologicalenvironment or to levels of development our objective is to measure whether the usabilityof cod data and the patterns of unusable codes are related to a country™s level of socioeconomic developmentmethodswe have assessed the quality of national cod datasets from the who mortality database by assessing their completeness of cod reporting and the extent pattern and severityof garbage codes ie codes that provide little or no information about the true underlyingcod garbage codes were classified into four groups based on the severity of the error inthe code the vital statistics performance index for quality vspiq was used to measurethe overall quality of each country™s mortality surveillance systemfindingsthe proportion of ˜garbage codes™ varied from to across the countries countrieswith a high sdi generally had a lower proportion of high impact ie more severe garbagecodes than countries with low sdi while the magnitude and pattern of garbage codes differed among countries the specific codes commonly used did notsthere is an inverse relationship between a country™s sociodemographic development andthe overall quality of its cause of death data but with important exceptions in particularsome low sdi countries have vital statistics systems that are as reliable as more developedcountries however in lowincome countries where most people die at home the proportion one 101371 pone0237539 august one 0cfollowing selected variables country referenceyear icd10 code 5years age group sex andnumber of deaths data were also obtained fromthe world population prospect unpopulation division populationunwppdownloadstandardpopulation with the followingselected variables country reference year years age group sex and population numberfunding this study was funded under an awardfrom bloomberg philanthropies to the university ofmelbourne to support the data for health initiativewwwbloombergprogrampublichealthdatahealth grant number not applicablethe funders had no role in study design datacollection and analysis decision to publish orpreparation of the manuscriptcompeting interests the authors have declaredthat no competing interests existare cause of death data fit for purposeof unusable codes often exceeds implying that half of all causespecific mortality datacollected is of little or no use in guiding public policy moreover the cause of death patternidentified from the data is likely to seriously underrepresent the true extent of the leadingcauses of death in the population with very significant consequences for health priority setting garbage codes are prevalent at all ages contrary to expectations further researchinto effective strategies deployed in these countries to improve data quality can informefforts elsewhere to improve cod reporting systemsintroductiona key source of evidence for targeting health interventions to improve population health ishigh quality cause of death cod data that reliably document trends in mortality for differentdiseases and injuries however several studies have demonstrated that policy and practicein many countries are based on data that are far from accurate [“] in order to target effortsto improve the utility of cod data for policy it is important to first understand what the keydiagnostic errors area major problem with cod data is poor cause of death certification practices that result in˜garbage codes™ ie codes that provide little or no information about the true underlying causeof death garbage codes include what are often called ˜illdefined causes but encompass alarger universe of uninformative diagnoses the major consequence of garbage codes is thatthey obscure the true mortality pattern in a population for example if a death certificate onlystates septicemia as the cause of death there is no way of knowing whether this resulted forexample from an infected wound following an accident from a postoperative amputationdue to diabetes or from meningitis or pneumonia each of which would require different preventive strategies if the underlying cause that led to septicemia is not indicated on the deathcertificate public policy to prevent these deaths would be misinformed potentially leading toinefficient resource allocation to prevent themcod data provide the essential health intelligence for health policies across countries atvarious levels of socioeconomic development our premise is that a better understanding ofgarbage codes ie their levels and patterns in countries at different stages of socioeconomicdevelopment will help to target improvements in cod reporting systems in this study weinvestigate whether the usability of cod data and the patterns of garbage codes are related toa country™s socioeconomic development using the anaconda software tool [ s1 file]to assess the quality of national cod datasets several countries have used this tool to assesshow fit for purpose their data are [“] but there has not been any crosscountry analysis ofdata quality across a range of socioeconomic development levels and cod reporting systemsusing the common anaconda frameworkthe implication of our findings for public policy to improve population health is that if therelationship is found to be very weak or nonexistent then efforts to improve national civilregistration and vital statistics systems can expect to make significant progress towardsimproving the evidence base for policy without depending on further socioeconomicdevelopmentdata and methodswe carried out a crosssectional study using publicly available data from the who mortalitydatabase which contains cod data reported by its member states the countries one 101371 pone0237539 august one 0care cause of death data fit for purposewere selected on the basis that they used icd10 had provided data to who for a relatively recent year “ were located in all major regions of the world and differed in levels of socioeconomic development population data were taken from the un worldpopulation prospects with the youngest age group of “ years divided into and14years age groups using sprague™s interpolation we classified a country™s level of development using the socio demographic index sdiscore from the global burden of disease study gbd into three levels high middle and lowthe sdi is a summary measure of development expressed on a scale from to taking intoaccount the total fertility rate years of schooling and gross national income for the lowestsdi level the who mortality database only contained the few countries we selected for themiddle and high sdi levels we selected countries with recent data from different regions of theworld our study included low sdi countries middle sdi and high sdi countrieson the basis of the country specific icd10 codes used in gbd the most severecertification and coding errors that can mislead policy and public health planning were identified and categorized into four groups the fourtier garbage code typology used in anaconda is based on the premise that some garbage codes are worse than others depending onhow serious their impact is for guiding or misguiding policy debates and will thus likely impactdisease and injury control strategies differently ¢ level very high“codes with serious policy implications these are causes for which thetrue underlying cod could in fact belong to any of three broad cause group ie it is impossible to establish whether the true cause was a communicable disease a noncommunicabledisease or because of an injury a good example being ˜septicaemia™ reported as the underlying cause of death these are the most serious misdiagnoses among the universe of unusable codes since they could potentially grossly misinform understanding of the extent ofepidemiological transition in the population¢ level high“codes with substantial implications for policy these are causes for whichthe true underlying cod is likely to belong to one or two of the three broad cause groupsfor example ˜essential primary hypertension™¢ level medium“codes with important implications for policy these are causes forwhich the true underlying cod is likely to be within the same icd chapter for example˜unspecified cancer™ and thus are of some policy value¢ level low“codes with limited implications for policy these are diagnoses for whichthe true underlying cod is likely to be confined to a single disease or injury category egunspecified stroke would still be assigned as a stroke death and not to some other diseasecategory the implications of unusable causes classified at this level will therefore be muchless important for public policy but a more specific code would have increased their utilityfor specific public health analysesa full list of the composition of specific icd10 garbage codes for each of the four severitylevels is given in s2 filegiven the considerable differences in population age structure between countries at highand low levels of socioeconomic development we age standardised the pattern of garbagecodes the point of agestandardising was to investigate whether countries with a comparatively old age structure and hence relatively high average age at death might expect to have agreater fraction of garbage codes simply because of the higher likelihood of multiple comorbidity in the elderly we used the global age distribution of deaths from the latest gbd studyas the standard one 101371 pone0237539 august one 0care cause of death data fit for purposein addition to diagnostic accuracy the ability of any dataset to describe the true mortalitypattern in a population also depends on how complete it is both in terms of capturing alldeaths that occur and in assigning each a cod completeness of the cod reporting ie thepercentage of actual deaths in a population that are assigned a cod for each of the countries was calculated using the adairlopez empirical method incorporated into anaconda the empirical method models the relationship between the crude death rate cdr andits principal determinants namely the age structure of the population and the overall level ofmortality as reflected by the level of child mortality the predicted cdr based on these inputvariables for a population is then compared with the observed cdr to estimate death registration completeness given that the model was built largely from historical data where the levelsof adult mortality and child mortality are closely correlated the predictions of completenessfor populations where this assumption is not valid such as those severely affected by hivshould be interpreted cautiouslydatasets that are both incomplete and have a high proportion of garbage codes provide limited insight into the true health status of a population we combined the proportion of unrecorded deaths with the amount of garbage codes to provide a summary measure of the utilityof the data for policy this indicator is particularly important when investigating data qualityin countries with low completeness where the data available may only come from hospitalsand other health facilities where diagnostic facilities and physician availability is greaterpotentially overstating the policy utility of the dataa key output of any mortality surveillance system is a table showing the leading causes ofdeath for the population in countries where garbage codes are commonly assigned they frequently appear among the or leading causes and can seriously impact the overall utilityof the cod data this is particularly the case when they permeate the top leading causesand are œhigh impact providing little or no useful information for policythe anaconda software tool specifically developed for assessing quality of mortalityand cod data was used to investigate each dataset s1 file to identify the pattern and extentof garbage codes in the data their frequency among the leading causes the completeness ofthe dataset and to provide an overall summary index of the quality of the output of the mortality data system namely the vital statistics performance index for quality vspiq resultsthe proportion of garbage codes in the country datasets varied substantially by countryranging from to see fig while the relationship between sdi and amount of garbage codes in the data is broadly apparent the relatively low r2 arises from the presenceof outliers particularly uzbekistan kyrgyzstan nicaragua and colombia it is quite possiblethat specific certification and coding procedures have been introduced in these countries toavoid the use of garbage codes if these countries are omitted the strength of the inverse relationship is much more apparent further insights into the general characteristics of populationand mortality of the selected countries can be found in s2 filethe mean values for each sdi group indicate that on average countries of high sdi statushad a lower proportion of garbage codes in their data than middle and low sdi countries table interestingly there was large intercountry variation in theuse of garbage codes within each sdi level for example of the high sdi countries finlandhad the lowest amount of garbage codes in their data whereas in japan and france the level was five times higher affecting about one in three deaths for the middle sdigroup argentina thailand and tunisia had more than half of all cods coded to a garbagecode in thailand close to of these were high impact errors while for other countries in one 101371 pone0237539 august one 0care cause of death data fit for purposefig percentage of total garbage codes versus socio demographic index selected countries high sdi middle sdi low sdi101371 pone0237539g001this group notably uzbekistan turkey and colombia the use of garbage codes was much lessprominent surprisingly among the low sdi countries kyrgyzstan and nicaragua had comparatively low levels of garbage and respectively whereas in egypt at a similarsdi level twothirds of all deaths are coded to garbage codes and of these over were ofhigh impact table importantly the fraction of high impact garbage codes ranged from a low of finlandto egypt and for low impact codes from finland to brazil countries withhigh sociodemographic development had a lower proportion of high impact garbage codesmean compared with middle and low sdi countries table rankingcountries according to their percent of high impact garbage codes reveals a very substantialgap between the best and worst performing cod information systems and a surprising mixture of sdi levels fig kyrgyzstan has the secondbest performing system after finlandwith cod data in colombia of almost equal quality to the uk and that in nicaragua fallingbetween australia and canada uzbekistan turkey brazil and jordan all assign less causes tohigh impact garbage codes than both japan and francethe impact of population age structure on the overall level of garbage codes varied acrosscountries but was generally small contrary to what might have been expected table injapan and argentina the ageadjusted fraction of garbage codes was “ points lower than one 101371 pone0237539 august one 0ctable levels of garbage codes standardised by age and registration completeness for select countries ranked by their sociodemographic index sdiare cause of death data fit for purposecountryfinlandcanadaaustraliajapanfranceyearsdivalueunited kingdom high sdimeanturkeyargentinairanjordanthailandsouth africatunisiabrazilcolombiauzbekistanmiddle sdimeankyrgyzstanegyptnicaraguatajikistanlow sdi meantotal meansdi levelshigh impact gclow impact gca total gc ageadjb completeness of codc deaths of no policygc registration value sdi three levels collapsed from gbd five sdi levelshigh highmiddle high middle middlelow low low middlegarbage code gc levelshigh impact gc levels “low impact gc level total gc high lowdeath of no value for policyc 1b a�bthis equation calculates unavailable deaths for policy ie unregistered deaths plus deaths with a garbage code101371 pone0237539t001the unadjusted fraction while in france and south africa it increased by a similar amountcolumn a agestandardisation therefore had no impact on the mean level of garbage codesin each development categoryagestandardisation however masks the age pattern of garbage coding particularly its relative importance at younger adult ages where accurate and specific diagnoses are critical fuiding policies designed to prevent premature deaths the perception that garbage codes arelargely confined to deaths among the elderly due to the presence of comorbidities at oraround the time of death is not confirmed by the agespecific fractions of garbage codes shown one 101371 pone0237539 august one 0care cause of death data fit for purposefig percentage of high impact garbage codes in total causes of death selected countries c high sdi middlesdi low sdi101371 pone0237539g002in fig with exact fractions reported in s3 file garbage codes are prevalent at all ages andoften in similar proportions to what is observed for the age group indeed in some highlydeveloped countries eg finland canada uk the proportion of garbage codes is significantly higher at ages “ for both sexes than at older ages indeed in tunisia for males andfig garbage codes as a percentage of all deaths by age selected countries101371 pone0237539g003 one 101371 pone0237539 august one 0care cause of death data fit for purposein jordan and kyrgyzstan for both sexes this pattern is already evident from age in uzbekistan garbage coding is more common for deaths of children and adolescents than at ages and abovegiven that the utility of a country™s mortality information system is reduced not only by theamount of garbage codes but also by how complete it is in terms of capturing all deaths wemeasured their combined impact for two countries namely jordan and tajikistan the consolidated indicator of registration completeness and fraction of garbage codes was close to suggesting that useful information on only about one quarter of all deaths that occur injordan and tajikistan is available for policy purposes col c table overall the combinedindicator showed the proportion of deaths for which information was either missing or of littleor no value for guiding health policy was much higher in low and middle sdicountries compared to high sdi countriestable shows the strong influence of garbage codes on the leading cause distribution whenthese are ranked with high impact garbage codes marked in red and low impact in orangethe presence of high impact garbage codes among the leading causes of death will substantially distort the true picture of what are the common cod that most people die from amonghigh sdi countries only japan and france have high impact garbage codes for males amongthe ten leading causes of death canada australia and uk had only low impact causes in thiscategory and finland had neither for the middle and low sdi countries there were manymore high impact garbage codes listed among the leading cod with egypt having seventajikistan five and thailand and tunisia each with four colombia and nicaragua had nonefor females egypt had seven iran tunisia and tajikistan five with the remaining countrieshaving between one and threenotwithstanding some variation in patterns and volume of garbage coding across countriesthe most common garbage codes were remarkably similar in particular other illdefined andunspecified causes of death senility heart failure unspecified neoplasm septicemia respiratory failure unknown cause of death hypertension and unspecified diabetes were observedacross all sdi levels in addition low sdi countries tended to report atherosclerosis hepaticfailure intracerebral hemorrhage and unattended deaths all garbage codes as leading causesfig ranks countries according to a single consolidated summary measure of system performance namely the vital statistics performance index for quality vspiq eleven countries scored and above a level where they could be considered as having wellfunctioningsystems six of the remaining countries achieved scores that would classify them as havingmedium performing systems with lower scores mostly arising from the high proportion ofgarbage codes that bias their cod distributions of the countries only jordan tajikistanand tunisia were classified as having poorly functioning systemsdiscussionin general one would expect that in high sdi countries where all deaths are medically certified with good quality clinical and diagnostic services comparatively few deaths would beassigned unusable garbage codes and certainly much less than in countries where such services are less common our findings based on an analysis of cod datasets from across theworld produced evidence both for and against this hypothesis france and japan do not seemto have more reliable cod data to guide policy than turkey colombia kyrgyzstan and nicaragua in france in deaths is assigned a garbage code with of all deaths being certifiedas due to œother illdefined and unspecified causes of death r99 heart failure i509 orrespiratory arrest r092 similarly in japan œold age senility r54 is a commonly assignedcause of death accounting for one fifth of all garbage codes other research has found that one 101371 pone0237539 august one 0cdeificepsnuesaesidtraehnoitcrafnilarberecdeificepsnusaercnapainomuenpdeificepsnuevitcurtsboyranomuplhtiwesaesidrewoletucayrotaripsernoitcefnideificepsnulnoocyrtnuocknarselamwolleynisenotcapmiwoldernidekramsedocegabragtcapmihgihxesdnayrtnuocybhtaedfosesuacgnidaelpotelbatcimeahcsicinorhcfoealeuqescinorhcsisohrriccilohoclarevilfoebolreppurosuhcnorbs™remiehzlaesaesidgnuldeificepsnulaidracoymnoitcrafnideificepsnuetatsorptesnofomsalpoenetalhtiwesaesidesaesidtraehetucatnangilams™remiehzlacitorelcsorehtadnalnifgnignahybmrahdnanoitalugnartsfleslanoitnetniecalpdeificepsnunoitacoffusdeificepsnusaercnaptonekortssadeificepsegahrromeahnoitcrafnirocitorelcsorehtaesaesidtraehnoitcrafnilarberecfoealeuqesllecrevileudsitinomuenpdeificepsnunoitcrafnilarberecamonicractimovdnadoofotevitcurtsboyranomuplesaesiddeificepsnulaidracoymnoitcrafnideificepsnuetucaaitnemedfomsalpoentraehcimeahcsietatsorpdeificepsnuesaesidytilineseruliaftraehdeificepsnudeificepsnuhcamotslaidracoymnoitcrafnideificepsnurosuhcnorbdeificepsnugnuldeificepsnugnulainomuenpdeificepsnunapajcinorhcdeificepsnutnangilamcinorhcetucarosuhcnorbneilartsuaainomuenpohcnorbdeificepsnutonekortssadeificepsainomuenpdeificepsnuroegahrromeahnoitcrafniesaesidyranomuplrewoletucahtiwyrotaripsernoitcefnietatsorpdeificepsnuesaesidevitcurtsboesaesidtraehfomsalpoenaitnemedtraehcimeahcsilaidracoymnoitcrafnideificepsnudeificepsnugnulmodgnikcinorhccitorelcsorehtatnangilamdeificepsnucinorhcetucarosuhcnorbdetinudeificepsnusaercnapdeificepsnudeificepsnuesaesidesaesiddeificepsnudeificepsnutraehcimeahcsitserralaidracoymnoitcrafnideificepsnufomsalpoendeificepsnudnaetatsorpfosesuacytilatromdeificepsnugnuls™remiehzlalnooceruliaftraehcinorhcyrotaripseretucatnangilamdenifedllirehtorosuhcnorbecnarfcinorhcralucsavorbereccitorelcsorehtacinorhceruliaftraehrosuhcnorbetucayekrutesaesidyranomuplroegahrromeahdeificepsnunoitcrafnievitcurtsbocinorhctonekortssadeificepsfomsalpoentnangilametatsorpdeificepsnucitorelcsorehtaaitnemedesaesidtraehlaidracoymnoitcrafnideificepsnudeificepsnugnuletucarosuhcnorbadanacdeificepsnulnoocdeificepsnuesaesidyranomupletatsorpevitcurtsbofomsalpoencinorhctnangilamrewoletucahtiwyrotaripsernoitcefniaimeacitpesdeificepsnuroegahrromeahnoitcrafnitonekortssadeificepsesaesids™remiehzlatesnoetalhtiwdeificepsnuhcamotsainomuenpdeificepsnuesaesidyranomupldeificepsnuevitcurtsboesaesidesaesidtraehevitcurtsboyranomuplesaesiddeificepsnudeificepsnudeificepsnugnullaidracoymnoitcrafnideificepsnudnadenifeddeificepsnufosesuacytilatromdeificepsnugnuldeificepsnullirehtorosuhcnorberuliaftraehetucaainomuenpanitnegraare cause of death data fit for purposeesaesidtraehdnasuhcnorbfodenifedllifomsalpoenesaesidtraehcimeahcsignulfosnoitpircsedhcamotsesaesidsetebaidsutillemdeunitnoceruliaftraehdeificepsnuainomuenpcitorelcsorehtayramirplaitnesserosuhcnorblarberecdeificepsnudeificepsnuesaesidtraehnoisnetrepyhdeificepsnugnulnoitcrafnideificepsnusnoitacilpmocsetebaidsutillemtuohtiwdeificepsnudnaevisnetrepyhesaesidtraehevitsegnoceruliaftraehhtiwfosesuacytilatrommsalpoentnangilamdnasnoitacilpmoctnangilamtraehevisnetrepyhcinorhcdeificepsnutserracaidracdenifedllirehtolaidracoymnoitcrafnideificepsnunoitcrafnilarberecdeificepsnutropsnarttnediccadeificepsnulaidracoymnoitcrafnideificepsnulaidracoymnoitcrafnietucanarietucanadroj one 101371 pone0237539 august one 0cniderunjinosrepnoitcrafnilarberecytilinestserrayrotaripserdeificepsnudeificepsnutnetniidenmretednuesaesidtraehtnevedeificepsnucimeahcsicinorhclaidracoymnoitcrafnietucalarberecartniegahrromeaheruliaftraehdnaaeohrraidlarivrehtonamuhfositiretneortsagtonsesaesidycneicifedonummifomsalpoentnangilamdnasuhcnorbgnultonekortssadeificepsnigirosuoitcefnideifissalcsesaesidcitisarapdnasuoitcefninignitlusernoitcrafnidemuserperehwesleesaesid]ivh[surivroegahrromeahaimeacitpesrehtofomsalpoentnangilamcitapehartnistcudelibdnarevilytilinesainomuenpdeificepsnumsinagrooterusopxedeificepsnurotcafdeificepsnuainomuenpyrotaripsersetebaidsutillemdeificepsnudemrifnocmsinagrotonlsisoucrebutlyllacigooiretcablyllacigootsihrodeunitnocelbatllirehtodnaliahtdnadenifeddeificepsnufosesuacytilatromdnadenifeddeificepsnullirehtofosesuacytilatromhtuosacirfarotomdeificepsnudeificepsnuoteudtnediccaelcihevronoisulccociffartlarberecfosisonetsseiretrahtaeddednettanuyramirplaitnessedeificepsnunoisnetrepyhsnoitacilpmocsetebaidsutillemtuohtiwetatsorpdeificepsnudeificepsnuegahrromeahmraerifnoitcrafnirofosesuacytilatromfomsalpoentnangilamrosuhcnorbgnulybtluassadnarehtoecalptonekortsdenifedllirehtosadeificepsdeificepsnudnaainomuenpdeificepsnulaidracoymnoitcrafnideificepsnuetucalizarbsutillemsetebaidtnevesnoitacilpmocdeificepsnurehtohtiwidenmretednuegahrromeahdeificepstnetninoitcrafnirotonekortssadeificepslaidracoymnoitcrafnideificepsnudeificepsnugnuldnadenifeddeificepsnufosesuacytilatrometucarosuhcnorbllirehtoaisinutdeificepsnueruliafdeificepsnudnagnullanercinorhcrehtoybtluassarosuhcnorbevitcurtsbocinorhcegrahcsidmraerifdeificepsnuesaesidyranomuplfomsalpoentnangilametatsorpsisorelcsorehtaeruliaftraehecalpdeificepsnutonekortssadeificepsegahrromeahnoitcrafnirorewoletucahtiwyrotaripsernoitcefnitraehcimeahcsietucarehtosesaesidlarberecartniegahrromeahgnignahybmrahdnanoitalugnartsemohnoitacoffusfleslanoitnetnideificepsnutraehcimeahcsiralucsavoidracdeificepsnuesaesidosesaesiddebircsedevitcurtsboyranomuplcinorhcesaesidhcamotscinorhccitorelcsorehtadeificepsrehtoegrahcsiddnateertsyawhgihdeificepsnuhcamotsdnasisorbiffosisohrricrevildnadenifeddeificepsnullirehtofosesuacytilatromainomuenpdeificepsnuevitcurtsbodeificepsnudnayranomuplmraerifesaesidteertsegrahcsiddeificepsnuyawhgihdnalaidracoymnoitcrafnideificepsnucinorhcrehtoybtluassaetucaaibmoloclaidracoymnoitcrafniesaesidtraehtraehcimeahcsiesaesidetucasirotcepanignaevisnetrepyhcinorhcnatsikebzunoitcrafnirevilfosisohrricroegahrromeahdeificepsnunoitcrafnilaidracoymetucadeificepsnudnarehtosadeificepsesaesidtraehtonekortscitorelcsorehtanatszygrykare cause of death data fit for purposemsalpoentnangilamnilusninonetatsorpfosetebaidtnednepedlaidracoymnoitcrafnifosisohrriccilohoclalanerhtiwsutillemsnoitacilpmocrevilrevilfosisohrricerehwesletonegahrromeaherehwesletonfosisohrricdeificepsnudeifissalcdnarehtoderunjinosrepdeificepsnunielcihevrotomciffarttnediccaevitcurtsboyranomuplcinorhcesaesiddeificepsnudeifissalctonekortssadeificepsegahrromeahnoitcrafnirorevilainomuenpdeificepsnusisorelcsorehtaytilinesetucaeruliafyrotaripserlarberecartnieruliafcitapehdnasisorbiftserracaidracnoisnetrepyhlanercinorhcdeificepsnueruliafyramirplaitnesseeruliaftraehtpygelaidracoymnoitcrafnideificepsnuetucaaugaracintraehfosnoitpircsedesaesidesaesidfosesuacytilatromnoitcrafnisesaesidroegahrromeahnoisnetrepyhnoitcrafnidenifedllisutillemsetebaidtraehcimeahcsideificepsnudnalaidracoymtraehcimeahcsidnasnoitacilpmocdeificepsnucinorhcsisorelcsorehtadenifedllirehtoetucaetucarehtoytilinestonekortssadeificepsyramirplaitnessenatsikijatdeunitnocknarselamefyrtnuoc one 101371 pone0237539 august one 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cause of death data fit for purposetraehfosnoitpircseddenifedlliesaesiddnadenifeddeificepsnufosesuacytilatromesaesidnoisnetrepyhtraehcimeahcsiesaesidtraehsutillemsetebaiddnasnoitacilpmocytilinesllirehtoyramirplaitnessecinorhctserracaidracevisnetrepyhdeificepsnunoitcrafnilarbereclaidracoymnoitcrafnietucanarideificepsnutraehfonoitamroflamesaesidtraehdeificepsnulatinegnoccitorelcsorehtaeruliaftraehainomuenpdeificepsnudeificepsnutsaerbnoisnetrepyhyramirplaitnesselaidracoymsutillemsetebaidnoitcrafnideificepsnusnoitacilpmoctuohtiwetucadeificepsnuevisnetrepyhesaesidtraehevitsegnoceruliaftraehhtiwnoitcrafnideificepsnularberecnadrojdeunitnoclarberecartniegahrromeahgnuldnasuhcnorbcitapehartnistcudelibdnarevilmetsyssuovrenerehwesletonfosesaesiddeifissalcfomsalpoenfomsalpoeneruliafevitarenegedsetebaidsutillemtnangilamtnangilamlanercinorhcrehtodeificepsnuaimeacitpesrehtoainomuenpdeificepsnumsinagroytilinesllirehtodnaliahtdnadenifeddeificepsnufosesuacytilatromdeificepsnulnooceruliafyrotaripserrosuhcnorbdeificepsnugnultonekortssadeificepsaimeacitpesdeificepsnudeificepsnuroegahrromeahnoitcrafnidnadenifeddeificepsn Answer:

Colon_Cancer

subcutaneous hydration and medicationsinfusions effectiveness safety acceptability asystematic review of systematic reviews one e0237572 101371 pone0237572editor jose´ das neves university of portoportugalreceived november accepted july published august copyright broadhurst this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation filesfunding this project was partially supported viaan unrestricted project grant provided by bectondickinson canada a portion of the work of dswas funded as salary by the sponsor grant db asa former employee of medical pharmacies duringthe conduct of this research reports the researchas work independent of her employer with noemployer contributions to the research db is soleobjectiveto synthesize the current evidence for subcutaneous hydration and medication infusionsfrom systematic reviews and to assess their methodological qualityintroductionperipheral intravascular cannulacatheter insertion is a common invasive procedure foradministering fluids and medications venous depletion is a growing concern for severalpatient populations subcutaneous access for the administration of isotonic solutions andmedications is an alternative however vascular access assessment and planning guidelines rarely consider this routemethodssystematic review of systematic reviews prospero crd42018046504 we searched databases published in english language from to june identifying subcutaneousinfusions an alternate route for fluids or medication methodological quality was evaluatedusing amstar criteria and data for mechanisms of infusion and outcomes related toeffectiveness safety efficiency and acceptability extracted the johanna briggs institute™sgrades of recommendation informed the strength of recommendationresultsthe search yielded potential systematic reviews were excluded through and duplicate screen of the remaining s were excluded and were includedoverall evidence is strong for recommending subcutaneous hydration infusions for olderadults weak for pediatric patients and inconclusive for palliative patients there is strongevidence for medications weak evidence supporting medications however there areeight medications with inconclusive evidence to make a recommendation and four medications not appropriate for subcutaneous delivery one 101371 pone0237572 august one 0cproprietor of infusion excellence consulting whoseservices are unrelated to this research bg ownsand is employed as senior consultant and directorof education by clinical pharmacy partners aneducational and consulting firm for infusiontherapy providers and clinicians the funders hadno role in study conception design data collectionand analysis decision to publish or preparation ofthe manuscript the specific roles of these authorsare articulated in the ˜author contributions™ sectioncompeting interests db reports personal andconsulting fees ie travel and honoraria and nonfinancial support from 3m including researchgrant in unrelated field angiodynamicscardiomed medical supplies covalon freseniusand excelsior medical db™s former employermedical pharmacies provides infusion serviceshowever db affirms research was conductedindependent of her employer™s servicesproductsand of her current consulting business mc reportsgriffith university has received unrestrictedinvestigator initiated research or educational grantson mc™s behalf from product manufacturersbaxter becton dickinson and company centurionmedical products and entrotech lifesciencesunrelated to this research ds reports no conflictsbg reports personal and consulting fees ieconsultancy travel and honoraria from nutrishareand covalon bg affirms no relationshipspresenting competing interests or influences thisdoes not alter our adherence to one policieson sharing data and materialssubcutaneous hydration and medication infusionssubcutaneous access should be considered alongside intravenous therapy for hydration inolder adults and several medications there are additional benefits in terms of ease of useand costeffectiveness of this mode inclusion of subcutaneous access in clinical guidelinesmay promote uptake of this route to help preserve vessel health of vulnerable patients further highquality research is needed to inform subcutaneous infusion therapy in a variety ofpopulations including pediatrics and palliative care and medications and clarifying themechanism of deliveryintroductioninfusion therapy is a common treatment modality to deliver medications and fluids in theacute and home care settings and is gaining prevalence in the longterm care setting traditionally these parenteral therapies have been delivered via the intravenous route howevervenous depletion is a growing concern with an increasing aging population and patients withlongterm complex comorbidities [“] the problems of venous depletion are compoundedby unnecessary peripheral venipuncture which add to patient physical and psychologicaltrauma compromised intravenousrelated outcomes suboptimal use of healthcare resourcesand increased costs [ “] additionally infusion therapy is moving beyond the boundariesof the acute care and home care sector to hospices and longterm care facilities recentlypublished vascular access planning tools are either oriented primarily to the acute care settingor do not address the option of subcutaneous access [ ]the infusion therapy standards of practice recommend consideration of subcutaneousaccess for the administration of isotonic solutions and for continuous opioid and other infusion therapiesmedications eg immunoglobulin therapy subcutaneous access isachieved through the placement of a small catheter in the subcutaneous tissue with the infusate absorbed from this space into the circulatory system this route has the advantages ofrequiring a small catheter and less technical insertion skills than that used for intravenousaccess with its ease of application lending to its use in multiple settings additionallytime to place the catheter is less and fewer complications are likely resulting in cost benefits[ ] although practiced since there has been a slow uptake in part due to a lack offamiliarity with the technique among physicians and healthcare professionals and perceivedsuboptimal outcomes eg hypovolemic shock due to inappropriate use of hydration solutions [ ] in our scoping literature search to prepare for a systematic review of primarystudies numerous systematic reviews were identified however each either had a narrowscope addressing one treatment eg fisher and colleagues™ review of iron overload management or provided limited literature search or limited evidence eg duemsnoreiga andblasco™s review of subcutaneous fluid and drug deliverygiven the world™s aging population health care systems rapidly changing diversity in practice setting and complexity of care and patient health conditions the uptake and use of subcutaneous continuous infusions may help address these issues and challenges the aim of ourstudy is to determine the effectiveness safety acceptability and efficiency related to the use ofsubcutaneous infusion sci as an alternate route to intravenous for the management of conditions or treatments such as dehydration and palliation for children and adults in all care settings through a synthesis of systematic reviews of studies a secondary objective is to identify one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsthe mechanisms of subcutaneous fluid and drug delivery that facilitated achieving these outcomes this will provide an uptodate and rigorous review of subcutaneous infusion therapymaterial and methodswe adopted the systematic review of systematic reviews methodology from the joannabriggs institute™s to conduct this review the study protocol was registered with theprospero database registration number crd42018046504 and we report the reviewaccording to the preferred reporting items for systematic review and metaanalysisprisma guidelines s1 table eligibility criteriawe considered all types of systematic reviews which included the following characteristics¢ a clearly stated set of objectives with an explicit reproducible methodology¢ a systematic search that attempts to identify all studies that would meet the eligibilitycriteria¢ an assessment of the validity of the findings of the included studies eg assessment of riskof bias and confidence in cumulative estimates and¢ systematic presentation and synthesis of the characteristics and findings of the includedstudies metaanalyses where some of the primary studies in the reviews were duplicated we did not exclude thereviews as they had different aims and objectives and thus added to the overall understandingof sci to meet the aims of this current systematic review we did however only include the primary studies™ findings once in our analysis and synthesis expert opinionconsensus andbench research s and editorialscorrespondence were excluded no restrictionsregarding age gender diagnosis geographical location or healthcare setting were appliedwe included reviews that assessed interventions that used subcutaneous infusion for aduration of around hours or more as an alternate route for fluid or medication therapy subcutaneous infusion is defined as the delivery of fluids or medication into the subcutaneousspace for absorption into the circulation via œperfusion diffusion balance between hydrostaticosmotic pressure and lymphatic drainage p118 an infusion of around hoursduration was determined from considering the studies in a review by caccialanza where the range of infusion was hours to greater than days during the full textscreening numerous intermittent sc insulin vs continuous sc insulin systematic reviewswere identified and excluded because they did not meet our inclusion criteria of sci as analternate route reviews that included other routes as comparators such as intravenous andintraosseous were excluded if data on subcutaneous infusions could not be extractedseparatelyoutcome measuresthe primary outcomes of interest investigated included¢ effectiveness defined as clinical response to therapy eg cureimproved clinical failure orno change completion of therapy within prescribed time frame¢ safety defined as medication or vascular access adverse event eg abscess erythema bruising electrolyte imbalance edema infection pain fluid overload vascular collapse and one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsroute failure survival status eg died of underlying condition other causes lost to followup or status unknown and complications related to treatment eg unplanned hospitalreadmission related to treatment¢ acceptability defined as patient andor health care provider preference satisfaction or perceived benefits of subcutaneous therapy¢ efficiency defined as healthcare resource utilization including costs of infusion therapy supplies and treatment timesecondary outcomes included indications for subcutaneous infusion therapy medicationsolution type infusion rates volumes and duration subcutaneous access sites dwell timesand infusion control devices usedsearch strategya systematic search was conducted november and updated in june of reviews from as recommended by aromataris from the following databases excerpta medica database embase pubmed cumulative index to nursing and allied health literaturecinahl cochrane database of systematic reviews joanna briggs institute of systematicreviews and database of s of reviews of effects dare search terms included combinations of medical subject headings mesh and key word terms [˜subcutaneous infusion™or hypodermoclysis or ˜subcutaneous therapy™] and the term ˜systematic review™ with theassistance of a university librarian study resources limited inclusion to only english languageas listed in s2 table one author of a relevant was contacted to establish the status ofthe review however the review had not been completedthe title and of each were scanned independently by one reviewer ds andfull copies of s of potentially eligible reviews were obtained full texts of these reviewswere then screened independently by two reviewers mc and db against the review selectioncriteria disagreements were resolved by discussion between these reviewers and with consultation by a fourth reviewer and subject expert bgdata extraction and quality appraisaldata were extracted and assessed independently by the reviewers in pairs [bg db] [mcds] with anomalies reconciled by agreement data were obtained primarily from the systematic reviews although primary studies were consulted for critical missing data eg missinginfusion properties data extracted included authoryear aim design and number of studiesincluded search strategy population interventioncomparator quality appraisal of primarystudies infusion characteristics outcomes as previously defined key findings study limitations funding and smethodological rigour of each review was independently assessed by four reviewers in pairsas above using the amstar tool this is a 16item tool used to appraise the quality ofsystematic reviews and is frequently used in cochrane overviews a score of overall confidence high moderate low and critically low was assigned by the reviewers to depict the accuracy and comprehensiveness of the data summary and critical methodological flaws thisassessment informed the final grading of the recommendationsdata analysis and synthesisdue to the different interventions outcomes and outcome measures a metaanalysis was notfeasible quantitative outcome data are provided and synthesized where possible only data one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsfrom randomised controlled trials rcts and prospective cohort studies were included todetermine subcutaneous hydration outcomes however reviews exploring medications alsoincluded retrospective studies and case reports the jbi grades of recommendation were usedto derive the grading score to inform the strength of recommendation for the interventionjoanna briggs institute levels of evidence and grades of recommendation working party data synthesis for hydration and medications are provided as a final summary ofrecommendations grade a indicates the intervention™s desirable effects outweigh undesirableeffects with adequate supporting evidence a weak recommendation grade b depictsdesirable effects outweigh undesirable effects although this is not as clear or evidence not ofhigh quality where there was insufficient evidence we determined that a recommendation isinconclusiveresultsthe search strategy yielded potential systematic reviews fig of which wereexcluded through and duplicate screen of the remaining s were excludedafter full text examination against inclusion criteria in the full text screening for reasonsdescribed as above as listed in s3 tablecharacteristics of included studiestable details the systematic reviews of subcutaneous infusion of medications andor fluidswith focused on medications only on fluids and on both types of therapies the publication dates of reviews ranged between and [ ] most reviews focused onadult populations generally or specifically for example pregnant women and adults withamyotrophic lateral sclerosis als two reviews however are specifically paediatric [ ]eight all ages [ “] two focused on older adults and paediatrics [ ] andone where age was not reported the systematic reviews incorporated a diversity of study designs and quality levels fromcase studies to systematic reviews and randomised controlled trials however only reviewsincluded only randomised controlled trials [ “] similarly sample sizes varieddepending on the number of sci studies included in the review synthesise of findings withvery few metaanalyses performed due to varied study designs outcomes and sizemethodological qualitythe amstar quality scores of the included reviews are described in s4 table while reviews achieved an overall confidence rating of high sixteen of the reviews had critical methodological weaknesses studies with moderate low and studies rated as criticallylow the main deficiencies noted were failure to report a priori protocol study design selection full details of excluded studies funding sources of primary studies and results reflectingrisk of bias assessment the cochrane risk of bias tool or an adaptation was the most commonly used tool by systematic review investigators to determine quality of the primary rctstudies n while only provided a narrative discussion of quality most reviewauthors noted that results should be interpreted with caution due to study limitationsmechanisms of subcutaneous accesslimited data were reported on the materials and techniques used to achieve subcutaneousaccess for hydration or medication therapy table describes the characteristics of sc devicedesign and dwelltime subcutaneous anatomical sites and insertion techniques and infusion one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsfig prisma flow chart the diagram details our literature search and screening process for selection of systematic reviews included in thesystematic review adapted from moher d liberati a tetzlaff j altman dg the prisma group preferred reporting items for systematicreviews and metaanalyses the prisma statement med e1000097 101371 pmed1000097101371 pone0237572g001delivery systems in relation to studies reporting on hydration therapy no studies comparedthe safety and efficacy of the various characteristics including mode of delivery the findingsfor hydration and medication sci are presented separatelysubcutaneous hydrations5 table describes the indications and contraindications for sci for hydration as described inthe studies the most common indicator was mild to moderate dehydration with the mostcommon contraindication being rapid high fluid volume requirements one 101371 pone0237572 august one 0ctnatropmiyllacinilcmumnmiidecneirepxedahohwstneitaphtlaehdnaefilfoytilauqsuhtgivinoseacimetsyslatipsohnehwnoitpecrepfostroperdesaercnievahyamemohotdegnahcgividesabelihwseacimetsysdnalacolseidutsgicsdesabdellorneevahyamseiduts¢™stneitapnitnemevorpmi¢gicsnostneitaperomderreferpdnadetarelotdnacirtaidepybgivinahtsnoitcejnigicsdesabemoh¢stneitaptludamuresggielbatpeccaseveihcayparehttnemecalpergics¢sleveldnaytienegoretehlaitnatsbusseidutsssorcanosirapmocsnoitatimilllacigoodohtemsaibfoksiretaredomecnediveytilauqwol¢mrettrohsnoylnoatadloq¢puwollofnotcefferettebaevahyamlanoitnevnocnahtloqyparehtdesisehtnysseidutsylno¢suoenatucbussuounitnoc¢icsottnavelerenositrocordyhfonoisufnicsotviderapmocstcron¢derosnopsyrtsudni¢daehotdaehecnereffidehtniecnacifingislacitsitatssawerehthguohtla¢aenpsydecuderdnafoesuehthtiwdwmnillarevoehtsnilcycatsorpehtteemtondidtceffenoitarudpuwolloftrohsllasedulcnisisylanaatemsnoitnevretnifosepyt¢¢caidracgenoitcnufcaidracerusserplairtathgirxednievorpmiyamlanitsorpertcs¢gnitarehtgnitagitsevnirofairetircfoytilauqllacigoodohtemytilauqwolseiduts“ytilauqetaredomytilauqhgihrnsgnittesnodesabairetirc”stcrnerdlihcdnastludatrohocborenarhcocyranibnodesabseidutsydobitnayramirphtiwseicneicifedgnisuselbairavonsey“segnareziselpmassesabatadotpuetadyraunajotelbacilppaseidutsseidutslatotweiveremoctuo]noitatic[snoitacidemfonoisufnicsseidutsginilubolgonummiinassahobalslairtlacinilcevitcepsorp¢¢strohocevitcepsorter¢strohocytefasdnassenevitceffevisusrevcs][saibfoksirhgih”muidem”seidutslanoitavresboelacsawattoeltsacwenseidutslanoitavresborofytilauqriafstcrnielpmasycneiciffusniylujotpuetadsuounitnoc“egnarezisrnsgnittesdnaegacsenositrocordyhstcrretnecitlumlacinilclebalnepo¢ydutsrevossorc¢noisufnissenevitceffestnemtaertlootborenarhcoc“stcrlanerdahtiwstneitapsesabataddedulcniseidutsdiocitrococugl][lafonlagnitroperdnanoitcelessisongaidsraey“sehcraesdnahicsottnavelerycaciffelinitsorpertfoksirwolborenarhcocegaegaeziselpmassesabatadstcrtcrnilcycatsorp][senrabsaiblairetrayranomuplotpusetadnoisnetrepyhrebmetpesytefasdnasnoitatimilnoisulcnocllarevos™rohtualootlasiarppaytilauqnoitalupopygetartshcraesforebmundnangisednoitnevretnimairohtuadaelsweivercitametsysfoscitsiretcarahcelbatsubcutaneous hydration and medication infusionsdeunitnocelbaileraedivorptonnacdnataienmatekfotceffeylekilemoctuoylekilfonoitacidniottnavudanajsaesodynawolsiecnediveytilauqwol¢ehtfonoitacidnielbaileron¢stceffeebnacniaprecnacnisdioipostroperoslatubnoitacidemssenderdnanoitatirrietissertemfodohserhtldetroperstneveesrevda¢ehtotdetaleryltsomevitingocdnasnoitanicullahrofdetroperecnabrutsidienmatekfosesodrehgihebotthguohttcrnidetroperydutsehtotdetalerylbissopstneveesrevdasuoiresowt¢gurdgestneveesrevda¢dedivorpborraelcnudahniaprecnaclairtlacinilcpuienmateksraeynaemegaeracevitaillapgnittespuobecalpeziselpmasotsetadceddnenajignnnigebdesiversretsigerweiverenarhcoctcr¢ytefasdnassenevitceffeicssdioipoottnavudajtcrlootborenarhcocyrotcarferhtiwstneitaptludalsupsesabatadottnavelerseidutsnasaienmatek][lleb one 101371 pone0237572 august one 0cdnadetimilsawsemoctuomretgnolfognitroper¢evisulcnocnisawecnedivefoytilauqllarevotaslairtoteudwolyrevotwolesicerpmisetamitseemoctuoborsuoiresyrevrosuoiresesuairetircnoisulcxeevitcirtserfokcaldnastnemerusaemloqdetadilavnonfoatadfognitroperaimeassalahthtiwelpoepsiofdnehwrojamrodetacidniartnocetauqedanifoesuroetuorsuonevartnirosrotalehcnorilarorehtorosuoenatucbusrehtieybdehsilbatseehtsniamerhtobcaidracesreverottnemtaertnorioteudnoitcnufsydtnemtaertofddeifisnetni¢daolrevohtiwderapmocenorpirefeddetaertstneitapnidnaofdenorpirefeddenibmochtiwhtiwderapmocofddnahtiwdetaertstneitapnienolaofddesaercnierastneveesrevda¢ecnerehdasevorpmiyparehtofdfoicsotderapmocnoitalehclarofiniatrecnu¢foksirehtsecuderyparehtnoitalehclarofiniatrecnu¢ofdfoicsotderapmocstceffeesrevdasuoiresytilatromsecuderyparehtofdfoicsotderapmocnoitalehclarofiniatrecnu¢otdesulootbordetadilavon¢elbaliavaivdehcraessesabatadylno¢niaprecnacyrotcarfertnemtaertrofnoitpollamsfonoitatimilgehtiwseidutsforebmunwefseziselpmasllamsesreviddnastcrdednilblseigoodohtem“egnarseidutsseidutsfoytilauqssessaelbaivaebyamenmateki¢“ecnediveehtfoytilauqehtcirtaideaprofeziselpmasicsrofonoitaredisnocasedulcni“egnarstludascirtaidepseidutsydutslebalnepolairtdellortnocnustroperesacweiverevitcepsortertcr¢¢¢¢¢atadlevelhgihdetimli¢erasegasoddednemmocer¢loottnemssessaonhtiwnerdlihcdnastludasesabatadicsottnavelerseidutstnemtaertienmatek][ualderbsnoitatimilnoisulcnocllarevos™rohtualootlasiarppaytilauqnoitalupopygetartshcraesforebmundnangisednoitnevretnimairohtuadaelgnitarotelbacilppaseidutsseidutslatotweiveremoctuo]noitatic[deunitnocelbatihgkgmotmorfnoissucsiddetroperelpmasniapdetalerrecnacrnsetadstludaytefasdnassenevitceffeweivernidaolrevonorignitaertrof”ksirwol”ksirraelcnutnednepednoisufsnartlacidemrehtoenorpirefedderapmocnidedulcnislairticsylno¢detacidnillitssienorpirefed¢lootborenarhcochtiwnerdlihcdnastludalsupsesabatadseidutstcrsusrevenorpirefed][rehsifrofpunidnaspuehtrofsraeydnanaemhtiwsrydnaydutsydutsyadcinilctneitaptuognittescirtaideapertneceraclatipsohelpmasaimeassalahtdnasesabatadnienolaofddnaenola“egnaregaezisseirtsigerlairtslairtesehtfosryydutsrnydutsotpusetadsuoenatucbusdetropernisryfonaemhtiwhcramofdfonoisufniienmaxoirrefseddnaytefasofdssenevitceffesubcutaneous 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infusionsdeunitnocdnaytidilavehtniecnedifnoc¢lduohsyparehtpmupfoesu¢siecnedivefoytilibicudorperhcraeserehtotdetimilebwolgnittesfoytefasgnidragersnrecnoc¢tsisrepyparehtsmrahfoelacstnemssessalanoitanadnasetisretnecsgnitarsaibfoksirllarevomargorplatanireptneitaptuoehtmorfsmetidetcelesdessessasaibnoitirttaytilauqretsammcelgnissgnittes±egareva“egnarrneziselpmas”muidem“hgih”wolesabataddnanoitcetedecnamrofrepsnoitcartnocmreterp“setadtrohocevitcepsortertrohocevitcepsorpseiresesacdezimodnarnonlairttcr¢¢¢¢¢ssenevitceffednaytefassisylocotrofpmupnoitcelesgnidnuofnochtiwnemowtnangerpsesabatadseidutsybenilatubretfoics][teduagegaeziselpmasgnittes“egnarcinorhccinilctneitaptuolatipsohcirtaidepscinilcertnecitlumertneceraclootborenarhcocrostnecselodanerdlihclsupsesabatad“ksirhgihraelcnuesaesidllecelkcishtiw”wolraelcnustludadnasrevigeracriehtlacidemrehtodnasesabatadstcrseidutsotecnerehdaevorpmitcrnoitnevretninoitacidemotsnoitnevretninoitacidem][nitrofaimeassalahtyraurbefyparehttnednepednoisufsnartnonotpusetadnoitalehclarohtiwssenevitcefferotnednepednoisufsnartseirtsigerlairtofdfoicsderapmocyparehtnoitalehcnori 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cancer7560

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: subcutaneous hydration and medicationsinfusions effectiveness safety acceptability asystematic review of systematic reviews one e0237572 101371 pone0237572editor jose´ das neves university of portoportugalreceived november accepted july published august copyright broadhurst this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation filesfunding this project was partially supported viaan unrestricted project grant provided by bectondickinson canada a portion of the work of dswas funded as salary by the sponsor grant db asa former employee of medical pharmacies duringthe conduct of this research reports the researchas work independent of her employer with noemployer contributions to the research db is soleobjectiveto synthesize the current evidence for subcutaneous hydration and medication infusionsfrom systematic reviews and to assess their methodological qualityintroductionperipheral intravascular cannulacatheter insertion is a common invasive procedure foradministering fluids and medications venous depletion is a growing concern for severalpatient populations subcutaneous access for the administration of isotonic solutions andmedications is an alternative however vascular access assessment and planning guidelines rarely consider this routemethodssystematic review of systematic reviews prospero crd42018046504 we searched databases published in english language from to june identifying subcutaneousinfusions an alternate route for fluids or medication methodological quality was evaluatedusing amstar criteria and data for mechanisms of infusion and outcomes related toeffectiveness safety efficiency and acceptability extracted the johanna briggs institute™sgrades of recommendation informed the strength of recommendationresultsthe search yielded potential systematic reviews were excluded through and duplicate screen of the remaining s were excluded and were includedoverall evidence is strong for recommending subcutaneous hydration infusions for olderadults weak for pediatric patients and inconclusive for palliative patients there is strongevidence for medications weak evidence supporting medications however there areeight medications with inconclusive evidence to make a recommendation and four medications not appropriate for subcutaneous delivery one 101371 pone0237572 august one 0cproprietor of infusion excellence consulting whoseservices are unrelated to this research bg ownsand is employed as senior consultant and directorof education by clinical pharmacy partners aneducational and consulting firm for infusiontherapy providers and clinicians the funders hadno role in study conception design data collectionand analysis decision to publish or preparation ofthe manuscript the specific roles of these authorsare articulated in the ˜author contributions™ sectioncompeting interests db reports personal andconsulting fees ie travel and honoraria and nonfinancial support from 3m including researchgrant in unrelated field angiodynamicscardiomed medical supplies covalon freseniusand excelsior medical db™s former employermedical pharmacies provides infusion serviceshowever db affirms research was conductedindependent of her employer™s servicesproductsand of her current consulting business mc reportsgriffith university has received unrestrictedinvestigator initiated research or educational grantson mc™s behalf from product manufacturersbaxter becton dickinson and company centurionmedical products and entrotech lifesciencesunrelated to this research ds reports no conflictsbg reports personal and consulting fees ieconsultancy travel and honoraria from nutrishareand covalon bg affirms no relationshipspresenting competing interests or influences thisdoes not alter our adherence to one policieson sharing data and materialssubcutaneous hydration and medication infusionssubcutaneous access should be considered alongside intravenous therapy for hydration inolder adults and several medications there are additional benefits in terms of ease of useand costeffectiveness of this mode inclusion of subcutaneous access in clinical guidelinesmay promote uptake of this route to help preserve vessel health of vulnerable patients further highquality research is needed to inform subcutaneous infusion therapy in a variety ofpopulations including pediatrics and palliative care and medications and clarifying themechanism of deliveryintroductioninfusion therapy is a common treatment modality to deliver medications and fluids in theacute and home care settings and is gaining prevalence in the longterm care setting traditionally these parenteral therapies have been delivered via the intravenous route howevervenous depletion is a growing concern with an increasing aging population and patients withlongterm complex comorbidities [“] the problems of venous depletion are compoundedby unnecessary peripheral venipuncture which add to patient physical and psychologicaltrauma compromised intravenousrelated outcomes suboptimal use of healthcare resourcesand increased costs [ “] additionally infusion therapy is moving beyond the boundariesof the acute care and home care sector to hospices and longterm care facilities recentlypublished vascular access planning tools are either oriented primarily to the acute care settingor do not address the option of subcutaneous access [ ]the infusion therapy standards of practice recommend consideration of subcutaneousaccess for the administration of isotonic solutions and for continuous opioid and other infusion therapiesmedications eg immunoglobulin therapy subcutaneous access isachieved through the placement of a small catheter in the subcutaneous tissue with the infusate absorbed from this space into the circulatory system this route has the advantages ofrequiring a small catheter and less technical insertion skills than that used for intravenousaccess with its ease of application lending to its use in multiple settings additionallytime to place the catheter is less and fewer complications are likely resulting in cost benefits[ ] although practiced since there has been a slow uptake in part due to a lack offamiliarity with the technique among physicians and healthcare professionals and perceivedsuboptimal outcomes eg hypovolemic shock due to inappropriate use of hydration solutions [ ] in our scoping literature search to prepare for a systematic review of primarystudies numerous systematic reviews were identified however each either had a narrowscope addressing one treatment eg fisher and colleagues™ review of iron overload management or provided limited literature search or limited evidence eg duemsnoreiga andblasco™s review of subcutaneous fluid and drug deliverygiven the world™s aging population health care systems rapidly changing diversity in practice setting and complexity of care and patient health conditions the uptake and use of subcutaneous continuous infusions may help address these issues and challenges the aim of ourstudy is to determine the effectiveness safety acceptability and efficiency related to the use ofsubcutaneous infusion sci as an alternate route to intravenous for the management of conditions or treatments such as dehydration and palliation for children and adults in all care settings through a synthesis of systematic reviews of studies a secondary objective is to identify one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsthe mechanisms of subcutaneous fluid and drug delivery that facilitated achieving these outcomes this will provide an uptodate and rigorous review of subcutaneous infusion therapymaterial and methodswe adopted the systematic review of systematic reviews methodology from the joannabriggs institute™s to conduct this review the study protocol was registered with theprospero database registration number crd42018046504 and we report the reviewaccording to the preferred reporting items for systematic review and metaanalysisprisma guidelines s1 table eligibility criteriawe considered all types of systematic reviews which included the following characteristics¢ a clearly stated set of objectives with an explicit reproducible methodology¢ a systematic search that attempts to identify all studies that would meet the eligibilitycriteria¢ an assessment of the validity of the findings of the included studies eg assessment of riskof bias and confidence in cumulative estimates and¢ systematic presentation and synthesis of the characteristics and findings of the includedstudies metaanalyses where some of the primary studies in the reviews were duplicated we did not exclude thereviews as they had different aims and objectives and thus added to the overall understandingof sci to meet the aims of this current systematic review we did however only include the primary studies™ findings once in our analysis and synthesis expert opinionconsensus andbench research s and editorialscorrespondence were excluded no restrictionsregarding age gender diagnosis geographical location or healthcare setting were appliedwe included reviews that assessed interventions that used subcutaneous infusion for aduration of around hours or more as an alternate route for fluid or medication therapy subcutaneous infusion is defined as the delivery of fluids or medication into the subcutaneousspace for absorption into the circulation via œperfusion diffusion balance between hydrostaticosmotic pressure and lymphatic drainage p118 an infusion of around hoursduration was determined from considering the studies in a review by caccialanza where the range of infusion was hours to greater than days during the full textscreening numerous intermittent sc insulin vs continuous sc insulin systematic reviewswere identified and excluded because they did not meet our inclusion criteria of sci as analternate route reviews that included other routes as comparators such as intravenous andintraosseous were excluded if data on subcutaneous infusions could not be extractedseparatelyoutcome measuresthe primary outcomes of interest investigated included¢ effectiveness defined as clinical response to therapy eg cureimproved clinical failure orno change completion of therapy within prescribed time frame¢ safety defined as medication or vascular access adverse event eg abscess erythema bruising electrolyte imbalance edema infection pain fluid overload vascular collapse and one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsroute failure survival status eg died of underlying condition other causes lost to followup or status unknown and complications related to treatment eg unplanned hospitalreadmission related to treatment¢ acceptability defined as patient andor health care provider preference satisfaction or perceived benefits of subcutaneous therapy¢ efficiency defined as healthcare resource utilization including costs of infusion therapy supplies and treatment timesecondary outcomes included indications for subcutaneous infusion therapy medicationsolution type infusion rates volumes and duration subcutaneous access sites dwell timesand infusion control devices usedsearch strategya systematic search was conducted november and updated in june of reviews from as recommended by aromataris from the following databases excerpta medica database embase pubmed cumulative index to nursing and allied health literaturecinahl cochrane database of systematic reviews joanna briggs institute of systematicreviews and database of s of reviews of effects dare search terms included combinations of medical subject headings mesh and key word terms [˜subcutaneous infusion™or hypodermoclysis or ˜subcutaneous therapy™] and the term ˜systematic review™ with theassistance of a university librarian study resources limited inclusion to only english languageas listed in s2 table one author of a relevant was contacted to establish the status ofthe review however the review had not been completedthe title and of each were scanned independently by one reviewer ds andfull copies of s of potentially eligible reviews were obtained full texts of these reviewswere then screened independently by two reviewers mc and db against the review selectioncriteria disagreements were resolved by discussion between these reviewers and with consultation by a fourth reviewer and subject expert bgdata extraction and quality appraisaldata were extracted and assessed independently by the reviewers in pairs [bg db] [mcds] with anomalies reconciled by agreement data were obtained primarily from the systematic reviews although primary studies were consulted for critical missing data eg missinginfusion properties data extracted included authoryear aim design and number of studiesincluded search strategy population interventioncomparator quality appraisal of primarystudies infusion characteristics outcomes as previously defined key findings study limitations funding and smethodological rigour of each review was independently assessed by four reviewers in pairsas above using the amstar tool this is a 16item tool used to appraise the quality ofsystematic reviews and is frequently used in cochrane overviews a score of overall confidence high moderate low and critically low was assigned by the reviewers to depict the accuracy and comprehensiveness of the data summary and critical methodological flaws thisassessment informed the final grading of the recommendationsdata analysis and synthesisdue to the different interventions outcomes and outcome measures a metaanalysis was notfeasible quantitative outcome data are provided and synthesized where possible only data one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsfrom randomised controlled trials rcts and prospective cohort studies were included todetermine subcutaneous hydration outcomes however reviews exploring medications alsoincluded retrospective studies and case reports the jbi grades of recommendation were usedto derive the grading score to inform the strength of recommendation for the interventionjoanna briggs institute levels of evidence and grades of recommendation working party data synthesis for hydration and medications are provided as a final summary ofrecommendations grade a indicates the intervention™s desirable effects outweigh undesirableeffects with adequate supporting evidence a weak recommendation grade b depictsdesirable effects outweigh undesirable effects although this is not as clear or evidence not ofhigh quality where there was insufficient evidence we determined that a recommendation isinconclusiveresultsthe search strategy yielded potential systematic reviews fig of which wereexcluded through and duplicate screen of the remaining s were excludedafter full text examination against inclusion criteria in the full text screening for reasonsdescribed as above as listed in s3 tablecharacteristics of included studiestable details the systematic reviews of subcutaneous infusion of medications andor fluidswith focused on medications only on fluids and on both types of therapies the publication dates of reviews ranged between and [ ] most reviews focused onadult populations generally or specifically for example pregnant women and adults withamyotrophic lateral sclerosis als two reviews however are specifically paediatric [ ]eight all ages [ “] two focused on older adults and paediatrics [ ] andone where age was not reported the systematic reviews incorporated a diversity of study designs and quality levels fromcase studies to systematic reviews and randomised controlled trials however only reviewsincluded only randomised controlled trials [ “] similarly sample sizes varieddepending on the number of sci studies included in the review synthesise of findings withvery few metaanalyses performed due to varied study designs outcomes and sizemethodological qualitythe amstar quality scores of the included reviews are described in s4 table while reviews achieved an overall confidence rating of high sixteen of the reviews had critical methodological weaknesses studies with moderate low and studies rated as criticallylow the main deficiencies noted were failure to report a priori protocol study design selection full details of excluded studies funding sources of primary studies and results reflectingrisk of bias assessment the cochrane risk of bias tool or an adaptation was the most commonly used tool by systematic review investigators to determine quality of the primary rctstudies n while only provided a narrative discussion of quality most reviewauthors noted that results should be interpreted with caution due to study limitationsmechanisms of subcutaneous accesslimited data were reported on the materials and techniques used to achieve subcutaneousaccess for hydration or medication therapy table describes the characteristics of sc devicedesign and dwelltime subcutaneous anatomical sites and insertion techniques and infusion one 101371 pone0237572 august one 0csubcutaneous hydration and medication infusionsfig prisma flow chart the diagram details our literature search and screening process for selection of systematic reviews included in thesystematic review adapted from moher d liberati a tetzlaff j altman dg the prisma group preferred reporting items for systematicreviews and metaanalyses the prisma statement med e1000097 101371 pmed1000097101371 pone0237572g001delivery systems in relation to studies reporting on hydration therapy no studies comparedthe safety and efficacy of the various characteristics including mode of delivery the findingsfor hydration and medication sci are presented separatelysubcutaneous hydrations5 table describes the indications and contraindications for sci for hydration as described inthe studies the most common indicator was mild to moderate dehydration with the mostcommon contraindication being rapid high fluid volume requirements one 101371 pone0237572 august one 0ctnatropmiyllacinilcmumnmiidecneirepxedahohwstneitaphtlaehdnaefilfoytilauqsuhtgivinoseacimetsyslatipsohnehwnoitpecrepfostroperdesaercnievahyamemohotdegnahcgividesabelihwseacimetsysdnalacolseidutsgicsdesabdellorneevahyamseiduts¢™stneitapnitnemevorpmi¢gicsnostneitaperomderreferpdnadetarelotdnacirtaidepybgivinahtsnoitcejnigicsdesabemoh¢stneitaptludamuresggielbatpeccaseveihcayparehttnemecalpergics¢sleveldnaytienegoretehlaitnatsbusseidutsssorcanosirapmocsnoitatimilllacigoodohtemsaibfoksiretaredomecnediveytilauqwol¢mrettrohsnoylnoatadloq¢puwollofnotcefferettebaevahyamlanoitnevnocnahtloqyparehtdesisehtnysseidutsylno¢suoenatucbussuounitnoc¢icsottnavelerenositrocordyhfonoisufnicsotviderapmocstcron¢derosnopsyrtsudni¢daehotdaehecnereffidehtniecnacifingislacitsitatssawerehthguohtla¢aenpsydecuderdnafoesuehthtiwdwmnillarevoehtsnilcycatsorpehtteemtondidtceffenoitarudpuwolloftrohsllasedulcnisisylanaatemsnoitnevretnifosepyt¢¢caidracgenoitcnufcaidracerusserplairtathgirxednievorpmiyamlanitsorpertcs¢gnitarehtgnitagitsevnirofairetircfoytilauqllacigoodohtemytilauqwolseiduts“ytilauqetaredomytilauqhgihrnsgnittesnodesabairetirc”stcrnerdlihcdnastludatrohocborenarhcocyranibnodesabseidutsydobitnayramirphtiwseicneicifedgnisuselbairavonsey“segnareziselpmassesabatadotpuetadyraunajotelbacilppaseidutsseidutslatotweiveremoctuo]noitatic[snoitacidemfonoisufnicsseidutsginilubolgonummiinassahobalslairtlacinilcevitcepsorp¢¢strohocevitcepsorter¢strohocytefasdnassenevitceffevisusrevcs][saibfoksirhgih”muidem”seidutslanoitavresboelacsawattoeltsacwenseidutslanoitavresborofytilauqriafstcrnielpmasycneiciffusniylujotpuetadsuounitnoc“egnarezisrnsgnittesdnaegacsenositrocordyhstcrretnecitlumlacinilclebalnepo¢ydutsrevossorc¢noisufnissenevitceffestnemtaertlootborenarhcoc“stcrlanerdahtiwstneitapsesabataddedulcniseidutsdiocitrococugl][lafonlagnitroperdnanoitcelessisongaidsraey“sehcraesdnahicsottnavelerycaciffelinitsorpertfoksirwolborenarhcocegaegaeziselpmassesabatadstcrtcrnilcycatsorp][senrabsaiblairetrayranomuplotpusetadnoisnetrepyhrebmetpesytefasdnasnoitatimilnoisulcnocllarevos™rohtualootlasiarppaytilauqnoitalupopygetartshcraesforebmundnangisednoitnevretnimairohtuadaelsweivercitametsysfoscitsiretcarahcelbatsubcutaneous 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one 101371 pone0237572 august one 0csubcutaneous hydration and medication 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one 101371 pone0237572 august one 0chtiwlyamedirohcmuissatopotdeddaylefasebnoituacstseggusecnedivedetimli¢icsrofdetiusyllaediebyamcdh¢noitardyhedfotnemtaertehtodsecnalabmicilobatemsadetcerrocebotevahtonyletaidemminodesabetuorrehtiegnisuylralimisdebrosbasidiufl¢stcrgnitaertrofnoitponasicdh¢stneitapnehwnoitardyhedekatnilaroetauqedanievahstceffeesrevdasnoituosleerfetylortcelediova¢etomorpotesadinorulayh¢sniamernoitprosbadevlosernufonoitaredisnocasedulcniecnediveehtfoytilauqehtroopsa“egnaregarngnittesseidutselbaliavafoytirojam¢etylortcelehtiwcdh¢ytilauqroopfoeraefassisdiulfignniatnocnoissucsiddetropereziselpmas“setadloottnemssessaonnoitardyhedhtiwstludaesabatadseidutsytefasnoitardyhcs][nohcorseiresstroperesa Answer:

Colon_Cancer

tenascinc tnc is an extracellular matrix ecm glycoprotein that plays an important rolein cell proliferation migration and tumour invasion in various cancers tnc is one of themain protein overexpressed in breast cancer indicating a role for this ecm molecule in cancer pathology in this study we have evaluated the tnc lossofffunction in breast cancercells in our approach we used dsrna sharing sequence homology with tnc mrna calledatnrna we present the data showing the effects of atnrna in mdamb231 cells bothin monolayer and threedimensional culture cells treated with atnrna were analyzed forphenotypic alterations in proliferation migration adhesion cell cycle multicaspase activation and the involvement in epithelial to mesenchymal transition emt processes as complementary analysis the oncogenomic portals were used to assess the clinical implication oftnc expression on breast cancer patient™s survival showing the tnc overexpression associated with a poor survival outcome our approach applied first in brain tumors and then inbreast cancer cell lines reveals that atnrna significantly diminishes the cell proliferationmigration and additionally reverses the mesenchymal cells phenotype to the epithelial onethus tnc could be considered as the universal target in different types of tumors wheretnc overexpression is associated with poor prognosisa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation wawrzyniak d grabowska m głodowiczp kuczyński k kuczyńska b fedorukwyszomirska a downregulation oftenascinc inhibits breast cancer cells developmentby cell growth migration and adhesionimpairment one e0237889 101371 pone0237889editor lucia r languino thomas jeffersonuniversity united statesreceived may accepted august published august copyright wawrzyniak this is anopen access distributed under the terms ofthe creative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportingintroductioninformation filesfunding this work was supported by the ministryof science and higher education of the republic ofpoland by know program kk was supported byncbr program powr03020000i03216competing interests the authors have declaredthat no competing interests existthe tumor microenvironment is composed of the surrounding stromal cells such as endothelialcells in blood vessels immune cells fibroblasts and the extracellular matrix ecm [ ] during carcinogenesis is often perturbed and deregulated while during embryonic development isstrictly controlled to maintain homeostasis in tumors the composition of the ecm differsfrom that of normal tissue and enables new interactions that affect the function of cancer cellsand are critical in modulating invasion associated with cell migration and growth the tumorassociated ecm presents several tumorassociated antigens that are generally more abundant one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentand possibly more stable than those of the cell surface [“] consequently these proteins represent possible valuable targets for tumor imaging and therapy [ ] ecm proteins such as fibronectin fn and tenascin have isoforms that are expressed in a tissue specific manner generatedby alternative splicing of their primary transcripts one of the most consistent isoform changesin the ecm of many tumors is the upregulation of the glycoprotein tenascinc tnc tncalongside tenascinx tnx tenascinr tnr and tenascinw tnn are members of wellconserved among vertebrates tenascin family tn [“] numerous isoforms of tnc can beproduced through alternative splicing of nine fibronectin type iii regions between repeats and at the premrna level there is a considerable amount of literature on the contribution of different splicingdependent tnc domains in specific biological functions changes in thetnc isoforms expression pattern have been then described in a number of malignancies andtheir nature appears to be tumortype specific recent studies have demonstrated that somesplice variants are specific to diseased tissues [“] in breast tissues expression of two tncvariants one containing domain d and the other both b and d was found to be associated withinvasive phenotype tnc promotes cell migration angiogenesis inhibit focal contact formation and also act as a cell survival factor [“] its importance was found in the development and progression of different types of neoplasm including colon and breast cancerfibrosarcoma lung cancer melanoma squamous cell carcinoma bladder cancer and prostaticadenocarcinoma [ ] tnc is also highly expressed in highgrade gliomas which correlatesas well with the invasiveness of glioma cells [“] in the brain it is important for the development of neural stem cells [ ] and moreover is suspected to be a potential marker for glioblastoma multiforme gbm stem cells gsc previously we have shown that tnc is overexpressed in gbm and can be a good target inrnai approach with 164nt long dsrna complementary to the mrna of tnc which wecalled atnrna we conducted the experimental therapy for gbm patients the discoverythat tnc presents a dominant epitope in glioblastoma prompted us to investigate the potentialof atnrna to block the tnc expression and its effect on the growth of human breast cancers where tnc overexpression was also established and linked with the highest malignancyinvasion capability and metastasis ability this view is supported by mock who showedthat gbm patients with antibodies against the egflike repeats of tnc antibody targetvcedgftgpdcae have a significantly better prognosis than other patients thus weassumed that in the light of the satisfactory results of brain tumors experimental therapy breastcancer could be the next possible object of interest to establish the atnrna approachhere we demonstrate that atnrna approach can be successfully used in breast cancercells impairing the basic hallmarks of tumor cells with the performed analysis of proliferation migration rate multicaspases induction pathway cell cycle analysis spheroids viabilityand the involvement of tnc in emt induction we have then interrogated the impact of tncon breast cancer growth showing its potency to be also the promising therapeutic target inbreast cancer treatmentresultsoncogenomic in silico analysis reveals the tnc correlation with poorsurvival of breastcancer patientsto look deeper into the tnc function we performed the analysis of genomewide breast cancerdata with available oncogenomic portals such as gepia the human protein atlas cbioportaland ppisurv based on the status of three important receptors conventionally used for breastcancer subtyping ie estrogen receptor er progesterone receptor pr and human epithelialreceptor her2 breast cancer is classified as luminal a luminal b her2 positive and triple one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentnegative œbasallike triplenegative and her2overexpressing breast cancer yields a poorpatient prognosis because of a high incidence of metastases disease progression and resistanceto current chemotherapy regimens we first compared the expression level of tenascincin subtypes of breast cancer using gepia program fig a in s1 file mrna level of tncwere higher in triplenegative and her2 subtypes compared to the luminal a and luminal bsubtypes which have a better prognosis for patient survival therefore we chose mdamb231cells as a model for in vitro experiments because it is the most invasive cell line from breast cancer models mdamb231 cell genome clusters with the basal subtype of breast cancer sincethe cells also lack the growth factor receptor her2 they represent a good model of triplenegative breast cancer what is important adams showed that only invasive cell linessuch as mdamb231 or mdamb468 express tenascinc whereas the tumor cell lines witha low invasive capacity mcf7 and t47d do notas a next step we compared the expression levels of tenascin genes tnc tnn tnrtnxb in invasive breast cancer using gepia program fig 1a mrna level of tnc washighly expressed in breast cancer tissue brca interestingly expression levels of tnn andtnxb were significantly lower in breast cancer tissues fig 1a there was no significant difference in tnr gene expression between breast cancer and nontumor tissueswe also examined the expression of tenascin proteins in normal and malignant tissues byquerying data from the human protein atlas tnc in most cases and partly tnn wereexpressed at medium levels whereas tnr was not detected fig 1b and 1c tnc and tnrlevels were undetectable in samples from normal breast adipocytes glandular and myoepithelial cells taken together our results demonstrate that mrna and protein levels of tnc is relatively higher in invasive breast cancer tissues than those in normal tissuesthe cbioportal analysis enabled to look for the mutations in the tnc gene it appeared thattnc gene mutations measured for breast cancer patients are present as somatic mutation only in cases since these mutations seem to be irrelevant for breast cancer wedid not perform any further analysiswith ppisurv portal we looked through the transcriptomic data to correlate the tncexpression with the different clinical parameters such as survival or prognosis of the canceras the initial step of analysis we performed the alignment of tnc and other proteins from thetenascin family such as tenascinx tnx looking for the homology between these two proteinsat the top of that we made the analysis of the homology between the tnc and tnx with the relation to atnrna sequence the alignment of these two ecm proteins shows that they shareonly limited number of nucleotides query cover and for short and long transcriptionalvariants in the protein nterminus region respectively the sequence alignment analysis clearlyshow the atnrna matching exclusively to the tnc sequence identity fig 2appisurv analysis based on the kaplanmeier statistics showed very clearly the strong correlation with tnc expression and patients survival the high expression level has a greatimpact on the shorter survival for the patients thus suggesting also that tnc can be also considered as the prognostic factor for breast cancers p fig 2b at the same time weanalyzed also the available data for the tnx the results showed an inverse correlation oftnxb mrna expression and survival p fig 2c analysis was carried out on thegroup of patient n for tnc and n for tnxbatnrna mediates the downregulation of tnc mrna and proteinexpression in breast cancer cellsto achieve downregulation of tnc expression in breast cancer cell line transfection withvarious concentration of atnrna and nm was performed h after one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig tenascin is highly expressed in breast invasive carcinoma tcgabrca a messenger rna levels of tnc tenascinctnn tenascinw tnr tenascinr tnxb tenascinx genes in specimens from patients with invasive carcinoma of the breast one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentvs nontumor samples rna sequencing data were retrieved from the database of tcga and analysed using the gepia geneexpression profiling interactive analysis online web server httpgepiacancerpkucn the red boxes represent cancer specimensgrey boxes represent healthy breast specimens significance value � p b summary of tenascin expression patterns in breastcancer tissues and healthy breast determined by immunohistochemical staining data were retrieved from the human protein atlasdatabase case numbers of invasive breast cancer are shown na not available nd not detectable in œhealthy breast column œmeans that tnc and tnr are not detected in nontumor samples results in normal breast are based on immunohistochemical stainingof a single sample c representative images of immunohistochemical staining for tnc tnn and tnr in breast healthy tissue andinvasive breast carcinoma specimens the images shown here are of the tissue sections from tissue microarray arrays tmas stainedwith appropriate antibodies tnc“cab004592 tnn“cab010907 tnr“cab022343 all the images were taken at × magnification101371 pone0237889g001transfection the expression level of tnc was examined by qrtpcr analyses significantdownregulation of tnc mrna expression was observed compared to control treated withscrambled rna the level of tnc was decreased from at a concentration of nmatnrna up to for cells treated with nm atnrna in comparison to the controlp fig 3afig the oncogenomic analysis of the survival association with tenascinc and tenascinx in breast cancer asequence alignment of tenascinc versus tenascinx with their relation to atnrna the sequence alignment analysisclearly show the atnrna matching exclusively to the tnc sequence identity relation of tnc b and tnxbc gene expression to survival of breast cancer patients survival analysis performed with the use of a dataset breastcancer geo gse7390 and gse3494 deposited in and tools available from the ppisurv web portal tnxbl longtranscription variant tnxbs short transcription variant101371 pone0237889g002 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig expression level of tnc and immune response genes after atnrna treatment in mdamb231 cell line a relative expression level of theexpression of tnc oas1 oas3 rig1 ifi16 and tlr3 established by qrtpcr relative expression was calculated using the“δδcp method statisticalevaluation of atnrna versus scrambled sirnas ccontrol cells was performed using oneway anova followed by tukey™s posthoc test effect of poly ic μgml on immune response genes oas1 oas3 rig1 ifi16 tlr3 in the figure presented as purple bars the results for hprtnormalized expressionof mrna are expressed as fold change of target gene expression relative to the control without poly ic treatment which is defined as b the proteinexpression levels of tnc and hprt c western blot analysis reveals efficient tnc silencing in mdamb231 cells with atnrna compared to cells treatedwith sirnas ccontrol the data represents the means ± sd from independent experiments significance value � p �� p ��� p 101371 pone0237889g003the qrtpcr analysis was also supported by direct analysis of the protein expression levelwe have observed already a decrease in tnc protein expression upon 50nm atnrnatreatment the highest concentration 100nm used led to the dramatic drop of the protein one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentexpression measured as the of the decrease fig 3b and 3c these observations were fullyconsistent with relative tnc expression level measured by qrtpcrinterferon response to atnrnato establish interferon induction in breast cancer mdamb231 cultured cells we looked forinterferon stimulated genes isg including oas1 oas3 rig1 tlr3 and ifi16 genes theanalysis was carried out with the qrtpcr fig 3a changes after atnrna measured byqrtpcr were not significant as shown basically for all of the genes in the concentrationrange of “ nm in parallel a synthetic form of dsrna polyipolyc poly ic wastransfected as a positive control poly ic has been used extensively as a tlr3 ligand to induceantiviral response [“] we showed that transfection with poly ic μgml efficientlyinduced the expression of immune response genes oas1 oas3 rig1 ifi16 tlr3 inmdamb231 cells this enhancement in mrna expression was “7fold higher in poly ictreated cells than in untreated cells fig 3a purple barstnc knockdown inhibits cells proliferation and leads to the changes inmigration rate and adhesion potential of breast cancer cellsin order to investigate the involvement of tnc on breast cancer cells proliferation mdamb cell line was treated with atnrna and the realtime cell proliferation assay was performed the cells ability to proliferate was measured for h we have noticed time and concentrationdependent decrease in proliferation rate the most effective concentration ofatnrna was nm with decrease from “ after and h respectively fig 4anoteworthy nm and nm of atnrna was already sufficient concentration for the efficient inhibition of breast cancer cells proliferation the dosedependent effect of atnrna inmdamb231 proliferation potential resulted in standard sigmoidal doseresponses with ic50of ± nm after h ± nm after h and ± nm after h of treatmentfig 4bto get more insight into the downregulation of tnc expression by atnrna on themobility of breast cancer cells realtime measurements of migration was carried out wefound that downregulation of tnc expression by atnrna significantly impaired the cellmigration in breast cancer cell lines fig 4c the results were quantitatively assessed during h of experiment and showed that mdamb231 cells transfected with atnrna had thelowest motility beginning from h post transfection it was established that atnrnadelayed the migration of mdamb231 cells by ± h ± h ± h and ± h with and 100nm concentration respectively notably the most effective concentration which affected the migration potential of the cells was 10nm when compared to the control the observed delay was ± hsince tnc is implicated also in cellmatrix attachment we further looked at the adhesionability of atnrna treated cells the cells were conducted to realtime adhesion assay withxcelligence system compared with the controls tnc knockdown resulted in increased cellsadhesion on average fig 4dtnc promotes apoptosis and is involved in cell cycle regulation in breastcancer cellsto determine additionally the effect of atnrna on the cell cycle progression themdamb231 cells were treated with different concentrations of atnrna for “ h andthe cell cycle analysis was assessed by muse1 cell analyzer cells transfected with atnrna one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig activity of atnrna in proliferation migration and adhesion proliferation of breast cancer in culture was monitored in realtime using xcelligencesystem a impedance was recorded every min but to improve the clarity of the graphs only every fourth readout was plotted data show the mean ± sd ofthree independent measurements b dosedependent effects of atnrna on proliferation was evaluated using nonlinear regression by fitting experimentalvalues to sigmoidal bellshaped equation c migration of mdamb231 cancer cells was studied using xcelligence system serumdepleted cells weretransfected with increasing concentrations of atnrna from to nm or scrambled sirnas ccontrol impedance ci values of each experimentalcondition was recorded over time plotted against time fitted to fourparameter logistic nonlinear regression model and et50 was calculated for each atnrnaconcentration to generate doseresponse curves et50 value was normalized to the data obtained for untreated cells and plotted as normalized half maximaleffective time et50 of cell migration against atnrna concentrations d adhesion of mdamb231 cell line was observed in realtime using xcelligencesystem graph shows the final impendence values minus the initial values for the respective samples differences between ci values for atnrna treated andcontrol cells were statistically evaluated using oneway anova followed by tukey™s posthoc test symbols above the bars significance value ��� p compared to cells treated with scrambled sirnas ccontrol101371 pone0237889g004showed the cell cycle distribution with the concentrationdependent decrease in cell numberin g0g1 phase increased in s phase and unchanged in g2m phase compared to the cellstransfected with unspecific control rna fig 5a atnrna impacted the cell cycle by almostdoubling the cells sphase fraction from to for the highest atnrna concentrationthus resulted with the cells arrest in s phase the cell cycle analysis proved the nontoxic effectof atnrna since we did not observe the increase in g1 population s phase arrest persistsfollowing up to h of atnrna treatment fig 5bthus to examine whether atnrnainduced apoptosis would be associated with the caspases activation the expression levels and activity of caspases such as caspase1 and in the atnrnatreated mdamb231 cells were assessed using muse1 cell one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on breast cancer cell cycle phase analysis mdamb231 cells were transiently transfected with increasing concentrations ofatnrna from to nm or scrambled sirna ccontrol for a and h b the cells were then fixed and added with propidium iodide pirnase a staining solution and analyzed in muse1 cell analyzer using modfit lttm software a percentage of cell distribution in each cell cycle phase wassummarized and shown the cell cycle distribution profile image is shown as a representative result of three independent experiments101371 pone0237889g005analyzer as shown in fig 6a breast cancer cells treated with atnrna exhibited enhancedmulticaspase activity in a concentrationdependent manner the multicaspase activity was ± ± ± and ± respectively at and nm atnrna concentration compared with the control fig 6b thus we observed almost5fold increase of the population of the apoptotic cells with the lowest atnrna concentration whereas almost 12fold with the highest oneatnrna has an impact on spheroids integrityto visualize the involvement of tnc in tumor formation the 3d culture model was appliedsince 3d cell culture models mimic better the in vivo behavior of cells in tumour tissues andare excellent surrogates to predict tumorigenic potential in vivo the ability to spheroid maintenance of breast cancer cells was assessed after atnrna treatment we have observed that one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on multiple caspase activation caspase1 and in mdamb231 cell linebreast cancer cells were transiently transfected with increasing concentrations of atnrna from to nm orscrambled sirnas ccontrol for h the transfected cells were then incubated with muse1 multicaspase reagent followedby analysis of the percentage of cell population in live caspase caspasedead and dead in muse1 cell analyzer a thepercentage of live caspase caspasedead and dead cells profile image is shown as a representative result from one of threeindependent experiments b the graphical representation of percentage of live and exhibiting caspase activity cellpopulation transfected with atnrna and scrambled sirnas statistical evaluation of atnrna versus scrambled cells one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenttreated with scrambled sirnas was performed using oneway anova followed by tukey™s posthoc test significance value��� p compared to scrambled control ccontrol error bars represent sd101371 pone0237889g006downregulation of tnc led to the disintegration of the spontaneously forming spheroids ofmdamb231 the clearly visible effect on the spheroid viability was observed even with thelowest atnrna concentration the increased concentrations of dsrna had a great impacton spheroids integrity resulting in structure disintegration at the highest concentration of nm fig 7a the atnrna application influenced the spheroid volume and shape displaying the total shrinking of the compact structure into the small fragments with the highestatnrna concentration fig 7ato have a better insight into the mdamb231 spheroids structure and the atnrnaimpact on their viability the confocal microscopy imaging was assessed the analysis of fluorescent labelling with greenfluorescent calceinam of living and dead cells within the spheroid with the livedead viabilitycytotoxicity kit was carried out as revealed by the image ofthe untreated mdamb231 cells compact multicellular spheroids were obtained fluorescence images revealed the overall morphology of the mdamb231 spheroids fig 7b thecell density in the core of the untreated spheroid was high and no dead cells were identifiedthe similar pictures were obtained for the control furthermore all conditions with differentatnrna concentrations resulted with losing the spheroid density increased dimensionsand appearing a higher population of dead cells it is worthy of note that the spheroids treatedwith increasing atnrna concentrations did not display a smooth contour following h oftreatment and subsequently their round shape was markedly altered by the treatments afterthe treatment with 50nm concentrations the spheroids showed the strong overrepresentationof dead cells fig 7b thus the 100nm concentration of atnrna was most likely too highfor the cells viability and we were not able to keep the spheroids in shape that would allow forthe imagingtnc is involved in emt processesas the consequence of these finding we analyzed the expression level of proteins involved inemt processes we took into account two main emt markers ecadherin and vimentinwestern blot analysis shows the significant increase of ecadherin level followed by the dropof the expression of vimentin protein fig 8a these observations were concentrationdependent showing the highest efficacy for atnrna at the concentration of 100nm for ecadherin expression similarly for vimentin we have observed the highest decrease of expressionupon atnrna transfection at 100nm concentration fig 8bdiscussiontnc is the main ecm protein of various tumors and its overexpression is repeatedlyobserved in breast cancer cells both in vitro and in vivo indicating a role for this extracellularmatrix glycoprotein in neoplastic pathology moreover its high expression correlates withworsened patient survival prognosis in several cancer types in breast cancers severalstudies demonstrate that high expression of tnc is not only an indicator of poor prognosisbut also correlates with metastasis to distinct ans such as lymph nodes liver and lung [“] tnc plays a substantial role in emt that is believed to be a key mechanism in cancer progression whereby cancer cells acquire more aggressive behavior [ ] in human breast cancer specimens tnc is coexpressed with the mesenchymal marker vimentin themechanistic role of tnc in the process of emt remains poorly defined however studies one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effects of atnrna on viability and spheroid structure in mdamb231 cells a monolayer cultures weretransfected with indicated amounts of atnrna oligonucleotides and after hrs cellular spheroids of mdamb cells were generated from cells in perfecta3d1 96well hanging drop plate and cultured for up to hoursscale bars μm scrambled sirnas cscr b the viability of the atnrna transfected cells within spheroidsusing livedead cell imaging kit left and middle panels present live cells green and dead cells red respectively one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentthe right panels show merge of two fluorescent images scrambled sirnas cscr concentrations ofatnrna used for transfection fluorescence images were taken using leica tcs sp5 confocal laser scanningmicroscope and plan apo × na oilimmersion objective scale bars μm101371 pone0237889g007suggest that tnc can induce an emt like phenotype in mcf7 breast cancer cells via theαvβ6 and αvβ1 integrins [ ] many studies on various cancer tissues have demonstrateddownregulation of epithelial markers including ecadherin plakoglobin and cytokeratin aswell as the upregulation of mesenchymal markers such as ncadherin and vimentin andexpression of emt transcription factors snai and twist since these changes towardsmesenchymal phenotype could correlate with invasiveness metastatic potential and poorpatient™s outcome we have investigated the effect of tnc knockdown on the expression levelsof emt markers our results show that down regulation of tnc reverses the malignant phenotype of the cancer cells as the experimental result we observed the downregulation of mesenchymal marker”vimentin followed by the upregulation of epithelial marker”ecadherinthis indicates atnrna as a potential therapeutic agent which could switch the mesenchymal phenotype of breast cancer cells to the epithelial one inhibiting the ability to metastasisand invasion additionally it has been also shown that tnc as the ecm component plays alsofig the effect of tnc downregulation on emt process of mdamb231 cells a the protein expression levelsof ecadherin vimentin and gapdh b western blot analysis of atnrna effects on the emt process revealed asignificant increase in ecadherin level followed by the drop of the expression of vimentin protein the data representsthe means ± sd from independent experiments101371 pone0237889g008 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenta role in cell to cell or cellmatrix attachment most probably inhibiting the cells™ migration inapproach with atnrna it seems that tnc in breast cancer cell line plays an antiadhesiverole which would affect the cell migration and invasion ability in addition to emt processesthus tnc downregulation seems to enhances the adhesiveness of cancer cells showing thedirect involvement of tnc in cell adhesive properties targeting the tnc in potential therapymight be also highly beneficial since it has been already established that tnc maintain a stemcell niche in the brain tumors thus could promote the tumor cell invasion therefore its overexpression largely contributes to radiochemotherapy resistance and tumor recurrence infact it has been shown that targeting gbm invasion increases tumor sensitivity to temozolomide tnc also promotes stromal events such as the angiogenic switch and the formationof more but leaky blood vessels involving wnt signaling and inhibition of dickkopf1 dkk1in a neuroendocrine tumor model we observed significant atnrna“mediated downregulation of tnc was in concordance with the observed changes in proliferation and migration rates the results show thatthe atnrna transfected cells lose their ability to migrate thus showing the involvement oftnc in breast cancer invasiveness our data indicate also that the downregulation of tncexpression in mdamb231 cancer cell lines inhibits proliferation along with induction of celldeath we were able to determine the tnc impact on the apoptosis by measuring level of bothcaspases initiating intracellular events caspase2 and effector caspases3 and orend demonstrated that tnc causes cdk2 inactivation and blocks cell cycle progression from g1 phase to s phase of anchoragedependent fibroblasts by interfering withfibronectin“syndecan4 interactions for the proliferation of anchoragedependent cellsattachment to the ecm is required detachment of fibroblasts by a pure tenascinc substratum results in g1phase arrest by inactivation of the cdk2 complex in a ckidependent manner in contrast to fibroblasts proliferation of most tumor cells is stimulated by tnc this shows that the effect of tnc on proliferation is cell typespecific and suggests that in cancer cells the cdk2 complex is not repressed in the presence of tnc in leukemia breast carcinoma and glioma were found subpopulations of stemlike cells that support tumor growth in such cells adhesion on the tnc substratum may override the g0g1 and gls cellcycle checkpoints which may explain the increased proliferation rate the g1s transitionis enforced by cyclin ecdk2 activation via syndecan4 related signalling it has been shownthat tnc in tumor cells binds to the syndecan4 binding site in fibronectin thereby blockingsyndecan4 ligation releasing the tumor cells from the suppressive effect of fibronectin ontheir proliferation this would a

cancer7561

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: tenascinc tnc is an extracellular matrix ecm glycoprotein that plays an important rolein cell proliferation migration and tumour invasion in various cancers tnc is one of themain protein overexpressed in breast cancer indicating a role for this ecm molecule in cancer pathology in this study we have evaluated the tnc lossofffunction in breast cancercells in our approach we used dsrna sharing sequence homology with tnc mrna calledatnrna we present the data showing the effects of atnrna in mdamb231 cells bothin monolayer and threedimensional culture cells treated with atnrna were analyzed forphenotypic alterations in proliferation migration adhesion cell cycle multicaspase activation and the involvement in epithelial to mesenchymal transition emt processes as complementary analysis the oncogenomic portals were used to assess the clinical implication oftnc expression on breast cancer patient™s survival showing the tnc overexpression associated with a poor survival outcome our approach applied first in brain tumors and then inbreast cancer cell lines reveals that atnrna significantly diminishes the cell proliferationmigration and additionally reverses the mesenchymal cells phenotype to the epithelial onethus tnc could be considered as the universal target in different types of tumors wheretnc overexpression is associated with poor prognosisa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation wawrzyniak d grabowska m głodowiczp kuczyński k kuczyńska b fedorukwyszomirska a downregulation oftenascinc inhibits breast cancer cells developmentby cell growth migration and adhesionimpairment one e0237889 101371 pone0237889editor lucia r languino thomas jeffersonuniversity united statesreceived may accepted august published august copyright wawrzyniak this is anopen access distributed under the terms ofthe creative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportingintroductioninformation filesfunding this work was supported by the ministryof science and higher education of the republic ofpoland by know program kk was supported byncbr program powr03020000i03216competing interests the authors have declaredthat no competing interests existthe tumor microenvironment is composed of the surrounding stromal cells such as endothelialcells in blood vessels immune cells fibroblasts and the extracellular matrix ecm [ ] during carcinogenesis is often perturbed and deregulated while during embryonic development isstrictly controlled to maintain homeostasis in tumors the composition of the ecm differsfrom that of normal tissue and enables new interactions that affect the function of cancer cellsand are critical in modulating invasion associated with cell migration and growth the tumorassociated ecm presents several tumorassociated antigens that are generally more abundant one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentand possibly more stable than those of the cell surface [“] consequently these proteins represent possible valuable targets for tumor imaging and therapy [ ] ecm proteins such as fibronectin fn and tenascin have isoforms that are expressed in a tissue specific manner generatedby alternative splicing of their primary transcripts one of the most consistent isoform changesin the ecm of many tumors is the upregulation of the glycoprotein tenascinc tnc tncalongside tenascinx tnx tenascinr tnr and tenascinw tnn are members of wellconserved among vertebrates tenascin family tn [“] numerous isoforms of tnc can beproduced through alternative splicing of nine fibronectin type iii regions between repeats and at the premrna level there is a considerable amount of literature on the contribution of different splicingdependent tnc domains in specific biological functions changes in thetnc isoforms expression pattern have been then described in a number of malignancies andtheir nature appears to be tumortype specific recent studies have demonstrated that somesplice variants are specific to diseased tissues [“] in breast tissues expression of two tncvariants one containing domain d and the other both b and d was found to be associated withinvasive phenotype tnc promotes cell migration angiogenesis inhibit focal contact formation and also act as a cell survival factor [“] its importance was found in the development and progression of different types of neoplasm including colon and breast cancerfibrosarcoma lung cancer melanoma squamous cell carcinoma bladder cancer and prostaticadenocarcinoma [ ] tnc is also highly expressed in highgrade gliomas which correlatesas well with the invasiveness of glioma cells [“] in the brain it is important for the development of neural stem cells [ ] and moreover is suspected to be a potential marker for glioblastoma multiforme gbm stem cells gsc previously we have shown that tnc is overexpressed in gbm and can be a good target inrnai approach with 164nt long dsrna complementary to the mrna of tnc which wecalled atnrna we conducted the experimental therapy for gbm patients the discoverythat tnc presents a dominant epitope in glioblastoma prompted us to investigate the potentialof atnrna to block the tnc expression and its effect on the growth of human breast cancers where tnc overexpression was also established and linked with the highest malignancyinvasion capability and metastasis ability this view is supported by mock who showedthat gbm patients with antibodies against the egflike repeats of tnc antibody targetvcedgftgpdcae have a significantly better prognosis than other patients thus weassumed that in the light of the satisfactory results of brain tumors experimental therapy breastcancer could be the next possible object of interest to establish the atnrna approachhere we demonstrate that atnrna approach can be successfully used in breast cancercells impairing the basic hallmarks of tumor cells with the performed analysis of proliferation migration rate multicaspases induction pathway cell cycle analysis spheroids viabilityand the involvement of tnc in emt induction we have then interrogated the impact of tncon breast cancer growth showing its potency to be also the promising therapeutic target inbreast cancer treatmentresultsoncogenomic in silico analysis reveals the tnc correlation with poorsurvival of breastcancer patientsto look deeper into the tnc function we performed the analysis of genomewide breast cancerdata with available oncogenomic portals such as gepia the human protein atlas cbioportaland ppisurv based on the status of three important receptors conventionally used for breastcancer subtyping ie estrogen receptor er progesterone receptor pr and human epithelialreceptor her2 breast cancer is classified as luminal a luminal b her2 positive and triple one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentnegative œbasallike triplenegative and her2overexpressing breast cancer yields a poorpatient prognosis because of a high incidence of metastases disease progression and resistanceto current chemotherapy regimens we first compared the expression level of tenascincin subtypes of breast cancer using gepia program fig a in s1 file mrna level of tncwere higher in triplenegative and her2 subtypes compared to the luminal a and luminal bsubtypes which have a better prognosis for patient survival therefore we chose mdamb231cells as a model for in vitro experiments because it is the most invasive cell line from breast cancer models mdamb231 cell genome clusters with the basal subtype of breast cancer sincethe cells also lack the growth factor receptor her2 they represent a good model of triplenegative breast cancer what is important adams showed that only invasive cell linessuch as mdamb231 or mdamb468 express tenascinc whereas the tumor cell lines witha low invasive capacity mcf7 and t47d do notas a next step we compared the expression levels of tenascin genes tnc tnn tnrtnxb in invasive breast cancer using gepia program fig 1a mrna level of tnc washighly expressed in breast cancer tissue brca interestingly expression levels of tnn andtnxb were significantly lower in breast cancer tissues fig 1a there was no significant difference in tnr gene expression between breast cancer and nontumor tissueswe also examined the expression of tenascin proteins in normal and malignant tissues byquerying data from the human protein atlas tnc in most cases and partly tnn wereexpressed at medium levels whereas tnr was not detected fig 1b and 1c tnc and tnrlevels were undetectable in samples from normal breast adipocytes glandular and myoepithelial cells taken together our results demonstrate that mrna and protein levels of tnc is relatively higher in invasive breast cancer tissues than those in normal tissuesthe cbioportal analysis enabled to look for the mutations in the tnc gene it appeared thattnc gene mutations measured for breast cancer patients are present as somatic mutation only in cases since these mutations seem to be irrelevant for breast cancer wedid not perform any further analysiswith ppisurv portal we looked through the transcriptomic data to correlate the tncexpression with the different clinical parameters such as survival or prognosis of the canceras the initial step of analysis we performed the alignment of tnc and other proteins from thetenascin family such as tenascinx tnx looking for the homology between these two proteinsat the top of that we made the analysis of the homology between the tnc and tnx with the relation to atnrna sequence the alignment of these two ecm proteins shows that they shareonly limited number of nucleotides query cover and for short and long transcriptionalvariants in the protein nterminus region respectively the sequence alignment analysis clearlyshow the atnrna matching exclusively to the tnc sequence identity fig 2appisurv analysis based on the kaplanmeier statistics showed very clearly the strong correlation with tnc expression and patients survival the high expression level has a greatimpact on the shorter survival for the patients thus suggesting also that tnc can be also considered as the prognostic factor for breast cancers p fig 2b at the same time weanalyzed also the available data for the tnx the results showed an inverse correlation oftnxb mrna expression and survival p fig 2c analysis was carried out on thegroup of patient n for tnc and n for tnxbatnrna mediates the downregulation of tnc mrna and proteinexpression in breast cancer cellsto achieve downregulation of tnc expression in breast cancer cell line transfection withvarious concentration of atnrna and nm was performed h after one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig tenascin is highly expressed in breast invasive carcinoma tcgabrca a messenger rna levels of tnc tenascinctnn tenascinw tnr tenascinr tnxb tenascinx genes in specimens from patients with invasive carcinoma of the breast one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentvs nontumor samples rna sequencing data were retrieved from the database of tcga and analysed using the gepia geneexpression profiling interactive analysis online web server httpgepiacancerpkucn the red boxes represent cancer specimensgrey boxes represent healthy breast specimens significance value � p b summary of tenascin expression patterns in breastcancer tissues and healthy breast determined by immunohistochemical staining data were retrieved from the human protein atlasdatabase case numbers of invasive breast cancer are shown na not available nd not detectable in œhealthy breast column œmeans that tnc and tnr are not detected in nontumor samples results in normal breast are based on immunohistochemical stainingof a single sample c representative images of immunohistochemical staining for tnc tnn and tnr in breast healthy tissue andinvasive breast carcinoma specimens the images shown here are of the tissue sections from tissue microarray arrays tmas stainedwith appropriate antibodies tnc“cab004592 tnn“cab010907 tnr“cab022343 all the images were taken at × magnification101371 pone0237889g001transfection the expression level of tnc was examined by qrtpcr analyses significantdownregulation of tnc mrna expression was observed compared to control treated withscrambled rna the level of tnc was decreased from at a concentration of nmatnrna up to for cells treated with nm atnrna in comparison to the controlp fig 3afig the oncogenomic analysis of the survival association with tenascinc and tenascinx in breast cancer asequence alignment of tenascinc versus tenascinx with their relation to atnrna the sequence alignment analysisclearly show the atnrna matching exclusively to the tnc sequence identity relation of tnc b and tnxbc gene expression to survival of breast cancer patients survival analysis performed with the use of a dataset breastcancer geo gse7390 and gse3494 deposited in and tools available from the ppisurv web portal tnxbl longtranscription variant tnxbs short transcription variant101371 pone0237889g002 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig expression level of tnc and immune response genes after atnrna treatment in mdamb231 cell line a relative expression level of theexpression of tnc oas1 oas3 rig1 ifi16 and tlr3 established by qrtpcr relative expression was calculated using the“δδcp method statisticalevaluation of atnrna versus scrambled sirnas ccontrol cells was performed using oneway anova followed by tukey™s posthoc test effect of poly ic μgml on immune response genes oas1 oas3 rig1 ifi16 tlr3 in the figure presented as purple bars the results for hprtnormalized expressionof mrna are expressed as fold change of target gene expression relative to the control without poly ic treatment which is defined as b the proteinexpression levels of tnc and hprt c western blot analysis reveals efficient tnc silencing in mdamb231 cells with atnrna compared to cells treatedwith sirnas ccontrol the data represents the means ± sd from independent experiments significance value � p �� p ��� p 101371 pone0237889g003the qrtpcr analysis was also supported by direct analysis of the protein expression levelwe have observed already a decrease in tnc protein expression upon 50nm atnrnatreatment the highest concentration 100nm used led to the dramatic drop of the protein one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentexpression measured as the of the decrease fig 3b and 3c these observations were fullyconsistent with relative tnc expression level measured by qrtpcrinterferon response to atnrnato establish interferon induction in breast cancer mdamb231 cultured cells we looked forinterferon stimulated genes isg including oas1 oas3 rig1 tlr3 and ifi16 genes theanalysis was carried out with the qrtpcr fig 3a changes after atnrna measured byqrtpcr were not significant as shown basically for all of the genes in the concentrationrange of “ nm in parallel a synthetic form of dsrna polyipolyc poly ic wastransfected as a positive control poly ic has been used extensively as a tlr3 ligand to induceantiviral response [“] we showed that transfection with poly ic μgml efficientlyinduced the expression of immune response genes oas1 oas3 rig1 ifi16 tlr3 inmdamb231 cells this enhancement in mrna expression was “7fold higher in poly ictreated cells than in untreated cells fig 3a purple barstnc knockdown inhibits cells proliferation and leads to the changes inmigration rate and adhesion potential of breast cancer cellsin order to investigate the involvement of tnc on breast cancer cells proliferation mdamb cell line was treated with atnrna and the realtime cell proliferation assay was performed the cells ability to proliferate was measured for h we have noticed time and concentrationdependent decrease in proliferation rate the most effective concentration ofatnrna was nm with decrease from “ after and h respectively fig 4anoteworthy nm and nm of atnrna was already sufficient concentration for the efficient inhibition of breast cancer cells proliferation the dosedependent effect of atnrna inmdamb231 proliferation potential resulted in standard sigmoidal doseresponses with ic50of ± nm after h ± nm after h and ± nm after h of treatmentfig 4bto get more insight into the downregulation of tnc expression by atnrna on themobility of breast cancer cells realtime measurements of migration was carried out wefound that downregulation of tnc expression by atnrna significantly impaired the cellmigration in breast cancer cell lines fig 4c the results were quantitatively assessed during h of experiment and showed that mdamb231 cells transfected with atnrna had thelowest motility beginning from h post transfection it was established that atnrnadelayed the migration of mdamb231 cells by ± h ± h ± h and ± h with and 100nm concentration respectively notably the most effective concentration which affected the migration potential of the cells was 10nm when compared to the control the observed delay was ± hsince tnc is implicated also in cellmatrix attachment we further looked at the adhesionability of atnrna treated cells the cells were conducted to realtime adhesion assay withxcelligence system compared with the controls tnc knockdown resulted in increased cellsadhesion on average fig 4dtnc promotes apoptosis and is involved in cell cycle regulation in breastcancer cellsto determine additionally the effect of atnrna on the cell cycle progression themdamb231 cells were treated with different concentrations of atnrna for “ h andthe cell cycle analysis was assessed by muse1 cell analyzer cells transfected with atnrna one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig activity of atnrna in proliferation migration and adhesion proliferation of breast cancer in culture was monitored in realtime using xcelligencesystem a impedance was recorded every min but to improve the clarity of the graphs only every fourth readout was plotted data show the mean ± sd ofthree independent measurements b dosedependent effects of atnrna on proliferation was evaluated using nonlinear regression by fitting experimentalvalues to sigmoidal bellshaped equation c migration of mdamb231 cancer cells was studied using xcelligence system serumdepleted cells weretransfected with increasing concentrations of atnrna from to nm or scrambled sirnas ccontrol impedance ci values of each experimentalcondition was recorded over time plotted against time fitted to fourparameter logistic nonlinear regression model and et50 was calculated for each atnrnaconcentration to generate doseresponse curves et50 value was normalized to the data obtained for untreated cells and plotted as normalized half maximaleffective time et50 of cell migration against atnrna concentrations d adhesion of mdamb231 cell line was observed in realtime using xcelligencesystem graph shows the final impendence values minus the initial values for the respective samples differences between ci values for atnrna treated andcontrol cells were statistically evaluated using oneway anova followed by tukey™s posthoc test symbols above the bars significance value ��� p compared to cells treated with scrambled sirnas ccontrol101371 pone0237889g004showed the cell cycle distribution with the concentrationdependent decrease in cell numberin g0g1 phase increased in s phase and unchanged in g2m phase compared to the cellstransfected with unspecific control rna fig 5a atnrna impacted the cell cycle by almostdoubling the cells sphase fraction from to for the highest atnrna concentrationthus resulted with the cells arrest in s phase the cell cycle analysis proved the nontoxic effectof atnrna since we did not observe the increase in g1 population s phase arrest persistsfollowing up to h of atnrna treatment fig 5bthus to examine whether atnrnainduced apoptosis would be associated with the caspases activation the expression levels and activity of caspases such as caspase1 and in the atnrnatreated mdamb231 cells were assessed using muse1 cell one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on breast cancer cell cycle phase analysis mdamb231 cells were transiently transfected with increasing concentrations ofatnrna from to nm or scrambled sirna ccontrol for a and h b the cells were then fixed and added with propidium iodide pirnase a staining solution and analyzed in muse1 cell analyzer using modfit lttm software a percentage of cell distribution in each cell cycle phase wassummarized and shown the cell cycle distribution profile image is shown as a representative result of three independent experiments101371 pone0237889g005analyzer as shown in fig 6a breast cancer cells treated with atnrna exhibited enhancedmulticaspase activity in a concentrationdependent manner the multicaspase activity was ± ± ± and ± respectively at and nm atnrna concentration compared with the control fig 6b thus we observed almost5fold increase of the population of the apoptotic cells with the lowest atnrna concentration whereas almost 12fold with the highest oneatnrna has an impact on spheroids integrityto visualize the involvement of tnc in tumor formation the 3d culture model was appliedsince 3d cell culture models mimic better the in vivo behavior of cells in tumour tissues andare excellent surrogates to predict tumorigenic potential in vivo the ability to spheroid maintenance of breast cancer cells was assessed after atnrna treatment we have observed that one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on multiple caspase activation caspase1 and in mdamb231 cell linebreast cancer cells were transiently transfected with increasing concentrations of atnrna from to nm orscrambled sirnas ccontrol for h the transfected cells were then incubated with muse1 multicaspase reagent followedby analysis of the percentage of cell population in live caspase caspasedead and dead in muse1 cell analyzer a thepercentage of live caspase caspasedead and dead cells profile image is shown as a representative result from one of threeindependent experiments b the graphical representation of percentage of live and exhibiting caspase activity cellpopulation transfected with atnrna and scrambled sirnas statistical evaluation of atnrna versus scrambled cells one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenttreated with scrambled sirnas was performed using oneway anova followed by tukey™s posthoc test significance value��� p compared to scrambled control ccontrol error bars represent sd101371 pone0237889g006downregulation of tnc led to the disintegration of the spontaneously forming spheroids ofmdamb231 the clearly visible effect on the spheroid viability was observed even with thelowest atnrna concentration the increased concentrations of dsrna had a great impacton spheroids integrity resulting in structure disintegration at the highest concentration of nm fig 7a the atnrna application influenced the spheroid volume and shape displaying the total shrinking of the compact structure into the small fragments with the highestatnrna concentration fig 7ato have a better insight into the mdamb231 spheroids structure and the atnrnaimpact on their viability the confocal microscopy imaging was assessed the analysis of fluorescent labelling with greenfluorescent calceinam of living and dead cells within the spheroid with the livedead viabilitycytotoxicity kit was carried out as revealed by the image ofthe untreated mdamb231 cells compact multicellular spheroids were obtained fluorescence images revealed the overall morphology of the mdamb231 spheroids fig 7b thecell density in the core of the untreated spheroid was high and no dead cells were identifiedthe similar pictures were obtained for the control furthermore all conditions with differentatnrna concentrations resulted with losing the spheroid density increased dimensionsand appearing a higher population of dead cells it is worthy of note that the spheroids treatedwith increasing atnrna concentrations did not display a smooth contour following h oftreatment and subsequently their round shape was markedly altered by the treatments afterthe treatment with 50nm concentrations the spheroids showed the strong overrepresentationof dead cells fig 7b thus the 100nm concentration of atnrna was most likely too highfor the cells viability and we were not able to keep the spheroids in shape that would allow forthe imagingtnc is involved in emt processesas the consequence of these finding we analyzed the expression level of proteins involved inemt processes we took into account two main emt markers ecadherin and vimentinwestern blot analysis shows the significant increase of ecadherin level followed by the dropof the expression of vimentin protein fig 8a these observations were concentrationdependent showing the highest efficacy for atnrna at the concentration of 100nm for ecadherin expression similarly for vimentin we have observed the highest decrease of expressionupon atnrna transfection at 100nm concentration fig 8bdiscussiontnc is the main ecm protein of various tumors and its overexpression is repeatedlyobserved in breast cancer cells both in vitro and in vivo indicating a role for this extracellularmatrix glycoprotein in neoplastic pathology moreover its high expression correlates withworsened patient survival prognosis in several cancer types in breast cancers severalstudies demonstrate that high expression of tnc is not only an indicator of poor prognosisbut also correlates with metastasis to distinct ans such as lymph nodes liver and lung [“] tnc plays a substantial role in emt that is believed to be a key mechanism in cancer progression whereby cancer cells acquire more aggressive behavior [ ] in human breast cancer specimens tnc is coexpressed with the mesenchymal marker vimentin themechanistic role of tnc in the process of emt remains poorly defined however studies one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effects of atnrna on viability and spheroid structure in mdamb231 cells a monolayer cultures weretransfected with indicated amounts of atnrna oligonucleotides and after hrs cellular spheroids of mdamb cells were generated from cells in perfecta3d1 96well hanging drop plate and cultured for up to hoursscale bars μm scrambled sirnas cscr b the viability of the atnrna transfected cells within spheroidsusing livedead cell imaging kit left and middle panels present live cells green and dead cells red respectively one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentthe right panels show merge of two fluorescent images scrambled sirnas cscr concentrations ofatnrna used for transfection fluorescence images were taken using leica tcs sp5 confocal laser scanningmicroscope and plan apo × na oilimmersion objective scale bars μm101371 pone0237889g007suggest that tnc can induce an emt like phenotype in mcf7 breast cancer cells via theαvβ6 and αvβ1 integrins [ ] many studies on various cancer tissues have demonstrateddownregulation of epithelial markers including ecadherin plakoglobin and cytokeratin aswell as the upregulation of mesenchymal markers such as ncadherin and vimentin andexpression of emt transcription factors snai and twist since these changes towardsmesenchymal phenotype could correlate with invasiveness metastatic potential and poorpatient™s outcome we have investigated the effect of tnc knockdown on the expression levelsof emt markers our results show that down regulation of tnc reverses the malignant phenotype of the cancer cells as the experimental result we observed the downregulation of mesenchymal marker”vimentin followed by the upregulation of epithelial marker”ecadherinthis indicates atnrna as a potential therapeutic agent which could switch the mesenchymal phenotype of breast cancer cells to the epithelial one inhibiting the ability to metastasisand invasion additionally it has been also shown that tnc as the ecm component plays alsofig the effect of tnc downregulation on emt process of mdamb231 cells a the protein expression levelsof ecadherin vimentin and gapdh b western blot analysis of atnrna effects on the emt process revealed asignificant increase in ecadherin level followed by the drop of the expression of vimentin protein the data representsthe means ± sd from independent experiments101371 pone0237889g008 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenta role in cell to cell or cellmatrix attachment most probably inhibiting the cells™ migration inapproach with atnrna it seems that tnc in breast cancer cell line plays an antiadhesiverole which would affect the cell migration and invasion ability in addition to emt processesthus tnc downregulation seems to enhances the adhesiveness of cancer cells showing thedirect involvement of tnc in cell adhesive properties targeting the tnc in potential therapymight be also highly beneficial since it has been already established that tnc maintain a stemcell niche in the brain tumors thus could promote the tumor cell invasion therefore its overexpression largely contributes to radiochemotherapy resistance and tumor recurrence infact it has been shown that targeting gbm invasion increases tumor sensitivity to temozolomide tnc also promotes stromal events such as the angiogenic switch and the formationof more but leaky blood vessels involving wnt signaling and inhibition of dickkopf1 dkk1in a neuroendocrine tumor model we observed significant atnrna“mediated downregulation of tnc was in concordance with the observed changes in proliferation and migration rates the results show thatthe atnrna transfected cells lose their ability to migrate thus showing the involvement oftnc in breast cancer invasiveness our data indicate also that the downregulation of tncexpression in mdamb231 cancer cell lines inhibits proliferation along with induction of celldeath we were able to determine the tnc impact on the apoptosis by measuring level of bothcaspases initiating intracellular events caspase2 and effector caspases3 and orend demonstrated that tnc causes cdk2 inactivation and blocks cell cycle progression from g1 phase to s phase of anchoragedependent fibroblasts by interfering withfibronectin“syndecan4 interactions for the proliferation of anchoragedependent cellsattachment to the ecm is required detachment of fibroblasts by a pure tenascinc substratum results in g1phase arrest by inactivation of the cdk2 complex in a ckidependent manner in contrast to fibroblasts proliferation of most tumor cells is stimulated by tnc this shows that the effect of tnc on proliferation is cell typespecific and suggests that in cancer cells the cdk2 complex is not repressed in the presence of tnc in leukemia breast carcinoma and glioma were found subpopulations of stemlike cells that support tumor growth in such cells adhesion on the tnc substratum may override the g0g1 and gls cellcycle checkpoints which may explain the increased proliferation rate the g1s transitionis enforced by cyclin ecdk2 activation via syndecan4 related signalling it has been shownthat tnc in tumor cells binds to the syndecan4 binding site in fibronectin thereby blockingsyndecan4 ligation releasing the tumor cells from the suppressive effect of fibronectin ontheir proliferation this would a Answer:

Colon_Cancer

environmental exposure to arsenite as3 has a strong association with the development ofhuman urothelial cancer uc and is the 5th most common cancer in men and the 12th mostcommon cancer in women muscle invasive urothelial cancer miuc are grouped into basalor luminal molecular subtypes based on their gene expression profile the basal subtype ismore aggressive and can be associated with squamous differentiation characterized byhigh expression of keratins krt1 and and epidermal growth factor receptoregfr within the tumors the luminal subtype is less aggressive and is predominatelycharacterized by elevated gene expression of peroxisome proliferatoractivated receptamma pparÎ and forkhead box protein a1 foxa1 we have previously shown thatas3transformed urothelial cells ast exhibit a basal subtype of uc expressing genesassociated with squamous differentiation we hypothesized that the molecular subtype ofthe ast cells could be altered by inducing the expression of pparÎ andor inhibiting theproliferation of the cells nontransformed and ast cells were treated with troglitazonetg pparg agonist μm pd153035 pd an egfr inhibitor μm or a combination oftg and pd for days the results obtained demonstrate that treatment of the ast cellswith tg upregulated the expression of pparÎ and foxa1 whereas treatment with pddecreased the expression of some of the basal keratins however a combined treatment oftg and pd resulted in a consistent decrease of several proteins associated with the basalsubtype of bladder cancers krt1 krt14 krt16 p63 and tfap2a our data suggeststhat activation of pparÎ while inhibiting cell proliferation facilitates the regulation of genesinvolved in maintaining the luminal subtype of uc in vivo animal studies are needed toaddress the efficacy of using pparÎ agonists andor proliferation inhibitors to reduce tumradestage of miuca1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation mehus aa bergum n knutson pshrestha s zhou xd garrett sh activation of pparÎ and inhibition of cellproliferation reduces key proteins associated withthe basal subtype of bladder cancer in as3transformed urotsa cells one e0237976 101371 pone0237976editor karl x chai university of central floridaunited statesreceived may accepted july published august copyright mehus this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the paper and its supporting informationfilesfunding seema somji und school of medicineand health sciences pilot grant das allundergraduate research and core facilities ndinbre idea program p20 gm103442 from thenational institute of general medical sciences nih one 101371 pone0237976 august one 0ccompeting interests the authors have declaredthat no competing interests existactivation of luminal pathway in basal bladder cancerintroductionbladder cancer bc is the ninth most common cancer diagnosed worldwide and in theamerican cancer society estimated that about new cases of bc would be identified inthe us and about deaths would occur from bladder cancer among bcs urothelialcell carcinomas uc are the most common being the second most diagnosed cancer of thegenitourinary tract behind prostate cancer [ ] it is the 5th most common cancer in menand the 12th most common cancer in women urothelial cancers are classified as muscle invasive miuc or nonmuscleinvasivenmiuc nonmuscleinvasive urothelial cancers have a lower tendency to progress whereasmiucs have a high rate of metastasis and a year survival rate of approximately bothmiuc and nmiuc have been subtyped into various groups with the basal and luminal subtype being the most prominent the luminal subtype of human uc includes the majority ofthe early stage noninvasive ucs and a significant number of miucs this subtype isenriched for papillary histology is less aggressive and has a more favorable patient outcome [ ] basal classified tumors have a poorer overall survival compared to luminal tumors they often exhibit squamous differentiation are aggressive and found exclusively inmiuc that metastasize and spread to distal ans about of miucs arise independent of the papillary pathway have poor outcomes and an overall year survival rate of environmental exposure to arsenite as3 has a strong association with the developmentof human uc the increased risk of uc correlates to the same endemic areas of the worldwhere populations have been identified with arsenicinduced skin cancer [“] we havedeveloped a cell culture model of arsenicinduced urothelial cancer by exposing the immortalized nontumorigenic urothelial cell line urotsa to arsenite these transformed cell lines produce tumors in athymic mice that express genes for keratin krt and asignature pattern highly similar to the basal subtype of human miuc [ ] the tumorshave a histology similar to urothelialtransitional cell carcinomas with focal areas of squamousdifferentiation [ ] which is associated with poor prognosis [ ]the molecular mechanism driving a tumor towards a basalsquamous subtype is currentlyunknown in a recent study yamashita show that transcription factor activatingprotein alpha tfap2a is expressed at high levels in basalsquamous bladder cancerenriched in areas of squamous differentiation and is associated with increase lymph nodemetastasis and distant recurrence of the disease the study also shows that increased expression of tfap2a can facilitate the expression of other transcription factors such as tumor protein p63 tp63p63 also known as p63 which is known to be associated with the basalsubtype of uc palmbos demonstrated that p63 binds to the transcriptional regulatory regions of the gene ataxiatelangiectasia group d complementing gene atdc alsoknown as trim29 and krt14 thus increasing their expression the study further showedthat both krt14 and trim29 promote the invasion of the basal subtype of uc in vitro and invivo the luminal subtype of uc is associated with the expression of the transcriptional factorsforkhead box protein a1 foxa1 gata binding protein gata3 and peroxisome proliferatoractivated receptor gamma pparÎ [ ] the activation of pparÎ with an agonistcan represses the expression of tfap2a and inhibit squamous differentiation in vitro the exact role of pparÎ signaling in carcinogenesis is somewhat unclear however theexpression of pparÎ in bladder cancers is a favorable prognostic marker both in vivoand in vitro studies indicate that pparÎ ligands such as troglitizone can promote differentiation inhibit cellular proliferation induce autophagy and enhance apoptosis in bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer[“] likewise suppressing cellular proliferation with epidermal growth factor receptoregfr inhibitors has been used preclinically to reduce basallike muscle invasive bladdertumor growth although the egfr inhibitors did not have the same efficacy in nonbasalliketumors the goal of this study was to determine if the activation of pparÎ and inhibition of cellproliferation in the urotsa parent and the as3transformed urotsa isolates would repressthe expression of genes involved in maintaining the basalsquamous type of bladder cancerand induce genes that were associated with the luminaldifferentiated state of bladder cancermaterials and methodsanimalsathymic nude ncrnunu mice purchased from envigo were used in these studies themice were housed four to a cage at ˚c under a 12hour lightdark cycle food and water wasavailable ad libitum mouse heterotransplants of the urotsa transformed cell lines as3 andas4 and the rt4 cell line were produced by subcutaneous injection at a dose of x cellsin the dorsal thoracic midline of athymic nude ncrnunu mice this study adhered to allrecommendation dictated in the guide for the care and use of laboratory animals of thenih tumor sizes were assessed weekly and the animals were sacrificed when the size of thetumor was approximately “ cm or when dictated by clinical conditions euthanizationwas done by co2 asphyxiation and conformed to the american veterinary medical association guideline on euthanasia death was confirmed by ascertaining cardiac and respiratoryarrest following which the ans and tumor were harvested care of taken to ensure thatthere was no distress to the animals during the procedure the protocol was approved by theuniversity of north dakota animal care committee iacuc16122ccell culturethe urotsa parent cells and two of the as3transformed isolates as3 and as4 were cultured in in dulbeco™s modified eagle™s medium dmem supplemented with vv fetalbovine serum as described previously the cells were subcultured at a ratio usingtrypsinedta and the cultures were fed fresh growth medium every three days the urotsaparent cell line has been authenticated using short tandem repeat str analysis the as3transformed isolates used in the current study have been previously characterized for its ability to form colonies in soft agar form spheroids when grown in ultralow attachment flasksand form tumors when injected subcutaneously in immunecompromised mice [ “]the as3 can also form tumors upon intraperitoneal injection for drug treatmentsurotsa parent and the as3transformed isolates as3 and as4 were grown to confluence inserum containing medium following which the cells were incubated with a serum freemedium consisting of a mixture of dmem and hams™s f12 growth medium for h thecells were then exposed to either dimethyl sulfoxide dmso the drug vehicle troglitizonetg μm a pparÎ agonist pd153035 pd μm an epidermal growth factor receptoregfr inhibitor or a combination of tg and pd tgpd for and hours the concentrations of the drugs were chosen based on preliminary studiesvisualization of dapistained cellstoxic effects of tg and pd on the urotsa cells was determined by visualization of 406diamidino2phenylindole dapistained nuclei as described previously by this laboratory atthe indicated time points the cell monolayers were washed twice with phosphate buffered one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancersaline pbs following which the cells were fixed for min with ethanol and rehydratedwith 1ml pbs the rehydrated cells were stained with 10μl dapi 10μgml in distilled waterrna isolation and realtime pcr analysistotal rna was isolated using tri reagent molecular research center as described previously the expression of various genes was assessed with realtime reverse transcriptionpolymerase chain reaction rtpcr using primers that were purchased commercially frombiorad laboratories the genes along with the catalog number of the primers are listed insupplemental material s1 table total rna μg was transcribed to cdna using theiscript cdna synthesis kit biorad laboratories the amplification of the cdna was performed using the itaq universal sybr green supermix biorad laboratories with μlcdna and μm primers in a total volume of μl in a cfx96 realtime detection systembiorad laboratories amplification was monitored by sybr green fluorescence cyclingparameters consisted of a s hotstart followed by cycles of denaturation at ˚c for sannealing at ˚c for s and extension at ˚c for s which gave optimal amplificationefficiency the resulting levels were normalized to βactin expression assessed by the sameassay the threshold cycles cts for βactin and the resulting delta cts for the target genes arereported in s2 tablewestern blot analysiswestern blot analysis was performed as described previously the cell pellets were dissolved in 1x radioimmunoassay precipitation assay ripa lysis buffer supplemented withpmsf protease inhibitor cocktail and sodium orthovandate santa cruz biotechnology thecell suspension was sonicated and the lysate was centrifuged to remove cellular debris proteinlysates were quantified using the pierce bca protein assay kit thermoscientific pierceprior to loading samples were reduced and denatured the protein extracts were separated ontgx anykd sdspolyacrylamide gels purchased from biorad laboratories and transferred toa μm hybondp polyvinylidene difluoride membrane using semidry transfer the blotswere blocked in trisbuffered saline tbs containing tween20 tbst and wvbovine serum albumin bsa for min at room temperature the membranes were probedovernight at ˚c with the primary antibody diluted in wv bovine serum albumin allantibodies were purchased from commercial suppliers and were validated against known positive and negative expressing cell lines by western analysis prior to use in experimental protocols the source of the antibody along with their catalog numbers are reported in s3 tableafter washing times for minutes each wash in tbst the membranes were incubated withthe antimouse or antirabbit secondary antibody for min at room temperaturethe blots were visualized using the clarity„¢ western ecl blotting substrate bioradlaboratoriesimmunohistochemical stainingserial sections were cut at “ μm and immersed in preheated target retrieval solutiondako in a steamer for min the sections were allowed to cool to room temperature andimmersed into tbst for min the primary antibodies used in this study along with theirdilutions and catalogue numbers are listed in s3 table the primary antibodies were localizedusing dako peroxidase conjugated envision plus for rabbit or mouse primary antibodies atroom temperature for min liquid diaminobenzidine dako was used for visualizationcounter staining was performed for “ sec at room temperature using readytousehematoxylin dako slides were rinsed in distilled water dehydrated in graded ethanol one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancercleared in xylene and coverslipped two pathologists judged the presence and degree ofimmunereactivity in the specimensstatistical analysisall experiments were performed in triplicate and the results are expressed as the mean ± semstatistical analyses were performed using graphpad prism1 software version using oneway anova with tukey™s or dunnett™s posthoc testing for gene expression statistics wererun on the delta cycle threshold δct values that were generated from normalization to βactin levels unless otherwise stated the level of significance was p005resultseffect of troglitizone and pd153035 on the viability and morphology ofurotsa parent and ast cellsthe urotsa parent and ast cells as3 and as4 were treated with either dmso controltroglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr as seenin fig 1a“1d there was no change in the morphology of the urotsa parent cells with varioustreatments there was a change in the morphology of the as3 and as4 cells when treatedwith tg fig 1f and 1j and tgpd fig 1h and 1l the cells looked more differentiatedformed mounds and resembled the intermediate cells of the bladder there was a decrease inthe number of urotsa parent cells treated with pd and tgpd when compared to the cellstreated with tg alone or with dmso fig 1m there was also a decrease in the number ofas4 cells when treated with tg and tgpd when compared to the dmso treated cells fig1o there was no significant decrease in the number of as3 cells in any of the treatmentgroups fig 1n an examination demonstrated the lack of dead cells in the treated groups andthe decrease in cell number compared to the dmso group could be due to lack of proliferationandor increased differentiation of cellseffect of pparÎ activation and egfr inhibition on the expression ofluminal genesthe transcription factors pparÎ foxa1 and gata3 play a role in the establishment of theluminal subtype of bladder cancer [ ] studies done by varley have shown thatagonistdependent activation of pparÎ with simultaneous inhibition of egfr phosphorylation in normal human urothelial cells increases the effectiveness of the pparÎ agonist in thepresent study we investigated the effect of the pparÎ agonist tg and an egfr inhibitor pdon the expression of luminal transcriptional factors in the urotsa parent cells and the astcells expression levels for the target genes in this study are reported for a hr hr and hr timecourse for the parent as3 and as4 cells s1 s2 and s3 figs respectively for simplicity the hr gene and protein levels are reported in the main body of the manuscripttreatment with tg increased the expression of pparÎ in the urotsa parent fig 2a i iv andv cell line a similar effect was seen in as3 fig 2b i iv and v and as4 fig 2c i iv and vcell lines treatment with pd did not induce the expression of pparÎ in the urotsa parentfig 2a iv and v or as3 fig 2b iv and v cells but there was a small increase in pparÎ protein in the as4 cells fig 2c iv and v treatment with both tg and pd tgpd increasedthe expression levels of pparÎ mrna in the urotsa parent cells but there was no increase inthe protein levels there was no increase in mrna expression in the as4 cells but there was aslight increase in the protein levels fig 2c i iv and v one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig morphology and viability of urotsa parent and ast cells the urotsa parent ad and ast cells as3 eh and as4 il were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr the measurements were performed in triplicatesand the values reported are mean percentage of control ± sem ordinary oneway anova was performed followed by tukey™s posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g001foxa1 gene and protein expression in the urotsa parent cells treated with tg wasreduced fig 2a ii iv and vi however the expression was increased in the as3 and as4cells fig 2b ii iv and vi and 2c ii iv and vi at the protein level treatment with pd decreasedfoxa1 protein in the parent cells but the levels were elevated in the as3 cells tgpdreduced the expression of foxa1 in the urotsa parent cells but it increased the expression offoxa1in the as4 cells at the protein leveltreatment of the urotsa parent cells with tg pd or tgpd did not increase the mrnalevels of gata3 but there was an increase in the protein levels after treatment with tg andtgpd when compared to the dmso treated group fig 2a iii iv and vii in as3 and as4cells there was an additive reduction of gata3 protein by using the combined tgpd treatment fig 2b iv and vii and 2c iv and vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression of luminal markers in urotsa parent and ast cells the urotsa parent aivii as3 bivii and as4 civii weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ciiii western blot analysis was used to measure protein levels a b civ and theintegrated optical density iod of each protein band was calculated a b cvvii gene expression was normalized to βactin and gene andprotein are plotted as foldchange relative to the dmso control amplification was below detectable levels for pparÎ in dmso as3 so adelta cycle threshold δct value of was assigned which is higher than the highest delta ct detected for pparÎ in that cell linetriplicate measurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova wasperformed followed by tukey™s posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g002effect of tg and pd on the phosphorylation and expression levels of egfrthe effect of tg and the egfr inhibitor pd was determined on the expression and phosphorylation of egfr in the urotsa parent and ast cells only the combination of tgpdreduced the expression of egfr mrna in the urotsa parent cells however all three treatments decreased the protein levels fig 3a i ii and iii there was no basal phosphorylation ofegfr in the urotsa parent cells and none of the treatments had any effect on the phosphorylation levels fig 3a ii the expression of egfr in the ast cells varied with a decrease inas3 cells and an increase in as4 cells fig 3b i ii and iv and fig 3c i ii and iv respectivelyboth the transformed cell lines had basal phosphorylation of the egfr pegfr and treatment with pd decreased the pegfr levels in as3 fig 3b ii and iii and as4 fig 3c ii andiii which indicates that the pd treatment was effectiveeffect of the pparÎ agonist and inhibition egfr phosphorylation on theexpression of keratinsstudies performed by varley have shown that inhibition of the egfr with simultaneous activation of pparÎ signaling switches normal human urothelial cells from a squamous metaplastic phenotype to a transitional differentiated state with the repression ofkrt14 and the upregulation of krt13 and krt20 therefore we wanted to determineif the urotsa parent and the ast cells would revert more from a basal phenotype to amore transitionalintermediate phenotype when treated with tg and pd the mrnaexpression data is shown in fig and the protein expression data is shown in fig for theurotsa parent cells there was a decrease in expression of krt1 krt5 and krt14 with alltreatments fig 4a i ii and vi and fig 5a i ii and iv the protein for krt1 was undetectedin the urotsa parent cells the expression of krt6 and krt16 increased with tg butdecreased with pd and tgpd fig 4a iii iv v and vii and fig 5a iii and v the krt6antibody does not distinguish between the krt6a krt6b and krt6c isoforms and recognizes protein made by these three genes thus it is not known which isoform is beingexpressed at the protein level there was a decrease in expression of krt13 in the urotsaparent cells fig 4a viii and fig 5a i and vi in the as3 cells the expression levels of thebasal krts with various treatments was similar to the urotsa parent cells fig 4b ivii andfig 5b ivi with the exception of krt1 protein which was expressed in the as3 cells andits expression decreased with tg and tgpd treatment in the as4 cells the expression ofthe krts was similar to as3 with the exception of krt16 fig 4c iviii treatment withtg decreased the expression of krt16 the protein levels for the all the krts was similarto the mrna level with the exception of krt5 krt13 and krt16 krt5 showed anincrease in expression with pd and tgpd treatment and krt16 which showed anincrease in expression with pd fig 5c ivii in the as3 and as4 cells krt13 geneexpression was reduced however the protein levels were elevated from all three treatmentsfig 4b viii fig 4c viii and fig 5b i vii fig 5c i vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression and phosphorylation of epidermal growth factor receptor the urotsa parent aiiii as3 biiv and as4 ciiv weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ci western blot analysis was used to measure protein levels a b cii and the integratedoptical density iod of each protein band was calculated aiii b and ciii and iv phosphorylatedegfr was not detected in the urotsa parentcell line gene expression was normalized to βactin and gene and protein are plotted as foldchange relative to the dmso control triplicatemeasurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed bytukey™s posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g003effect of pparÎ activation and egfr inhibition on expression oftranscriptional factors p63 and tfap2a and the oncogene trim29associated with squamous differentiationrecently tfap2a has been implicated in the development of squamous differentiation inbasal cancers and activation of pparÎ is shown to represses the expression of tfap2a we therefore investigated the effects of pparÎ activation and egfr inhibition on the expression of tfap2a in urotsa parent and ast cells our results demonstrate that tg reducedtfap2a protein within the parent and as3 cells while the mrna and protein was elevatedin the as4 cells from tg exposure treatment with pd as well as tgpd decreased the one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig gene expression of keratins the urotsa parent aiviii as3 biviii and as4 civiii were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real timertpcr analysis was performed to verify gene expression a b civiii gene expression was normalized to βactin andare plotted as foldchange relative to the dmso control triplicate measurements of gene levels were performed and arereported as mean ± sem ordinary oneway anova was performed followed by tukey™s posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g004expression of tfap2a in the urotsa parent fig 6a i iv and v as3 fig 6b i iv and v andas4 cells fig 6c i iv and vtranscription factor p63 is another protein associated with human bladder cancersenriched in basalsquamous markers [ ] there was a decrease in the expression of p63 inthe urotsa parent cells with all treatments fig 6a ii iv and vi in the as3 cells the expression of p63 was low and treatment with tg increased its expression however treatment withpd and tgpd decreased its expression fig 6b ii iv and vi the expression of p63 in as4cells increased at the mrna level with tg and tgpd treatments however the protein levelswere decreased when compared to the dmso control fig 6c ii iv and vithe expression of trim29 a gene associated with the basal gene expression program was also determined in the urotsa parent and the as3transformed cells for the urotsaparent and as3 cells there was a decrease in the expression of trim29 in cells treated withpd and tgpd fig 6a and 6b iii iv and vii for as4 pd and tgpd treatments increasedtrim29 protein fig 6c iv and viiexpression of trim29 tfap2a and p63 within tumors formed fromurotsa as3 as4 and rt4 cellsurotsa as3 and as4 are considered to be of the basal molecular subtype while rt4 cellsare considered to be of the luminal molecular subtype of bladder cancer cells therefore wewanted to confirm the in vivo expression of trim29 tfap2a and p63 within the nondifferentiated basalsquamous areas of tumors originating from urotsa as3 and as4 cells incomparison to the expression in tumors originating from the luminal rt4 cells all three ofthese proteins were enriched within the nondifferentiated areas of tumors developed from theurotsa as3 and as4 cells fig 7a 7b 7d 7e 7g and 7h signs a lower expression wasobserved in the welldifferentiated areas of the urotsa as3 and as4 tumors fig 7a 7b7d 7e 7g and 7h� asterisks there was low to no staining in the rt4 cells for trim29tfap2a and p63 fig 7c 7f and 7idiscussionthe classification of uc into various subtypes has implications in the overall patient management of the disease with the basal subtype having a worse outcome when compared to theluminal subtype the molecular mechanisms involved in the development of these subtypesare not yet established however the role of some transcriptional factors is established withpparÎ playing an important role in the activation of the luminal specific genes [ ] andtfap2a playing a role in the development of the basal subtype of uc in addition studieswith normal human urothelial cells show that activation of pparÎ with an agonist along withinhibition of cellular proliferation via the egfr pathway can switch cells from a squamousmetaplastic phenotype to a more transitional differentiated phenotype combination therapiesusing egfr inhibitors and pparÎ agonists show promising results against some urothelialtumors in vivo as well as against other cancers based on these studies we sought todetermine if the urotsa parent and the as3transformed urotsa cell lines that are one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig protein expression of keratins the urotsa parent aivi as3 bivii and as4 civii were treated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr western blot analysis was used to measure protein levels ai bi ci and the integratedoptical density iod of each protein band was calculated aiivi b and ciivii protein levels are plotted as foldchange relative to the dmso controltriplicate measurements of protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed by tukey™sposthoc test bars with differing letters indicate significant differences p 101371 pone0237976g005molecularly characterized as a basal subtype of bc could convert to a luminal transitionalcell type when treated with a pparÎ agonist andor an egfr inhibitor as this could affect theoverall outcome of the diseasethe urotsa parent cells when grown in serum expresses many genes that are associatedwith the basal subtype in this study treatment of these cells with the pparÎ agonist tginduced the expression of pparÎ as well as gata3 but not foxa1 in mortal human urothelial cells treatment with the pparÎ agonist shows an increase in the expression of the transcription factor foxa1 and this is contrary to what is seen in our study these differencescould be due to the cell type since we are using immortalized cells the as3transformedurotsa cells showed an induction in the expression of pparÎ and foxa1 when treated withtg however the expression of gata3 in these transformed lines was not consistent in bccell lines studies by others have shown that have shown that gata3 and foxa1 cooperatewith pparÎ activation to drive the differentiation of a basal bladder cancer subtype to a moredifferentiated luminal subtype other studies have also linked the expression of foxa1 tothe development of the luminal subtype of urothelial cancer [“] in our studies treatmentwith tg did result in the differentiation of the as3transformed cells based upon morphological changes and induced the expression of pparÎ as well as foxa1 both of which are factorsknown to drive luminal differentiation of bladder cancer cell linessignaling via the pparÎ pathway is essential for the growth arrest and terminal differentiation of adipocytes and other normal epithelial [“] and cancer cells [“] whereassignaling via the egfr receptor plays a role in cell proliferation the keratins play an essentialrole in the differentiation of epithelial cells and different keratin profiles are expressed at different stages of differentiation the normal bladder has three different stages of differentiationand these stages are marked by the expression of krt14 krt5 and krt20 krt14 isexpressed in a subset of basal cells that are thought to play a role in regeneration as well astumorigenesis whereas other basal cells and intermediate cells in the normal bladderexpress krt5 the expression of krt20 is restricted to the most differentiated cells theumbrella cells these differentiation stages of the bladder are shared by bladder cancersand malignant transformation can occur in any of the different cell types of the bladder theexpression of krt14 is seen in the least of the differentiated tumors and its expression correlates to poor prognosis krt14 whereas the expression of krt20 is restricted to differentiated bladder cancers and its expression is associated with good prognosis a study doneby varley showed that normal human urothelial cells in culture express krt14 andlack the expression of krt13 and krt20 activation of pparÎ in these cells induced theexpression of krt13 and decreased the expression of krt14 this effect was significantlyenhanced when

cancer7562

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: environmental exposure to arsenite as3 has a strong association with the development ofhuman urothelial cancer uc and is the 5th most common cancer in men and the 12th mostcommon cancer in women muscle invasive urothelial cancer miuc are grouped into basalor luminal molecular subtypes based on their gene expression profile the basal subtype ismore aggressive and can be associated with squamous differentiation characterized byhigh expression of keratins krt1 and and epidermal growth factor receptoregfr within the tumors the luminal subtype is less aggressive and is predominatelycharacterized by elevated gene expression of peroxisome proliferatoractivated receptamma pparÎ and forkhead box protein a1 foxa1 we have previously shown thatas3transformed urothelial cells ast exhibit a basal subtype of uc expressing genesassociated with squamous differentiation we hypothesized that the molecular subtype ofthe ast cells could be altered by inducing the expression of pparÎ andor inhibiting theproliferation of the cells nontransformed and ast cells were treated with troglitazonetg pparg agonist μm pd153035 pd an egfr inhibitor μm or a combination oftg and pd for days the results obtained demonstrate that treatment of the ast cellswith tg upregulated the expression of pparÎ and foxa1 whereas treatment with pddecreased the expression of some of the basal keratins however a combined treatment oftg and pd resulted in a consistent decrease of several proteins associated with the basalsubtype of bladder cancers krt1 krt14 krt16 p63 and tfap2a our data suggeststhat activation of pparÎ while inhibiting cell proliferation facilitates the regulation of genesinvolved in maintaining the luminal subtype of uc in vivo animal studies are needed toaddress the efficacy of using pparÎ agonists andor proliferation inhibitors to reduce tumradestage of miuca1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation mehus aa bergum n knutson pshrestha s zhou xd garrett sh activation of pparÎ and inhibition of cellproliferation reduces key proteins associated withthe basal subtype of bladder cancer in as3transformed urotsa cells one e0237976 101371 pone0237976editor karl x chai university of central floridaunited statesreceived may accepted july published august copyright mehus this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the paper and its supporting informationfilesfunding seema somji und school of medicineand health sciences pilot grant das allundergraduate research and core facilities ndinbre idea program p20 gm103442 from thenational institute of general medical sciences nih one 101371 pone0237976 august one 0ccompeting interests the authors have declaredthat no competing interests existactivation of luminal pathway in basal bladder cancerintroductionbladder cancer bc is the ninth most common cancer diagnosed worldwide and in theamerican cancer society estimated that about new cases of bc would be identified inthe us and about deaths would occur from bladder cancer among bcs urothelialcell carcinomas uc are the most common being the second most diagnosed cancer of thegenitourinary tract behind prostate cancer [ ] it is the 5th most common cancer in menand the 12th most common cancer in women urothelial cancers are classified as muscle invasive miuc or nonmuscleinvasivenmiuc nonmuscleinvasive urothelial cancers have a lower tendency to progress whereasmiucs have a high rate of metastasis and a year survival rate of approximately bothmiuc and nmiuc have been subtyped into various groups with the basal and luminal subtype being the most prominent the luminal subtype of human uc includes the majority ofthe early stage noninvasive ucs and a significant number of miucs this subtype isenriched for papillary histology is less aggressive and has a more favorable patient outcome [ ] basal classified tumors have a poorer overall survival compared to luminal tumors they often exhibit squamous differentiation are aggressive and found exclusively inmiuc that metastasize and spread to distal ans about of miucs arise independent of the papillary pathway have poor outcomes and an overall year survival rate of environmental exposure to arsenite as3 has a strong association with the developmentof human uc the increased risk of uc correlates to the same endemic areas of the worldwhere populations have been identified with arsenicinduced skin cancer [“] we havedeveloped a cell culture model of arsenicinduced urothelial cancer by exposing the immortalized nontumorigenic urothelial cell line urotsa to arsenite these transformed cell lines produce tumors in athymic mice that express genes for keratin krt and asignature pattern highly similar to the basal subtype of human miuc [ ] the tumorshave a histology similar to urothelialtransitional cell carcinomas with focal areas of squamousdifferentiation [ ] which is associated with poor prognosis [ ]the molecular mechanism driving a tumor towards a basalsquamous subtype is currentlyunknown in a recent study yamashita show that transcription factor activatingprotein alpha tfap2a is expressed at high levels in basalsquamous bladder cancerenriched in areas of squamous differentiation and is associated with increase lymph nodemetastasis and distant recurrence of the disease the study also shows that increased expression of tfap2a can facilitate the expression of other transcription factors such as tumor protein p63 tp63p63 also known as p63 which is known to be associated with the basalsubtype of uc palmbos demonstrated that p63 binds to the transcriptional regulatory regions of the gene ataxiatelangiectasia group d complementing gene atdc alsoknown as trim29 and krt14 thus increasing their expression the study further showedthat both krt14 and trim29 promote the invasion of the basal subtype of uc in vitro and invivo the luminal subtype of uc is associated with the expression of the transcriptional factorsforkhead box protein a1 foxa1 gata binding protein gata3 and peroxisome proliferatoractivated receptor gamma pparÎ [ ] the activation of pparÎ with an agonistcan represses the expression of tfap2a and inhibit squamous differentiation in vitro the exact role of pparÎ signaling in carcinogenesis is somewhat unclear however theexpression of pparÎ in bladder cancers is a favorable prognostic marker both in vivoand in vitro studies indicate that pparÎ ligands such as troglitizone can promote differentiation inhibit cellular proliferation induce autophagy and enhance apoptosis in bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer[“] likewise suppressing cellular proliferation with epidermal growth factor receptoregfr inhibitors has been used preclinically to reduce basallike muscle invasive bladdertumor growth although the egfr inhibitors did not have the same efficacy in nonbasalliketumors the goal of this study was to determine if the activation of pparÎ and inhibition of cellproliferation in the urotsa parent and the as3transformed urotsa isolates would repressthe expression of genes involved in maintaining the basalsquamous type of bladder cancerand induce genes that were associated with the luminaldifferentiated state of bladder cancermaterials and methodsanimalsathymic nude ncrnunu mice purchased from envigo were used in these studies themice were housed four to a cage at ˚c under a 12hour lightdark cycle food and water wasavailable ad libitum mouse heterotransplants of the urotsa transformed cell lines as3 andas4 and the rt4 cell line were produced by subcutaneous injection at a dose of x cellsin the dorsal thoracic midline of athymic nude ncrnunu mice this study adhered to allrecommendation dictated in the guide for the care and use of laboratory animals of thenih tumor sizes were assessed weekly and the animals were sacrificed when the size of thetumor was approximately “ cm or when dictated by clinical conditions euthanizationwas done by co2 asphyxiation and conformed to the american veterinary medical association guideline on euthanasia death was confirmed by ascertaining cardiac and respiratoryarrest following which the ans and tumor were harvested care of taken to ensure thatthere was no distress to the animals during the procedure the protocol was approved by theuniversity of north dakota animal care committee iacuc16122ccell culturethe urotsa parent cells and two of the as3transformed isolates as3 and as4 were cultured in in dulbeco™s modified eagle™s medium dmem supplemented with vv fetalbovine serum as described previously the cells were subcultured at a ratio usingtrypsinedta and the cultures were fed fresh growth medium every three days the urotsaparent cell line has been authenticated using short tandem repeat str analysis the as3transformed isolates used in the current study have been previously characterized for its ability to form colonies in soft agar form spheroids when grown in ultralow attachment flasksand form tumors when injected subcutaneously in immunecompromised mice [ “]the as3 can also form tumors upon intraperitoneal injection for drug treatmentsurotsa parent and the as3transformed isolates as3 and as4 were grown to confluence inserum containing medium following which the cells were incubated with a serum freemedium consisting of a mixture of dmem and hams™s f12 growth medium for h thecells were then exposed to either dimethyl sulfoxide dmso the drug vehicle troglitizonetg μm a pparÎ agonist pd153035 pd μm an epidermal growth factor receptoregfr inhibitor or a combination of tg and pd tgpd for and hours the concentrations of the drugs were chosen based on preliminary studiesvisualization of dapistained cellstoxic effects of tg and pd on the urotsa cells was determined by visualization of 406diamidino2phenylindole dapistained nuclei as described previously by this laboratory atthe indicated time points the cell monolayers were washed twice with phosphate buffered one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancersaline pbs following which the cells were fixed for min with ethanol and rehydratedwith 1ml pbs the rehydrated cells were stained with 10μl dapi 10μgml in distilled waterrna isolation and realtime pcr analysistotal rna was isolated using tri reagent molecular research center as described previously the expression of various genes was assessed with realtime reverse transcriptionpolymerase chain reaction rtpcr using primers that were purchased commercially frombiorad laboratories the genes along with the catalog number of the primers are listed insupplemental material s1 table total rna μg was transcribed to cdna using theiscript cdna synthesis kit biorad laboratories the amplification of the cdna was performed using the itaq universal sybr green supermix biorad laboratories with μlcdna and μm primers in a total volume of μl in a cfx96 realtime detection systembiorad laboratories amplification was monitored by sybr green fluorescence cyclingparameters consisted of a s hotstart followed by cycles of denaturation at ˚c for sannealing at ˚c for s and extension at ˚c for s which gave optimal amplificationefficiency the resulting levels were normalized to βactin expression assessed by the sameassay the threshold cycles cts for βactin and the resulting delta cts for the target genes arereported in s2 tablewestern blot analysiswestern blot analysis was performed as described previously the cell pellets were dissolved in 1x radioimmunoassay precipitation assay ripa lysis buffer supplemented withpmsf protease inhibitor cocktail and sodium orthovandate santa cruz biotechnology thecell suspension was sonicated and the lysate was centrifuged to remove cellular debris proteinlysates were quantified using the pierce bca protein assay kit thermoscientific pierceprior to loading samples were reduced and denatured the protein extracts were separated ontgx anykd sdspolyacrylamide gels purchased from biorad laboratories and transferred toa μm hybondp polyvinylidene difluoride membrane using semidry transfer the blotswere blocked in trisbuffered saline tbs containing tween20 tbst and wvbovine serum albumin bsa for min at room temperature the membranes were probedovernight at ˚c with the primary antibody diluted in wv bovine serum albumin allantibodies were purchased from commercial suppliers and were validated against known positive and negative expressing cell lines by western analysis prior to use in experimental protocols the source of the antibody along with their catalog numbers are reported in s3 tableafter washing times for minutes each wash in tbst the membranes were incubated withthe antimouse or antirabbit secondary antibody for min at room temperaturethe blots were visualized using the clarity„¢ western ecl blotting substrate bioradlaboratoriesimmunohistochemical stainingserial sections were cut at “ μm and immersed in preheated target retrieval solutiondako in a steamer for min the sections were allowed to cool to room temperature andimmersed into tbst for min the primary antibodies used in this study along with theirdilutions and catalogue numbers are listed in s3 table the primary antibodies were localizedusing dako peroxidase conjugated envision plus for rabbit or mouse primary antibodies atroom temperature for min liquid diaminobenzidine dako was used for visualizationcounter staining was performed for “ sec at room temperature using readytousehematoxylin dako slides were rinsed in distilled water dehydrated in graded ethanol one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancercleared in xylene and coverslipped two pathologists judged the presence and degree ofimmunereactivity in the specimensstatistical analysisall experiments were performed in triplicate and the results are expressed as the mean ± semstatistical analyses were performed using graphpad prism1 software version using oneway anova with tukey™s or dunnett™s posthoc testing for gene expression statistics wererun on the delta cycle threshold δct values that were generated from normalization to βactin levels unless otherwise stated the level of significance was p005resultseffect of troglitizone and pd153035 on the viability and morphology ofurotsa parent and ast cellsthe urotsa parent and ast cells as3 and as4 were treated with either dmso controltroglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr as seenin fig 1a“1d there was no change in the morphology of the urotsa parent cells with varioustreatments there was a change in the morphology of the as3 and as4 cells when treatedwith tg fig 1f and 1j and tgpd fig 1h and 1l the cells looked more differentiatedformed mounds and resembled the intermediate cells of the bladder there was a decrease inthe number of urotsa parent cells treated with pd and tgpd when compared to the cellstreated with tg alone or with dmso fig 1m there was also a decrease in the number ofas4 cells when treated with tg and tgpd when compared to the dmso treated cells fig1o there was no significant decrease in the number of as3 cells in any of the treatmentgroups fig 1n an examination demonstrated the lack of dead cells in the treated groups andthe decrease in cell number compared to the dmso group could be due to lack of proliferationandor increased differentiation of cellseffect of pparÎ activation and egfr inhibition on the expression ofluminal genesthe transcription factors pparÎ foxa1 and gata3 play a role in the establishment of theluminal subtype of bladder cancer [ ] studies done by varley have shown thatagonistdependent activation of pparÎ with simultaneous inhibition of egfr phosphorylation in normal human urothelial cells increases the effectiveness of the pparÎ agonist in thepresent study we investigated the effect of the pparÎ agonist tg and an egfr inhibitor pdon the expression of luminal transcriptional factors in the urotsa parent cells and the astcells expression levels for the target genes in this study are reported for a hr hr and hr timecourse for the parent as3 and as4 cells s1 s2 and s3 figs respectively for simplicity the hr gene and protein levels are reported in the main body of the manuscripttreatment with tg increased the expression of pparÎ in the urotsa parent fig 2a i iv andv cell line a similar effect was seen in as3 fig 2b i iv and v and as4 fig 2c i iv and vcell lines treatment with pd did not induce the expression of pparÎ in the urotsa parentfig 2a iv and v or as3 fig 2b iv and v cells but there was a small increase in pparÎ protein in the as4 cells fig 2c iv and v treatment with both tg and pd tgpd increasedthe expression levels of pparÎ mrna in the urotsa parent cells but there was no increase inthe protein levels there was no increase in mrna expression in the as4 cells but there was aslight increase in the protein levels fig 2c i iv and v one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig morphology and viability of urotsa parent and ast cells the urotsa parent ad and ast cells as3 eh and as4 il were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr the measurements were performed in triplicatesand the values reported are mean percentage of control ± sem ordinary oneway anova was performed followed by tukey™s posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g001foxa1 gene and protein expression in the urotsa parent cells treated with tg wasreduced fig 2a ii iv and vi however the expression was increased in the as3 and as4cells fig 2b ii iv and vi and 2c ii iv and vi at the protein level treatment with pd decreasedfoxa1 protein in the parent cells but the levels were elevated in the as3 cells tgpdreduced the expression of foxa1 in the urotsa parent cells but it increased the expression offoxa1in the as4 cells at the protein leveltreatment of the urotsa parent cells with tg pd or tgpd did not increase the mrnalevels of gata3 but there was an increase in the protein levels after treatment with tg andtgpd when compared to the dmso treated group fig 2a iii iv and vii in as3 and as4cells there was an additive reduction of gata3 protein by using the combined tgpd treatment fig 2b iv and vii and 2c iv and vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression of luminal markers in urotsa parent and ast cells the urotsa parent aivii as3 bivii and as4 civii weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ciiii western blot analysis was used to measure protein levels a b civ and theintegrated optical density iod of each protein band was calculated a b cvvii gene expression was normalized to βactin and gene andprotein are plotted as foldchange relative to the dmso control amplification was below detectable levels for pparÎ in dmso as3 so adelta cycle threshold δct value of was assigned which is higher than the highest delta ct detected for pparÎ in that cell linetriplicate measurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova wasperformed followed by tukey™s posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g002effect of tg and pd on the phosphorylation and expression levels of egfrthe effect of tg and the egfr inhibitor pd was determined on the expression and phosphorylation of egfr in the urotsa parent and ast cells only the combination of tgpdreduced the expression of egfr mrna in the urotsa parent cells however all three treatments decreased the protein levels fig 3a i ii and iii there was no basal phosphorylation ofegfr in the urotsa parent cells and none of the treatments had any effect on the phosphorylation levels fig 3a ii the expression of egfr in the ast cells varied with a decrease inas3 cells and an increase in as4 cells fig 3b i ii and iv and fig 3c i ii and iv respectivelyboth the transformed cell lines had basal phosphorylation of the egfr pegfr and treatment with pd decreased the pegfr levels in as3 fig 3b ii and iii and as4 fig 3c ii andiii which indicates that the pd treatment was effectiveeffect of the pparÎ agonist and inhibition egfr phosphorylation on theexpression of keratinsstudies performed by varley have shown that inhibition of the egfr with simultaneous activation of pparÎ signaling switches normal human urothelial cells from a squamous metaplastic phenotype to a transitional differentiated state with the repression ofkrt14 and the upregulation of krt13 and krt20 therefore we wanted to determineif the urotsa parent and the ast cells would revert more from a basal phenotype to amore transitionalintermediate phenotype when treated with tg and pd the mrnaexpression data is shown in fig and the protein expression data is shown in fig for theurotsa parent cells there was a decrease in expression of krt1 krt5 and krt14 with alltreatments fig 4a i ii and vi and fig 5a i ii and iv the protein for krt1 was undetectedin the urotsa parent cells the expression of krt6 and krt16 increased with tg butdecreased with pd and tgpd fig 4a iii iv v and vii and fig 5a iii and v the krt6antibody does not distinguish between the krt6a krt6b and krt6c isoforms and recognizes protein made by these three genes thus it is not known which isoform is beingexpressed at the protein level there was a decrease in expression of krt13 in the urotsaparent cells fig 4a viii and fig 5a i and vi in the as3 cells the expression levels of thebasal krts with various treatments was similar to the urotsa parent cells fig 4b ivii andfig 5b ivi with the exception of krt1 protein which was expressed in the as3 cells andits expression decreased with tg and tgpd treatment in the as4 cells the expression ofthe krts was similar to as3 with the exception of krt16 fig 4c iviii treatment withtg decreased the expression of krt16 the protein levels for the all the krts was similarto the mrna level with the exception of krt5 krt13 and krt16 krt5 showed anincrease in expression with pd and tgpd treatment and krt16 which showed anincrease in expression with pd fig 5c ivii in the as3 and as4 cells krt13 geneexpression was reduced however the protein levels were elevated from all three treatmentsfig 4b viii fig 4c viii and fig 5b i vii fig 5c i vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression and phosphorylation of epidermal growth factor receptor the urotsa parent aiiii as3 biiv and as4 ciiv weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ci western blot analysis was used to measure protein levels a b cii and the integratedoptical density iod of each protein band was calculated aiii b and ciii and iv phosphorylatedegfr was not detected in the urotsa parentcell line gene expression was normalized to βactin and gene and protein are plotted as foldchange relative to the dmso control triplicatemeasurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed bytukey™s posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g003effect of pparÎ activation and egfr inhibition on expression oftranscriptional factors p63 and tfap2a and the oncogene trim29associated with squamous differentiationrecently tfap2a has been implicated in the development of squamous differentiation inbasal cancers and activation of pparÎ is shown to represses the expression of tfap2a we therefore investigated the effects of pparÎ activation and egfr inhibition on the expression of tfap2a in urotsa parent and ast cells our results demonstrate that tg reducedtfap2a protein within the parent and as3 cells while the mrna and protein was elevatedin the as4 cells from tg exposure treatment with pd as well as tgpd decreased the one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig gene expression of keratins the urotsa parent aiviii as3 biviii and as4 civiii were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real timertpcr analysis was performed to verify gene expression a b civiii gene expression was normalized to βactin andare plotted as foldchange relative to the dmso control triplicate measurements of gene levels were performed and arereported as mean ± sem ordinary oneway anova was performed followed by tukey™s posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g004expression of tfap2a in the urotsa parent fig 6a i iv and v as3 fig 6b i iv and v andas4 cells fig 6c i iv and vtranscription factor p63 is another protein associated with human bladder cancersenriched in basalsquamous markers [ ] there was a decrease in the expression of p63 inthe urotsa parent cells with all treatments fig 6a ii iv and vi in the as3 cells the expression of p63 was low and treatment with tg increased its expression however treatment withpd and tgpd decreased its expression fig 6b ii iv and vi the expression of p63 in as4cells increased at the mrna level with tg and tgpd treatments however the protein levelswere decreased when compared to the dmso control fig 6c ii iv and vithe expression of trim29 a gene associated with the basal gene expression program was also determined in the urotsa parent and the as3transformed cells for the urotsaparent and as3 cells there was a decrease in the expression of trim29 in cells treated withpd and tgpd fig 6a and 6b iii iv and vii for as4 pd and tgpd treatments increasedtrim29 protein fig 6c iv and viiexpression of trim29 tfap2a and p63 within tumors formed fromurotsa as3 as4 and rt4 cellsurotsa as3 and as4 are considered to be of the basal molecular subtype while rt4 cellsare considered to be of the luminal molecular subtype of bladder cancer cells therefore wewanted to confirm the in vivo expression of trim29 tfap2a and p63 within the nondifferentiated basalsquamous areas of tumors originating from urotsa as3 and as4 cells incomparison to the expression in tumors originating from the luminal rt4 cells all three ofthese proteins were enriched within the nondifferentiated areas of tumors developed from theurotsa as3 and as4 cells fig 7a 7b 7d 7e 7g and 7h signs a lower expression wasobserved in the welldifferentiated areas of the urotsa as3 and as4 tumors fig 7a 7b7d 7e 7g and 7h� asterisks there was low to no staining in the rt4 cells for trim29tfap2a and p63 fig 7c 7f and 7idiscussionthe classification of uc into various subtypes has implications in the overall patient management of the disease with the basal subtype having a worse outcome when compared to theluminal subtype the molecular mechanisms involved in the development of these subtypesare not yet established however the role of some transcriptional factors is established withpparÎ playing an important role in the activation of the luminal specific genes [ ] andtfap2a playing a role in the development of the basal subtype of uc in addition studieswith normal human urothelial cells show that activation of pparÎ with an agonist along withinhibition of cellular proliferation via the egfr pathway can switch cells from a squamousmetaplastic phenotype to a more transitional differentiated phenotype combination therapiesusing egfr inhibitors and pparÎ agonists show promising results against some urothelialtumors in vivo as well as against other cancers based on these studies we sought todetermine if the urotsa parent and the as3transformed urotsa cell lines that are one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig protein expression of keratins the urotsa parent aivi as3 bivii and as4 civii were treated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr western blot analysis was used to measure protein levels ai bi ci and the integratedoptical density iod of each protein band was calculated aiivi b and ciivii protein levels are plotted as foldchange relative to the dmso controltriplicate measurements of protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed by tukey™sposthoc test bars with differing letters indicate significant differences p 101371 pone0237976g005molecularly characterized as a basal subtype of bc could convert to a luminal transitionalcell type when treated with a pparÎ agonist andor an egfr inhibitor as this could affect theoverall outcome of the diseasethe urotsa parent cells when grown in serum expresses many genes that are associatedwith the basal subtype in this study treatment of these cells with the pparÎ agonist tginduced the expression of pparÎ as well as gata3 but not foxa1 in mortal human urothelial cells treatment with the pparÎ agonist shows an increase in the expression of the transcription factor foxa1 and this is contrary to what is seen in our study these differencescould be due to the cell type since we are using immortalized cells the as3transformedurotsa cells showed an induction in the expression of pparÎ and foxa1 when treated withtg however the expression of gata3 in these transformed lines was not consistent in bccell lines studies by others have shown that have shown that gata3 and foxa1 cooperatewith pparÎ activation to drive the differentiation of a basal bladder cancer subtype to a moredifferentiated luminal subtype other studies have also linked the expression of foxa1 tothe development of the luminal subtype of urothelial cancer [“] in our studies treatmentwith tg did result in the differentiation of the as3transformed cells based upon morphological changes and induced the expression of pparÎ as well as foxa1 both of which are factorsknown to drive luminal differentiation of bladder cancer cell linessignaling via the pparÎ pathway is essential for the growth arrest and terminal differentiation of adipocytes and other normal epithelial [“] and cancer cells [“] whereassignaling via the egfr receptor plays a role in cell proliferation the keratins play an essentialrole in the differentiation of epithelial cells and different keratin profiles are expressed at different stages of differentiation the normal bladder has three different stages of differentiationand these stages are marked by the expression of krt14 krt5 and krt20 krt14 isexpressed in a subset of basal cells that are thought to play a role in regeneration as well astumorigenesis whereas other basal cells and intermediate cells in the normal bladderexpress krt5 the expression of krt20 is restricted to the most differentiated cells theumbrella cells these differentiation stages of the bladder are shared by bladder cancersand malignant transformation can occur in any of the different cell types of the bladder theexpression of krt14 is seen in the least of the differentiated tumors and its expression correlates to poor prognosis krt14 whereas the expression of krt20 is restricted to differentiated bladder cancers and its expression is associated with good prognosis a study doneby varley showed that normal human urothelial cells in culture express krt14 andlack the expression of krt13 and krt20 activation of pparÎ in these cells induced theexpression of krt13 and decreased the expression of krt14 this effect was significantlyenhanced when Answer:

Colon_Cancer

purposeconjunctival squamous cell carcinoma scc is primarily treated with surgical resectionscc has various stages and local recurrence is common the purpose of this study was todetermine molecular localization of epidermal growth factor receptor egfr and the possibility of egfr as a biomarker for the management of conjunctival sccmethodsin this retrospective study we performed immunohistochemistry to evaluate egfr expression and localization in tumor cells egfr mutationspecific expression e746a750del andl858r and human papillomavirus expression in a series of conjunctival sccsresultsall tumors in our cohort were egfr positive twentyone of tumors showed focal egfr staining and seven showed diffuse egfr staining in additionwe calculated the percentages of the two most important mutations in egfr exon a750del exon l858r mutant in conjunctival sccs weobserved that the translocation of egfr from the membrane into the cytoplasm was relatedto clinical prognosis as we detected correlations between egfr cytoplasmic staining andfinal orbital exenteration and between decreased egfr membrane staining and progressionfree survivala1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation sakai a tagami m kakehashi akatsuyamayoshikawa a misawa n wanibuchi h expression intracellular localizationand mutation of egfr in conjunctival squamouscell carcinoma and the association with prognosisand treatment one e0238120 101371 pone0238120editor sanjoy bhattacharya bascom palmer eyeinstitute united statesreceived april accepted august published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0238120copyright sakai this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and supporting informationfiles one 101371 pone0238120 august one 0cfunding none of the authors have any proprietaryor financial interests to declarecompeting interests none of the authors haveany proprietary or financial interests to declaresegfr is important in the pathology of ocular surface squamous neoplasia including sccand is a prognostic factor increased understanding of egfr mutations may have importantimplications for future treatment optionsegfr in conjunctival squamous cell carcinomaintroductionocular surface squamous neoplasia ossn includes several diseases such as conjunctival premalignant dysplasia carcinoma in situ and invasive conjunctival squamous cell carcinomascc the annual incidence of ossn was casesmillionyear conjunctival intraepithelial neoplasia casesmillionyear scc casesmillionyear in the united kingdom [ ] in the united states the rate of scc is 5fold higher among males and whites other previous research revealed that the risk increases with exposure to direct daylightand in outdoor workers metaanalysis demonstrated an association with human immunodeficiency virus odds ratio and human papillomavirus hpv odds ratio howeverno large epidemiological studies have been performed on people living in the far eastscholz examined clinicopathological factors and biomarkers and identified promotermutations in telomerase reverse transcriptase in of samples of conjunctival ossn associated with ultraviolet light induction recent research demonstrated that pdl1 isexpressed in almost half of conjunctival scc cases and noted the potential application ofimmune checkpoint blockade as a treatment strategy for conjunctival scc molecular targeted therapy is now used to treat most carcinomas and its use is continuingto increase uveal melanoma also has recently been reported in the ocular oncology area gefitinib is a relatively old tyrosine kinase inhibiter tki that is used as a molecular targeted therapy and its effects have been reported in various carcinomas on the other hand nobasic clinical studies on ocular tumors have been reported [“] in our current study weinvestigated epidermal growth factor receptor egfr expression in our cases to assess thepossible effect of gefitinib we also examined the molecular expression and intracellular localization of egfr in conjunctival scc in east asian patientsmaterials and methodsselection of cases and collation of clinicopathologic datathis study was approved by the institutional review boards of osaka city university andkobe kaisei hospital and adhered to the tenets of the declaration of helsinki writteninformed consent was obtained from all patients before enrollment we identified patientstreated by ophthalmologists aa mt between november and july from whom wewere able to procure tissue blocks with residual tumor for each patient we collected demographic features age at initial diagnosis and at presentation to our institution and sex andprimary tumor features disease status at presentation [primary or recurrent] and in situ versusinvasive disease the american joint committee on cancer ajcc stage local recurrenceanatomic site and date metastases regional or distant and date vital status at last followup cause of death types of surgery and adjuvant therapy were also recordedimmunohistochemistry ihcimmunohistochemical studies for egfr and hpv were performed on 6μmthick tissue sections using the following antibodies antihuman egfr rabbit monoclonal antibody clone one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomasp84 414r14 cell marque rocklin ca usa antihpv mouse monoclonal antibodyclone k1h8 ab75574 abcam cambridge uk horseradish peroxidaseconjugated antirabbit igg hl goat polyclonal antibody histofine nichirei corporation tokyojapan and horseradish peroxidaseconjugated antimouse igg hl goat polyclonal antibody histofine nichirei corporationegfr mutationspecific immunohistochemical staining was performed on cases as primary antibodies we used egfr e746a750del cell signaling technologies danversma usa and egfr l858r cell signaling technologies which were manuallyapplied to the slides stained sections were viewed with an olympus bx53dp74as controls for staining benign conjunctival lesions were also stained for egfr and coloncancer samples were stained as a positive controlimage analysisslides immunostained for egfr egfr mutations and hpv were evaluated in a blinded manner by two specialists mt and ak egfr expression was visually estimated as the percentageof tumor cells with complete or partial membranous staining tumors with egfr staining in� of tumor cells were considered the diffuse staining type diffuse type and those with of tumor cells were considered the focal staining type focal type the presence orabsence and intensity of cell membrane staining were semiquantitatively divided into groupswith a score of “ none weak strong very strong the presence or absence andintensity of cell cytoplasmic staining were also divided into groups with a score of “ andsemiquantitatively analyzed none weak strong very strong egfr mutationspecific immunostaining was divided into two groups those with immunostaining that wasclearly present and those without immunostainingslides immunostained for hpv were assessed with visual evaluation for the presence ofpunctate nuclear signals within tumor nuclei at — magnification and were scored as positive or negativeegfr expression in tumorsegfr expression in the tumor was analyzed with nanostring analysis archival formalinfixedparaffinembedded tumor tissue was retrieved and manually macrodissected total mrnawas isolated from the macrodissected tumor tissues using a qiagen mirneasy kit qiagenvalencia ca usa according to the manufacturer™s instructions the rna sample was quantified with nanodrop thermo scientific wilmington de usa and regarded as adequate ifit contained ng at minimum the sample was subsequently analyzed with the ncounterpancancer progression panel nanostring seattle wa usa according to the manufacturer™s instructions nanostring data processing was done with the r statistical programmingenvironment v342 considering the counts obtained for positive control probe sets rawnanostring counts for each gene were subjected to technical factorial normalization whichwas carried out by subtracting the mean counts plus two times the standard deviation from thecodeset inherent negative controls subsequently biological normalization using the includedmrna reference genes was performed additionally all counts with p after a onesidedttest versus negative controls plus two times the standard deviation were interpreted as notexpressed over basal noisestatistical analysisthe clinical and histopathologic characteristics were summarized using descriptive statisticscorrelations between immunohistochemical demographic and clinicopathologic factors were one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomaassessed using the wilcoxon rank sum and fisher™s exact tests progressionfree survival pfswas defined as the time from surgery to disease recurrence or death from any cause coxregression modeling was used to evaluate correlations between clinicopathologic and immunohistochemical features and survival outcomes statistical analyses were performed usingspss statistics version software ibm japan tokyo japan values of p were considered statistically significantresultsclinicopathologic findings of our cohort are summarized in table all patients in ourcohort were east asian and included men and women with a mean age at presentation of years fourteen patients had invasive scc and had an in situtumor primary orbital exenteration was necessary for local disease control in two patients and two patients underwent additional orbital exenteration nine patients table clinicopathologic findings of cases of conjunctival squamous cell carcinomaage years mean rangesexmalefemalefollowup after primary surgery months rangetstage ajccall n n “ “tist1t2t3t4primary surgery typelocal excisionorbital exenterationadjuvant therapynoyesadditional excisiontopical chemotherapyradiation therapyimmunohistochemical markershpv status in tumor cellsnegativepositiveegfr expression in tumorsdiffuse stainingfocal stainingnegativecell membrane egfr expression in tumorsvery strongstrongweak one 101371 pone0238120 august continued one 0ctable continuednegativecell cytoplasm egfr expression in tumorsvery strongstrongweaknegativeoutcomeorbital exenterationyesnolocal recurrence after curative therapyyesnometastasisdistantregional distantregionalnonevital status at last followupdeadalivecause of deathconjunctival scc metastasisother101371 pone0238120t001egfr in conjunctival squamous cell carcinomaall n n underwent adjuvant therapy most commonly additional local surgery topical chemotherapyand radiation therapy were performed in one patient in the adjuvant therapy group of thisgroup one patient died with disease months after diagnosis of regional and lung metastasesthe other patient was alive without disease at months after diagnosis of regional metastasestwo patients died one of which was due to conjunctival scc described above ninepatients experienced local recurrence after curative surgeryall tumors were egfr positive in our cohort twentyone of tumors showed focal egfr staining and seven showed diffuse egfr staining fig analysisof egfr intracellular staining patterns showed scores of for membrane staining and for cytoplasmic staining no significant difference was found between carcinoma in situ tisand invasive carcinoma tadv table no significant difference was found in the scoredepending on the stage egfr expression in colon cancer was used as a positive control fig2aon the other hand seven benign conjunctival lesions three pinguecula three pterygiumone dermoid cyst showed partial weak positive staining in conjunctival squamous epithelialcells especially on the cell membrane fig 2b in addition cytoplasmic staining was seen inonly one case benign cases showed scores of for membrane staining and for cytoplasmic staining cytoplasmic staining patterns were significantly different in benign compared to scc cases p table the correlation between egfr staining focal ordiffuse and egfr localization cytoplasmic staining group was not significantly different one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig egfr expression in conjunctival scc focal egfr staining a and diffuse egfr staining b scale bar μm inset corresponding field in ahematoxylineosinstained section membrane staining very strong c and cytoplasm staining very strong d scale bar μm101371 pone0238120g001but the diffuse egfr group tended to have a higher score p and respectivelytable egfr e746a750 del and egfr l858r expression were assessed with immunohistochemistry in all patients fig the mutation at exon egfr e7446a750 del was confirmedin cases and that at exon egfr l858r point mutation was confirmed in cases with ihc table the relationship between egfr mutation and egfr stainingtable staining patterns of egfrcell membranetis in situtadv invasiven n benign tumorn cell cytoplasmtis in situtadv invasiven n benign tumorn �p value based on the nonpaired ttest101371 pone0238120t002staining patterns n totaltotalaverageaveragepp� one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig a egfr expression in colon cancer as a positive control scale bar μm b egfr expression in a control benign lesion pinguecula scale bar μminset corresponding field in a hematoxylineosinstained section101371 pone0238120g002focal or diffuse was determined using univariate linear regression analysis with correctionfor age p regarding egfr expression in tumors we compared the tis and tadv groups according toajcc t grading n4 no significant difference was found p fig the majority of patients in our cohort were hpv negative n table the positive rate of hpv immunoreactivity increased with increases in ajcc t grading but the correlation was not statistically significantthe cox regression model was used to examine and analyze the relationship between longterm prognosis including orbital exenteration and pfs and the clinicopathological statusegfr staining pattern and egfr mutation univariate cox regression analyses revealed significant correlations between egfr cytoplasmic staining and final orbital exenteration hazard ratio hr p table additionally a significant correlation was seenbetween the t stage ajcc and pfs and between egfr membrane staining and pfs hr p p respectively table local recurrence distant metastasisrate and overall survival rate were not statistically significant in addition the egfr mutationwas not significantly correlated with final orbital exenteration or pfs tables and discussionto the best of our knowledge this is one of the first studies to survey the prevalence of egfrmutations and intracellular localization in conjunctival scc and to evaluate the prognostic significance of tumor cells that express egfr in conjunctival sccin this study we found that the tumor tissue of all conjunctival sccs expressedegfr in addition we determined the percentages of the two most important mutations intable egfr staining and localizationcell membranecell cytoplasmicegfr focaln ±±egfr diffusen ±±p101371 pone0238120t003 one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig egfr mutationspecific expression in conjunctival scc a basement membrane staining in a tumor with egfr e746a750 del bwhole tumor staining in an egfr e746a750 del mutant c conjunctival scc layer cells with strong staining in an egfr l858r mutant scalebar μm101371 pone0238120g003egfr exon 746a750del exon l858r mutant in conjunctival sccs we also showed that the translocation of egfr from the membrane into the cytoplasm was related to clinical activation of cancer as correlations between egfr cytoplasmicstaining and final orbital exenteration and between decreased egfr membrane staining andpfs were noted although the number of cases examined was small the expression of cytoplasmic staining of egfr was weak but significantly different from membrane staining in thebenign disease group our hypothesis is that as egfr transitions from the membrane into thecytoplasm malignant changes progress in addition a correlation between egfr stainingfocal or diffuse and egfr cytoplasmic staining was seen and a higher score tended to bepresent in the diffuse egfr staining grouptable summary of egfr e746a750 del and egfr l858r point mutationsmutationn age y sex mt stage egfr staining patterns diffuseegfr localization score membraneexon egfr e746a750 del n fexon egfr l858r point mutationn t3 t2 tis t3 tis focalcytoplasmicm male f female101371 pone0238120t004 one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig for egfr expression in tumors we compared carcinoma in situ tis and invasive carcinoma tadv groups according toajcc t grading n4 ns not significant101371 pone0238120g004intracellular transfer of egfr in the group with diffuse staining may indicate progressionand although no statistical differences were observed in this study significant findings mayemerge by increasing the number of cases in the futurein the past especially in african countries several studies on conjunctival sccs and egfrexpression have been reported they suggested a potential association with hpv [ ]other previous studies reported that posttranslational modification can promote egfrtable relationship between orbital exenteration and clinicopathologic and molecular factorsunivariate analysisvariablesagesextstage ajccegfr stainingn mean yearsmale female tis t1 t2 21t3 �focal 21diffuse egfr membrane stainingvery strong 1strong 21weak 7negative egfr cytoplasmic stainingegfr mutationhpv positivevery strong 4strong 6weak 19negative exon e746a750 del 8exon l858r point mutation positive 7negative hr — ci““““ — ““““ — p�ci indicates confidence interval hr hazard ratestatistically significant differences are underlined�p value based on the cox proportional hazard model101371 pone0238120t005 one 101371 pone0238120 august one 0ctable relationship between pfs and clinicopathologic and molecular factorsunivariate analysisvariablesagesextstage ajccegfr stainingn mean yearsmale 15female tis t1 t2 21t3 �focal 21diffuse egfr membrane stainingvery strong 1strong 21weak 7negative egfr cytoplasmic stainingegfr mutationhpv positivevery strong 4strong 6weak 19negative exon e746a750del 8exon l858r point mutation positive 7negative ci indicates confidence interval hr hazard ratestatistically significant differences are underlined�p value based on the cox proportional hazard model101371 pone0238120t006egfr in conjunctival squamous cell carcinomahr ci“““ — ““““ — “p��endocytosis and lysosomal degradation of egfr thereby ensuring termination of receptor signaling [ ]in our cohort expression and localization of egfr and its association with prognosis werefirst reported in the asian race additionally intracellular translocation of egfr from membrane staining to cytoplasm staining likely by endocytosis was associated with the percent offinal orbital exenteration cytoplasmic staining hr p and pfs membranestaining hr p in our cohort regarding the difference in local changes inegfr immunoreactivity in patients without egfr expression in the tumor we compared thetis and tadv groups according to ajcc t grading a recent study showed that feedback regulatory loops can modulate growth factors and receptor tyrosine kinases such as egfr to regulate cellular functions including abnormal states such as cancer our study examined thisphenomenon clinically and confirmed a pathological difference without changes in geneexpressionegfr mutations in ossn including invasive sccs have not been examined in asianpatients since approximately egfr mutations in lung cancer had been registeredin the cosmic the catalog of somatic mutations in cancer database most are concentrated in the exon “ region of the intracellular tyrosine kinase domain the most frequentone is at codon of exon a deletion mutation is present at a site centered on five aminoacids elrea near amino acid and a point mutation changes leucine to argininel858r at codon of exon shigematsu in and mitsudomi in reported that egfr mutations are common in asians females nonsmokers and adenocarcinomas in lung cancer [ ] generally when egfr mutation occurs the tyrosine kinaseactivity of egfr at the atp binding site is constantly active even without growth factor cancer cell growth and survival depend on this pathway oncogene addiction egfr tkis competitively inhibit atp binding in the kinase domain and suppress autophosphorylation ofegfr blockade of signal transmission has antitumor effects previous reports of egfractivating mutations common mutations described the frequency of exon deletion mutations as and for l858r mutations in lung cancer [ ]egfr mutations were examined to verify the effect of gefitinib on positive nonsmall celllung carcinoma in two phase iii clinical trials from japan in the nej002 trial and thewjtog3405 trial gefitinib was the test treatment group the standard treatment in the former one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig schematic of movement of egfr into the cytoplasm by endocytosis to avoid excessive signaling and for recycling101371 pone0238120g005was carboplatin paclitaxel and in the latter was cisplatin in all studies the gefitinib groupshowed superior pfs [ ] in view of these findings in lung cancer in asians our findingsregarding egfr expression and mutations will provide further options for potential treatmentof ossn for pre and postsurgical treatmentthe association of scc with hpv was not confirmed because the number of cases wassmall in addition our results may not be accurate because we did not use multiplex pcrwhich is currently the most suitable genotyping method ours is the first report to show that differences in the expression form and mutations inegfr in ossn are associated with prognosis and treatmentin an animal model egfr inhibition affected epithelial cell proliferation and stratificationduring corneal epithelial wound healing and may play a role in maintaining normal cornealepithelial thickness gefitinib is an egfr inhibitor and is the first approved molecular targeted therapy for cancer treatment in japan thus understanding the pathological role of egfr in ossn andapplying it to treatment are of great significance for seeking new treatment indications inossn including conjunctival sccs in this study egfr may translocate from the cell membrane into the cytoplasm tumor cells may transfer egfr into the cytoplasm by endocytosisto avoid excessive signaling by the feedback system fig furthermore in this study theegfr mutation was present in many patients with ossn this finding may suggest a courseof treatment in the future in addition the method we used for identification of egfr mutations was not general genotyping but was a judgment of immunohistochemically stained sections although the sensitivity and specificity were high in a previous report this is still alimitation one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomathis study has important limitations first regarding egfr expression on the ocular surface changes in benign diseases and agerelated changes in normal tissues may not have beensufficiently investigated our study found that egfr mutations were also present in conjunctival scc in east asians however we did not obtain results that correlated with the final prognosis further studies including further multiinstitutional studies and an increase in thenumber of cases will be needed in the future another limitation is that double testing of formalinfixed paraffinembedded specimens and plasma with realtime pcr for detection ofegfr mutations is more common than ihc in actual clinical practice according to the literature both the sensitivity and specificity were satisfactory for these two types of mutations in addition the size of our study cohort was small n and the length of followup lessthan year in some patients may not have been sufficient for longterm outcome analysestherefore additional studies will be needed to corroborate our findingsin the results of this study indicate that egfr is an active molecular target inthe pathology of ossn including scc and is a prognostic factor the finding also suggests thatdiscovery of mutations may have important implications for future treatment optionssupporting informations1 filexlsxacknowledgmentswe gratefully acknowledge the technical assistance of the research support platform osakacity university graduate school of medicine and the clinical laboratory department of kobekaisei hospitalauthor contributionsconceptualization mizuki tagami atsushi azumidata curation atsushi sakai mizuki tagami atsuko katsuyamayoshikawa norihiko misawa atsushi azumiformal analysis mizuki tagami anna kakehashi norihiko misawafunding acquisition mizuki tagamiinvestigation mizuki tagami atsuko katsuyamayoshikawa atsushi azumimethodology mizuki tagami anna kakehashi atsuko katsuyamayoshikawa atsushiazumiproject administration mizuki tagamisupervision anna kakehashi hideki wanibuchi atsushi azumi shigeru hondavisualization atsushi sakai mizuki tagami anna kakehashiwriting “ original draft atsushi sakai mizuki tagamiwriting “ review editing mizuki tagami shigeru hondareferenceslee ga hirst lw ocular surface squamous neoplasia surv ophthalmol “ 101016s0039625705800542 pmid one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinoma kiire ca stewart rmk srinivasan s heimann h kaye sb dhillon b a prospective study of the incidence associations and outcomes of ocular surface squamous neoplasia in the united kingdom eyelond “ mcclellan aj mcclellan al pezon cf karp cl feuer w galor a epidemiology of ocular surfacesquamous neoplasia in a veterans affairs population cornea “ 101097ico0b013e31829e3c80 pmid sun ec fears tr goedert jj epidemiology of squamous cell conjunctival cancer cancer epidemiolbiomarkers prev “ pmid scholz sl thomasen h reis h frequent tert promoter mutations in ocular surface squamousneoplasia invest ophthalmol vis sci “ 101167iovs1517469pmid nagarajan p elhadad c gruschkus sk ning j hudgens cw sagiv o pdl1pd1 expressioncomposition of tumorassociated immune infiltrate and hpv status in conjunctival squamous cellcarcinoma invest ophthalmol vis sci “ 101167iovs1926894pmid le tourneau c delord jp gonc¸alves a gavoille c dubot c isambert n molecularly targetedtherapy based on tumour molecular profiling versus conventional therapy for advanced cancershiva a multicentre openlabel proofofconcept randomised controlled phase trial lancetoncol “ 101016s1470204515001886 pmid el zaoui i bucher m rimoldi d nicolas m kaya g pescini gobert r conjunctival melanomatargeted therapy mapk and pi3kmtor pathways inhibition invest ophthalmol vis sci “ 101167iovs1826508 pmid ciardiello f tortora g a novel approach in the treatment of cancer targeting the epidermal growth factor receptor clin cancer res “ pmid lynch tj bell dw sordella r gurubhagavatula s okimoto ra brannigan bw activating mutations in the epidermal growth factor receptor underlying responsiveness of nonsmallcell lung cancer togefitinib n engl j med “ 101056nejmoa040938 pmid paez jg ja¨nne pa lee jc tracy s greulich h gabriel s egfr mutations in lung cancer correlation with clinical response to gefitinib therapy science “ 101126science1099314 pmid cesano a ncounter® pancancer immune profiling panel nanostring technologies inc seattlewa j immunother cancer 101186s4042501500887 pmid yu jj fu p pink jj dawson d wasman j orem j hpv infection and egfr activationalteration in hivinfected east african patients with conjunctival carcinoma one e10477101371 pone0010477 pmid mwololo a nyagol j rogena e ochuk w kimani m onyango n correlation of egfr pegfrand p16ink4 expressions and high risk hpv infection in hivaidsrelated squamous cell carcinoma ofconjunctiva infect agent cancer 1011861750937897 pmid haglund k dikic i the role of ubiquitylation in receptor endocytosis and endosomal sorting j cell sci “ 101242jcs091280 pmid zhang x gureasko j shen k cole pa kuriyan j an allosteric mechanism for activation of the kinasedomain of epidermal growth factor receptor cell “ 101016jcell pmid avraham r yarden y feedback regulation of egfr signalling decision making by early and delayedloops nat rev mol cell biol “ 101038nrm3048 pmid kobayashi y mitsudomi t not all epidermal growth factor receptor mutations in lung cancer are created equal perspectives for individualized treatment strategy cancer sci “101111cas12996 pmid shigematsu h lin l takahashi t nomura m suzuki m wistuba ii clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers j natl cancerinst “ 101093jncidji055 pmid mitsudomi t yatabe y mutations of the epidermal growth factor receptor gene and related genes asdeterminants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancercancer sci “ 101111j13497006200700607x pmid yun ch mengwasser ke toms av woo ms greulich h wong kk the t790m mutation inegfr kinase causes drug resistance by increasing the affinity for atp proc natl acad sci u s a “ 101073pnas0709662105 pmid one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinoma kobayashi y togashi y yatabe y mizuuchi h jangchul p kondo c egfr exon mutationsin lung cancer molecular predictors of augmented sensitivity to afatinib or neratinib as comparedwith first or thirdgeneration tkis clin cancer res “ 10115810780432ccr151046 pmid wu jy yu cj chang yc yang ch shih jy yang pc effectiveness of tyrosine kinase inhibitors onuncommon epidermal growth factor receptor mutations of unknown clinical significance in nonsmallcell lung cancer clin cancer res “ 10115810780432ccr10 pmid maemondo m inoue a kobayashi k sugawara s oizumi s isobe h gefitinib or chemotherapyfor nonsmallcell lung cancer with mutated egfr n engl j med “ 101056nejmoa0909530 pmid mitsudomi t morita s yatabe y negoro s okamoto i tsurutani j gefitinib versus cisplatin plusdocetaxel in patients with nonsmallcell lung cancer harbouring mutations of the epidermal growth factor receptor wjtog3405 an open label randomised phase trial lancet oncol “101016s147020450970364x pmid nishiwaki m yamamoto t tone s murai t ohkawara t matsunami t genotyping of humanpapillomaviruses by a novel onestep typing method with multiplex pcr and clinical applications j clinmicrobiol “ 101128jcm0079307 pmid nakamura y sotozono c kinoshita s the epidermal growth factor receptor egfr role in cornealwound healing and homeostasis exp eye res “ 101006exer2000 pmid fukuoka m yano s giaccone g tamura t nakagawa k douillard jy multiinstitutional randomized phase ii trial of gefitinib for previously treated patients with advanced nonsmallcell lung cancer the ideal trial [corrected] j clin oncol “ 101200jco pmid oldrini b hsieh wy erdjumentbromage h codega p carro ms curielgarcı´a a egfr feedbackinh

cancer7563

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: purposeconjunctival squamous cell carcinoma scc is primarily treated with surgical resectionscc has various stages and local recurrence is common the purpose of this study was todetermine molecular localization of epidermal growth factor receptor egfr and the possibility of egfr as a biomarker for the management of conjunctival sccmethodsin this retrospective study we performed immunohistochemistry to evaluate egfr expression and localization in tumor cells egfr mutationspecific expression e746a750del andl858r and human papillomavirus expression in a series of conjunctival sccsresultsall tumors in our cohort were egfr positive twentyone of tumors showed focal egfr staining and seven showed diffuse egfr staining in additionwe calculated the percentages of the two most important mutations in egfr exon a750del exon l858r mutant in conjunctival sccs weobserved that the translocation of egfr from the membrane into the cytoplasm was relatedto clinical prognosis as we detected correlations between egfr cytoplasmic staining andfinal orbital exenteration and between decreased egfr membrane staining and progressionfree survivala1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation sakai a tagami m kakehashi akatsuyamayoshikawa a misawa n wanibuchi h expression intracellular localizationand mutation of egfr in conjunctival squamouscell carcinoma and the association with prognosisand treatment one e0238120 101371 pone0238120editor sanjoy bhattacharya bascom palmer eyeinstitute united statesreceived april accepted august published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0238120copyright sakai this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and supporting informationfiles one 101371 pone0238120 august one 0cfunding none of the authors have any proprietaryor financial interests to declarecompeting interests none of the authors haveany proprietary or financial interests to declaresegfr is important in the pathology of ocular surface squamous neoplasia including sccand is a prognostic factor increased understanding of egfr mutations may have importantimplications for future treatment optionsegfr in conjunctival squamous cell carcinomaintroductionocular surface squamous neoplasia ossn includes several diseases such as conjunctival premalignant dysplasia carcinoma in situ and invasive conjunctival squamous cell carcinomascc the annual incidence of ossn was casesmillionyear conjunctival intraepithelial neoplasia casesmillionyear scc casesmillionyear in the united kingdom [ ] in the united states the rate of scc is 5fold higher among males and whites other previous research revealed that the risk increases with exposure to direct daylightand in outdoor workers metaanalysis demonstrated an association with human immunodeficiency virus odds ratio and human papillomavirus hpv odds ratio howeverno large epidemiological studies have been performed on people living in the far eastscholz examined clinicopathological factors and biomarkers and identified promotermutations in telomerase reverse transcriptase in of samples of conjunctival ossn associated with ultraviolet light induction recent research demonstrated that pdl1 isexpressed in almost half of conjunctival scc cases and noted the potential application ofimmune checkpoint blockade as a treatment strategy for conjunctival scc molecular targeted therapy is now used to treat most carcinomas and its use is continuingto increase uveal melanoma also has recently been reported in the ocular oncology area gefitinib is a relatively old tyrosine kinase inhibiter tki that is used as a molecular targeted therapy and its effects have been reported in various carcinomas on the other hand nobasic clinical studies on ocular tumors have been reported [“] in our current study weinvestigated epidermal growth factor receptor egfr expression in our cases to assess thepossible effect of gefitinib we also examined the molecular expression and intracellular localization of egfr in conjunctival scc in east asian patientsmaterials and methodsselection of cases and collation of clinicopathologic datathis study was approved by the institutional review boards of osaka city university andkobe kaisei hospital and adhered to the tenets of the declaration of helsinki writteninformed consent was obtained from all patients before enrollment we identified patientstreated by ophthalmologists aa mt between november and july from whom wewere able to procure tissue blocks with residual tumor for each patient we collected demographic features age at initial diagnosis and at presentation to our institution and sex andprimary tumor features disease status at presentation [primary or recurrent] and in situ versusinvasive disease the american joint committee on cancer ajcc stage local recurrenceanatomic site and date metastases regional or distant and date vital status at last followup cause of death types of surgery and adjuvant therapy were also recordedimmunohistochemistry ihcimmunohistochemical studies for egfr and hpv were performed on 6μmthick tissue sections using the following antibodies antihuman egfr rabbit monoclonal antibody clone one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomasp84 414r14 cell marque rocklin ca usa antihpv mouse monoclonal antibodyclone k1h8 ab75574 abcam cambridge uk horseradish peroxidaseconjugated antirabbit igg hl goat polyclonal antibody histofine nichirei corporation tokyojapan and horseradish peroxidaseconjugated antimouse igg hl goat polyclonal antibody histofine nichirei corporationegfr mutationspecific immunohistochemical staining was performed on cases as primary antibodies we used egfr e746a750del cell signaling technologies danversma usa and egfr l858r cell signaling technologies which were manuallyapplied to the slides stained sections were viewed with an olympus bx53dp74as controls for staining benign conjunctival lesions were also stained for egfr and coloncancer samples were stained as a positive controlimage analysisslides immunostained for egfr egfr mutations and hpv were evaluated in a blinded manner by two specialists mt and ak egfr expression was visually estimated as the percentageof tumor cells with complete or partial membranous staining tumors with egfr staining in� of tumor cells were considered the diffuse staining type diffuse type and those with of tumor cells were considered the focal staining type focal type the presence orabsence and intensity of cell membrane staining were semiquantitatively divided into groupswith a score of “ none weak strong very strong the presence or absence andintensity of cell cytoplasmic staining were also divided into groups with a score of “ andsemiquantitatively analyzed none weak strong very strong egfr mutationspecific immunostaining was divided into two groups those with immunostaining that wasclearly present and those without immunostainingslides immunostained for hpv were assessed with visual evaluation for the presence ofpunctate nuclear signals within tumor nuclei at — magnification and were scored as positive or negativeegfr expression in tumorsegfr expression in the tumor was analyzed with nanostring analysis archival formalinfixedparaffinembedded tumor tissue was retrieved and manually macrodissected total mrnawas isolated from the macrodissected tumor tissues using a qiagen mirneasy kit qiagenvalencia ca usa according to the manufacturer™s instructions the rna sample was quantified with nanodrop thermo scientific wilmington de usa and regarded as adequate ifit contained ng at minimum the sample was subsequently analyzed with the ncounterpancancer progression panel nanostring seattle wa usa according to the manufacturer™s instructions nanostring data processing was done with the r statistical programmingenvironment v342 considering the counts obtained for positive control probe sets rawnanostring counts for each gene were subjected to technical factorial normalization whichwas carried out by subtracting the mean counts plus two times the standard deviation from thecodeset inherent negative controls subsequently biological normalization using the includedmrna reference genes was performed additionally all counts with p after a onesidedttest versus negative controls plus two times the standard deviation were interpreted as notexpressed over basal noisestatistical analysisthe clinical and histopathologic characteristics were summarized using descriptive statisticscorrelations between immunohistochemical demographic and clinicopathologic factors were one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomaassessed using the wilcoxon rank sum and fisher™s exact tests progressionfree survival pfswas defined as the time from surgery to disease recurrence or death from any cause coxregression modeling was used to evaluate correlations between clinicopathologic and immunohistochemical features and survival outcomes statistical analyses were performed usingspss statistics version software ibm japan tokyo japan values of p were considered statistically significantresultsclinicopathologic findings of our cohort are summarized in table all patients in ourcohort were east asian and included men and women with a mean age at presentation of years fourteen patients had invasive scc and had an in situtumor primary orbital exenteration was necessary for local disease control in two patients and two patients underwent additional orbital exenteration nine patients table clinicopathologic findings of cases of conjunctival squamous cell carcinomaage years mean rangesexmalefemalefollowup after primary surgery months rangetstage ajccall n n “ “tist1t2t3t4primary surgery typelocal excisionorbital exenterationadjuvant therapynoyesadditional excisiontopical chemotherapyradiation therapyimmunohistochemical markershpv status in tumor cellsnegativepositiveegfr expression in tumorsdiffuse stainingfocal stainingnegativecell membrane egfr expression in tumorsvery strongstrongweak one 101371 pone0238120 august continued one 0ctable continuednegativecell cytoplasm egfr expression in tumorsvery strongstrongweaknegativeoutcomeorbital exenterationyesnolocal recurrence after curative therapyyesnometastasisdistantregional distantregionalnonevital status at last followupdeadalivecause of deathconjunctival scc metastasisother101371 pone0238120t001egfr in conjunctival squamous cell carcinomaall n n underwent adjuvant therapy most commonly additional local surgery topical chemotherapyand radiation therapy were performed in one patient in the adjuvant therapy group of thisgroup one patient died with disease months after diagnosis of regional and lung metastasesthe other patient was alive without disease at months after diagnosis of regional metastasestwo patients died one of which was due to conjunctival scc described above ninepatients experienced local recurrence after curative surgeryall tumors were egfr positive in our cohort twentyone of tumors showed focal egfr staining and seven showed diffuse egfr staining fig analysisof egfr intracellular staining patterns showed scores of for membrane staining and for cytoplasmic staining no significant difference was found between carcinoma in situ tisand invasive carcinoma tadv table no significant difference was found in the scoredepending on the stage egfr expression in colon cancer was used as a positive control fig2aon the other hand seven benign conjunctival lesions three pinguecula three pterygiumone dermoid cyst showed partial weak positive staining in conjunctival squamous epithelialcells especially on the cell membrane fig 2b in addition cytoplasmic staining was seen inonly one case benign cases showed scores of for membrane staining and for cytoplasmic staining cytoplasmic staining patterns were significantly different in benign compared to scc cases p table the correlation between egfr staining focal ordiffuse and egfr localization cytoplasmic staining group was not significantly different one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig egfr expression in conjunctival scc focal egfr staining a and diffuse egfr staining b scale bar μm inset corresponding field in ahematoxylineosinstained section membrane staining very strong c and cytoplasm staining very strong d scale bar μm101371 pone0238120g001but the diffuse egfr group tended to have a higher score p and respectivelytable egfr e746a750 del and egfr l858r expression were assessed with immunohistochemistry in all patients fig the mutation at exon egfr e7446a750 del was confirmedin cases and that at exon egfr l858r point mutation was confirmed in cases with ihc table the relationship between egfr mutation and egfr stainingtable staining patterns of egfrcell membranetis in situtadv invasiven n benign tumorn cell cytoplasmtis in situtadv invasiven n benign tumorn �p value based on the nonpaired ttest101371 pone0238120t002staining patterns n totaltotalaverageaveragepp� one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig a egfr expression in colon cancer as a positive control scale bar μm b egfr expression in a control benign lesion pinguecula scale bar μminset corresponding field in a hematoxylineosinstained section101371 pone0238120g002focal or diffuse was determined using univariate linear regression analysis with correctionfor age p regarding egfr expression in tumors we compared the tis and tadv groups according toajcc t grading n4 no significant difference was found p fig the majority of patients in our cohort were hpv negative n table the positive rate of hpv immunoreactivity increased with increases in ajcc t grading but the correlation was not statistically significantthe cox regression model was used to examine and analyze the relationship between longterm prognosis including orbital exenteration and pfs and the clinicopathological statusegfr staining pattern and egfr mutation univariate cox regression analyses revealed significant correlations between egfr cytoplasmic staining and final orbital exenteration hazard ratio hr p table additionally a significant correlation was seenbetween the t stage ajcc and pfs and between egfr membrane staining and pfs hr p p respectively table local recurrence distant metastasisrate and overall survival rate were not statistically significant in addition the egfr mutationwas not significantly correlated with final orbital exenteration or pfs tables and discussionto the best of our knowledge this is one of the first studies to survey the prevalence of egfrmutations and intracellular localization in conjunctival scc and to evaluate the prognostic significance of tumor cells that express egfr in conjunctival sccin this study we found that the tumor tissue of all conjunctival sccs expressedegfr in addition we determined the percentages of the two most important mutations intable egfr staining and localizationcell membranecell cytoplasmicegfr focaln ±±egfr diffusen ±±p101371 pone0238120t003 one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig egfr mutationspecific expression in conjunctival scc a basement membrane staining in a tumor with egfr e746a750 del bwhole tumor staining in an egfr e746a750 del mutant c conjunctival scc layer cells with strong staining in an egfr l858r mutant scalebar μm101371 pone0238120g003egfr exon 746a750del exon l858r mutant in conjunctival sccs we also showed that the translocation of egfr from the membrane into the cytoplasm was related to clinical activation of cancer as correlations between egfr cytoplasmicstaining and final orbital exenteration and between decreased egfr membrane staining andpfs were noted although the number of cases examined was small the expression of cytoplasmic staining of egfr was weak but significantly different from membrane staining in thebenign disease group our hypothesis is that as egfr transitions from the membrane into thecytoplasm malignant changes progress in addition a correlation between egfr stainingfocal or diffuse and egfr cytoplasmic staining was seen and a higher score tended to bepresent in the diffuse egfr staining grouptable summary of egfr e746a750 del and egfr l858r point mutationsmutationn age y sex mt stage egfr staining patterns diffuseegfr localization score membraneexon egfr e746a750 del n fexon egfr l858r point mutationn t3 t2 tis t3 tis focalcytoplasmicm male f female101371 pone0238120t004 one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig for egfr expression in tumors we compared carcinoma in situ tis and invasive carcinoma tadv groups according toajcc t grading n4 ns not significant101371 pone0238120g004intracellular transfer of egfr in the group with diffuse staining may indicate progressionand although no statistical differences were observed in this study significant findings mayemerge by increasing the number of cases in the futurein the past especially in african countries several studies on conjunctival sccs and egfrexpression have been reported they suggested a potential association with hpv [ ]other previous studies reported that posttranslational modification can promote egfrtable relationship between orbital exenteration and clinicopathologic and molecular factorsunivariate analysisvariablesagesextstage ajccegfr stainingn mean yearsmale female tis t1 t2 21t3 �focal 21diffuse egfr membrane stainingvery strong 1strong 21weak 7negative egfr cytoplasmic stainingegfr mutationhpv positivevery strong 4strong 6weak 19negative exon e746a750 del 8exon l858r point mutation positive 7negative hr — ci““““ — ““““ — p�ci indicates confidence interval hr hazard ratestatistically significant differences are underlined�p value based on the cox proportional hazard model101371 pone0238120t005 one 101371 pone0238120 august one 0ctable relationship between pfs and clinicopathologic and molecular factorsunivariate analysisvariablesagesextstage ajccegfr stainingn mean yearsmale 15female tis t1 t2 21t3 �focal 21diffuse egfr membrane stainingvery strong 1strong 21weak 7negative egfr cytoplasmic stainingegfr mutationhpv positivevery strong 4strong 6weak 19negative exon e746a750del 8exon l858r point mutation positive 7negative ci indicates confidence interval hr hazard ratestatistically significant differences are underlined�p value based on the cox proportional hazard model101371 pone0238120t006egfr in conjunctival squamous cell carcinomahr ci“““ — ““““ — “p��endocytosis and lysosomal degradation of egfr thereby ensuring termination of receptor signaling [ ]in our cohort expression and localization of egfr and its association with prognosis werefirst reported in the asian race additionally intracellular translocation of egfr from membrane staining to cytoplasm staining likely by endocytosis was associated with the percent offinal orbital exenteration cytoplasmic staining hr p and pfs membranestaining hr p in our cohort regarding the difference in local changes inegfr immunoreactivity in patients without egfr expression in the tumor we compared thetis and tadv groups according to ajcc t grading a recent study showed that feedback regulatory loops can modulate growth factors and receptor tyrosine kinases such as egfr to regulate cellular functions including abnormal states such as cancer our study examined thisphenomenon clinically and confirmed a pathological difference without changes in geneexpressionegfr mutations in ossn including invasive sccs have not been examined in asianpatients since approximately egfr mutations in lung cancer had been registeredin the cosmic the catalog of somatic mutations in cancer database most are concentrated in the exon “ region of the intracellular tyrosine kinase domain the most frequentone is at codon of exon a deletion mutation is present at a site centered on five aminoacids elrea near amino acid and a point mutation changes leucine to argininel858r at codon of exon shigematsu in and mitsudomi in reported that egfr mutations are common in asians females nonsmokers and adenocarcinomas in lung cancer [ ] generally when egfr mutation occurs the tyrosine kinaseactivity of egfr at the atp binding site is constantly active even without growth factor cancer cell growth and survival depend on this pathway oncogene addiction egfr tkis competitively inhibit atp binding in the kinase domain and suppress autophosphorylation ofegfr blockade of signal transmission has antitumor effects previous reports of egfractivating mutations common mutations described the frequency of exon deletion mutations as and for l858r mutations in lung cancer [ ]egfr mutations were examined to verify the effect of gefitinib on positive nonsmall celllung carcinoma in two phase iii clinical trials from japan in the nej002 trial and thewjtog3405 trial gefitinib was the test treatment group the standard treatment in the former one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig schematic of movement of egfr into the cytoplasm by endocytosis to avoid excessive signaling and for recycling101371 pone0238120g005was carboplatin paclitaxel and in the latter was cisplatin in all studies the gefitinib groupshowed superior pfs [ ] in view of these findings in lung cancer in asians our findingsregarding egfr expression and mutations will provide further options for potential treatmentof ossn for pre and postsurgical treatmentthe association of scc with hpv was not confirmed because the number of cases wassmall in addition our results may not be accurate because we did not use multiplex pcrwhich is currently the most suitable genotyping method ours is the first report to show that differences in the expression form and mutations inegfr in ossn are associated with prognosis and treatmentin an animal model egfr inhibition affected epithelial cell proliferation and stratificationduring corneal epithelial wound healing and may play a role in maintaining normal cornealepithelial thickness gefitinib is an egfr inhibitor and is the first approved molecular targeted therapy for cancer treatment in japan thus understanding the pathological role of egfr in ossn andapplying it to treatment are of great significance for seeking new treatment indications inossn including conjunctival sccs in this study egfr may translocate from the cell membrane into the cytoplasm tumor cells may transfer egfr into the cytoplasm by endocytosisto avoid excessive signaling by the feedback system fig furthermore in this study theegfr mutation was present in many patients with ossn this finding may suggest a courseof treatment in the future in addition the method we used for identification of egfr mutations was not general genotyping but was a judgment of immunohistochemically stained sections although the sensitivity and specificity were high in a previous report this is still alimitation one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomathis study has important limitations first regarding egfr expression on the ocular surface changes in benign diseases and agerelated changes in normal tissues may not have beensufficiently investigated our study found that egfr mutations were also present in conjunctival scc in east asians however we did not obtain results that correlated with the final prognosis further studies including further multiinstitutional studies and an increase in thenumber of cases will be needed in the future another limitation is that double testing of formalinfixed paraffinembedded specimens and plasma with realtime pcr for detection ofegfr mutations is more common than ihc in actual clinical practice according to the literature both the sensitivity and specificity were satisfactory for these two types of mutations in addition the size of our study cohort was small n and the length of followup lessthan year in some patients may not have been sufficient for longterm outcome analysestherefore additional studies will be needed to corroborate our findingsin the results of this study indicate that egfr is an active molecular target inthe pathology of ossn including scc and is a prognostic factor the finding also suggests thatdiscovery of mutations may have important implications for future treatment optionssupporting informations1 filexlsxacknowledgmentswe gratefully acknowledge the technical assistance of the research support platform osakacity university graduate school of medicine and the clinical laboratory department of kobekaisei hospitalauthor contributionsconceptualization mizuki tagami atsushi azumidata curation atsushi sakai mizuki tagami atsuko katsuyamayoshikawa norihiko misawa atsushi azumiformal analysis mizuki tagami anna kakehashi norihiko misawafunding acquisition mizuki tagamiinvestigation mizuki tagami atsuko katsuyamayoshikawa atsushi azumimethodology mizuki tagami anna kakehashi atsuko katsuyamayoshikawa atsushiazumiproject administration mizuki tagamisupervision anna kakehashi hideki wanibuchi atsushi azumi shigeru hondavisualization atsushi sakai mizuki tagami anna kakehashiwriting “ original draft atsushi sakai mizuki tagamiwriting “ review editing mizuki tagami shigeru hondareferenceslee ga hirst lw ocular surface squamous neoplasia surv ophthalmol “ 101016s0039625705800542 pmid one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinoma kiire ca stewart rmk srinivasan s heimann h kaye sb dhillon b a prospective study of the incidence associations and outcomes of ocular surface squamous neoplasia in the united kingdom eyelond “ mcclellan aj mcclellan al pezon cf karp cl feuer w galor a epidemiology of ocular surfacesquamous neoplasia in a veterans affairs population cornea “ 101097ico0b013e31829e3c80 pmid sun ec fears tr goedert jj epidemiology of squamous cell conjunctival cancer cancer epidemiolbiomarkers prev “ pmid scholz sl thomasen h reis h frequent tert promoter mutations in ocular surface squamousneoplasia invest ophthalmol vis sci “ 101167iovs1517469pmid nagarajan p elhadad c gruschkus sk ning j hudgens cw sagiv o pdl1pd1 expressioncomposition of tumorassociated immune infiltrate and hpv status in conjunctival squamous cellcarcinoma invest ophthalmol vis sci “ 101167iovs1926894pmid le tourneau c delord jp gonc¸alves a gavoille c dubot c isambert n molecularly targetedtherapy based on tumour molecular profiling versus conventional therapy for advanced cancershiva a multicentre openlabel proofofconcept randomised controlled phase trial lancetoncol “ 101016s1470204515001886 pmid el zaoui i bucher m rimoldi d nicolas m kaya g pescini gobert r conjunctival melanomatargeted therapy mapk and pi3kmtor pathways inhibition invest ophthalmol vis sci “ 101167iovs1826508 pmid ciardiello f tortora g a novel approach in the treatment of cancer targeting the epidermal growth factor receptor clin cancer res “ pmid lynch tj bell dw sordella r gurubhagavatula s okimoto ra brannigan bw activating mutations in the epidermal growth factor receptor underlying responsiveness of nonsmallcell lung cancer togefitinib n engl j med “ 101056nejmoa040938 pmid paez jg ja¨nne pa lee jc tracy s greulich h gabriel s egfr mutations in lung cancer correlation with clinical response to gefitinib therapy science “ 101126science1099314 pmid cesano a ncounter® pancancer immune profiling panel nanostring technologies inc seattlewa j immunother cancer 101186s4042501500887 pmid yu jj fu p pink jj dawson d wasman j orem j hpv infection and egfr activationalteration in hivinfected east african patients with conjunctival carcinoma one e10477101371 pone0010477 pmid mwololo a nyagol j rogena e ochuk w kimani m onyango n correlation of egfr pegfrand p16ink4 expressions and high risk hpv infection in hivaidsrelated squamous cell carcinoma ofconjunctiva infect agent cancer 1011861750937897 pmid haglund k dikic i the role of ubiquitylation in receptor endocytosis and endosomal sorting j cell sci “ 101242jcs091280 pmid zhang x gureasko j shen k cole pa kuriyan j an allosteric mechanism for activation of the kinasedomain of epidermal growth factor receptor cell “ 101016jcell pmid avraham r yarden y feedback regulation of egfr signalling decision making by early and delayedloops nat rev mol cell biol “ 101038nrm3048 pmid kobayashi y mitsudomi t not all epidermal growth factor receptor mutations in lung cancer are created equal perspectives for individualized treatment strategy cancer sci “101111cas12996 pmid shigematsu h lin l takahashi t nomura m suzuki m wistuba ii clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers j natl cancerinst “ 101093jncidji055 pmid mitsudomi t yatabe y mutations of the epidermal growth factor receptor gene and related genes asdeterminants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancercancer sci “ 101111j13497006200700607x pmid yun ch mengwasser ke toms av woo ms greulich h wong kk the t790m mutation inegfr kinase causes drug resistance by increasing the affinity for atp proc natl acad sci u s a “ 101073pnas0709662105 pmid one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinoma kobayashi y togashi y yatabe y mizuuchi h jangchul p kondo c egfr exon mutationsin lung cancer molecular predictors of augmented sensitivity to afatinib or neratinib as comparedwith first or thirdgeneration tkis clin cancer res “ 10115810780432ccr151046 pmid wu jy yu cj chang yc yang ch shih jy yang pc effectiveness of tyrosine kinase inhibitors onuncommon epidermal growth factor receptor mutations of unknown clinical significance in nonsmallcell lung cancer clin cancer res “ 10115810780432ccr10 pmid maemondo m inoue a kobayashi k sugawara s oizumi s isobe h gefitinib or chemotherapyfor nonsmallcell lung cancer with mutated egfr n engl j med “ 101056nejmoa0909530 pmid mitsudomi t morita s yatabe y negoro s okamoto i tsurutani j gefitinib versus cisplatin plusdocetaxel in patients with nonsmallcell lung cancer harbouring mutations of the epidermal growth factor receptor wjtog3405 an open label randomised phase trial lancet oncol “101016s147020450970364x pmid nishiwaki m yamamoto t tone s murai t ohkawara t matsunami t genotyping of humanpapillomaviruses by a novel onestep typing method with multiplex pcr and clinical applications j clinmicrobiol “ 101128jcm0079307 pmid nakamura y sotozono c kinoshita s the epidermal growth factor receptor egfr role in cornealwound healing and homeostasis exp eye res “ 101006exer2000 pmid fukuoka m yano s giaccone g tamura t nakagawa k douillard jy multiinstitutional randomized phase ii trial of gefitinib for previously treated patients with advanced nonsmallcell lung cancer the ideal trial [corrected] j clin oncol “ 101200jco pmid oldrini b hsieh wy erdjumentbromage h codega p carro ms curielgarcı´a a egfr feedbackinh Answer:

Colon_Cancer

"peroxisome proliferatoractivated receptorsppars asligandactivated transcription factors belong to the steroidreceptor superfamily which includes three isoforms pparalpha ppar betadelta and ppar gamma ppars formheterodimers with retinoic x receptors and regulate theexpression of various genes upon ligand binding ppars alsointeract with corepressors or coactivators to modulate thetranscription of its downstream target genes ppars asimportant transcriptional regulators have been suggested tobe involved in lipid metabolism and multiple cellular functions for instance ppar alpha also functions in fatty acidbetaoxidation and vascular ‚ammation ppar gammaacts as a regulator in adipocyte diï¬erentiation and type diabetes ppar betadelta is a key player in cardiac energyproduction angiogenesis and particularly in cancer progression ppar alpha and ppar gamma exert predominantly anantiangiogenic eï¬ect [“] but there still exist conflictingstudies showing opposite results [ ] on the contraryppar betadelta produces more obviously proangiogeniceï¬ects [“] in this review we will focus on the promotingrole of ppar betadelta in angiogenesis especially in tumorangiogenesis the network of interplay between ppar betadelta and its various downstream signal molecules and alsobetween those key molecules will be further discussed andestablished remarkably diverse important signal moleculesinvolved in tumor angiogenesis and progression and cancercell metabolism have been identified as direct ppar betadelta target genes angiogenesisangiogenesis is the physiological process through which anew capillary network forms from the preexisting vasculature[ ] whereas vasculogenesis denotes de novo bloodvessel formation mostly during embryogenesis in whichendothelial progenitor cells epc migrate to sites of vascularization then diï¬erentiate into endothelial cells ec andcoalesce into the initial vascular plexus [ ] besides theinteraction between proangiogenic factors and antiangiogenic factors angiogenesis is also a multiple step biologicalprocess during which a variety of molecules cooperateincluding cell adhesion molecules matrix metalloproteinases 0cppar researchmmps extracellular matrix ecm and basement membrane componentsangiogenesis is a physiological and vital process in development and growth an imbalance of proangiogenic andantiangiogenic factors causes angiogenesis in pathologicalconditions such as diabetic retinopathy and tumor growththus when the imbalance comes to a point at which angiogenesis is triggered by tumor cells then an œangiogenicswitch of tumor cells is turned on during tumor progression the œangiogenic switch is often activated and remainson [“] inducing angiogenesis is known as a hallmarkof cancer and angiogenesis is also a fundamental stepby which most benign tumors transition into malignant ones tumor angiogenesis tumor needs to sprout new vesselsand further develop a vascular network in order to supplynutrients and oxygen remove waste products support a continually high proliferative rate and ultimately expand neoplastic growth [ ] hence angiogenesis is essential forhelping sustain tumor growth and facilitate tumor progression besides being a requirement for angiogenesis an abnormal vasculature also helps to promote tumor progression andmetastasis the tumor vascular wall is imperfect and prone toleakage so it is much easier for tumor cells to directly penetrate into the blood vessels or lymphatic vessels and then proliferate at another distant site to form metastasis due to intensive abnormal neovascularization in tumortissues most malignant tumors grow rapidly and acquirethe ability to spread to adjacent and distant ans whichmakes them more malignant and even life threateningtherefore angiogenesis indeed plays an important role intumor progression and metastasis and to intervene with thisprocess would obviously prevent tumor development andspread thus this has been regarded as a critical target forantitumor therapy ppar alpha and angiogenesisit was reported firstly that a selective ppar alpha agonistwy14643 did not show any eï¬ect on angiogenesis or ec proliferation but some subsequent studies showed that theactivation of ppar alpha inhibited angiogenesis in vitro byusing fenofibrate a clinically used ppar alpha agonist moreover fenofibrate suppressed ec proliferation migration and tube formation through inhibition of protein kinaseb akt and disruption of the cytoskeleton furthermore ppar alpha activation was shown to inhibit vascularendothelial growth factor vegf induced ec migrationand basic fibroblast growth factor bfgffgf2 inducedcorneal angiogenesis in vitro and in vivo especiallyin vivo reduced tumor growth and microvessel numberswere observed in mice implanted with melanoma lewis lungcarcinoma llc fibrosarcoma and glioblastoma due to asystemic treatment of ppar alpha ligand and the antiangiogenic state induced through activation of ppar alpha withelevated thrombospondin1 tsp1 and endostatin expression howeverit was demonstrated inanother observation that activation of ppar alpha stimuin that same yearlated neovascularization in vivo with increased phosphorylation of endothelial nitric oxide synthase enos and akt via avegfdependent manner furthermore zhang andward also suggested that ppar alpha activation inducedproangiogenic responses in human ocular cells inanother study it was shown that a new ppar alpha agonistrk13675 had no eï¬ect on angiogenesis recentlyppar alpha activation is further shown to have antineovascularization eï¬ects with downregulation of vegf and angiopoietin expression in a rat alkali burn model in summary the role of ppar alpha in angiogenesis isstill controversial some observations showed that ligandactivation of ppar alpha had antiangiogenic eï¬ects mediated either through upregulation of antiangiogenic factorssuch as tsp1 and endostatin or downregulation of proangiogenic factors including vegf fgf2 akt and angiopoietins others also reported opposite results showing aproangiogenic role upon ppar alpha activation thus thespecific molecular mechanism is still unclear and needs tobe further studied ppar gamma and angiogenesisligand activation of ppar gamma was previously shown toinhibit human umbilical vein endothelial cell huvec tubeformation in collagen gels and vegfinduced choroidalneovascularization in vitro and in vivo another studyalso demonstrated that ec apoptosis was induced throughtreatment with the ppar gamma ligand 15dpgj2 furthermore rosiglitazone a potent ppar gamma agonist wasshown to inhibit primary tumor growth and metastasisthrough both direct and indirect antiangiogenic eï¬ectsin vitro and bfgfinduced corneal neovascularizationin vivo moreover a similar observation also displayedthe inhibition of vegfinduced angiogenesis in a chickchorioallantonic membrane model in a mouse modelwith ischemiainduced retinopathy pioglitazone a ppargamma agonist also showed a protective eï¬ect against pathological neoangiogenesis through upregulation of anti‚ammatory adipokine adiponectin additionally theppar gamma antagonist gw9662 was shown to reverseomega3 polyunsaturated fatty acidinduced reduction ofeselectin angiopoietin2 vascular cell adhesion molecule and intracellular adhesion molecule1 implicatingan antiangiogenic potential of ppar gamma itself howeveropposite results also showed that pioglitazone enhanced neovascularization and inhibited apoptosis of epc in vitro andin vivo via a phosphoinositide3kinase pi3k dependentmanner nadra observed that ppar gammanull embryosdisplayed a vascular structural defect at e95 moreover disanized placental layers and an altered placental microvasculature were observed in pregnant wildtype mice treatedwith the ppar gamma agonist rosiglitazone as well asreduced expression of proangiogenic factorsincludingvegf proliferin and plateletendothelial cell adhesionmolecule1 pecam1cd31 suggesting a crucial roleof ppar gamma in placental vascular development the 0cppar researchmajor antiangiogenic properties on ppar gamma activationwere also reviewed here notablyin most cancersthe canonical wntbetacatenin pathway is upregulated while on the contrary ppargamma is downregulated interestingly in numerous tissuesthe activation of ppar gamma inhibits the betacateninpathway whereascanonicalwntbetacatenin signal cascade also inactivates ppargamma implicating a negative regulatory role of ppargamma in carcinogenesis where tumor angiogenesis mightbe a fundamental stepstimulation ofthethein summary ppar gamma predominantly displays anantiangiogenic eï¬ect that may be mediated through the inhibition of vegf or bfgfinduced neovascularization andreduction of the expression level of some proangiogenicfactors ppar betadelta and angiogenesisunlike ppar alpha and ppar gamma on the contrarymany studies have explicitly shown the proangiogenic eï¬ectsof ppar betadelta on physiological and pathological angiogenesis the first evidence provided in a study is that activation of ppar betadelta with gw501516 a highly selectiveppar betadelta agonistinduces huvec proliferationand an increased expression of vegf and its receptorvegfr1 flt1 besides inducing ec proliferationppar betadelta activation by itsligand prostacyclinpgi2 also stimulates upregulation of alpha expression an antiapoptotic and anti‚ammatory protein whichthereby protects ecs from h2o2induced apoptosis and oxidant injury moreover a subsequent study further provides evidence that activation of ppar betadelta withgw501516 induces angiogenesis during which vegfrelease is considered as a major trigger factor firstlysuggesting the promotion for angiogenesis upon pparbetadelta activationmüllerbrüsselbach show that ppar betadelta mice implanted with llc and b16 melanoma exhibit diminished blood flow and immature microvascular structurescompared with wildtype mice moreover reexpression ofppar betadelta into the matrigelinvading cells triggersmicrovessel maturation and restores normal vascularization indicating a crucial role of ppar betadelta in tumorvascularization additionally another study also observedreduced levels of calcium intracellular channel protein clic4 but it observed enhanced expression of cellular retinol binding protein crbp1 in migrating ecs from pparbetadeltanull mice both of which play a role in tumorvascularization [ ] it was reported that ppar betadeltawas required for placentation and most of the pparbetadeltanull mutant embryos died at e95 to e105 due toabnormal celltocell communication atthe placentaldecidual interface however in these studies [“] adefect in angiogenesis was not observed during normal development in ppar betadeltaknockout micesome observations also show the important role of pparbetadelta in physiological angiogenesis for instance skeletal musclespecific ppar betadelta overexpression leads toppar betadeltaan increase in the number of oxidative muscle fibers andrunning endurance in adult mice [“] moreover pparbetadelta activation promotes a rapid muscle remodelingvia a calcineurindependent manner and induces muscleangiogenesis in highly selective ppar betadelta agonistgw0742treated animals furthermore in the heartpharmacologicalstimulation withgw0742 induces rapid cardiac growth and cardiac angiogenesis through direct transcriptional activation of calcineurin interestingly the same cardiac phenotype wasalso observed after treatment with the ppar betadelta agonist gw501516implicating a response specificity forppar betadelta stimulation calcineurin activationfurther leads to the stimulation of nuclear factoractivatedt cell c3 nfatc3 and an enhanced expression of hypoxiainducible factor alpha hif1alpha and cyclindependentkinase cdk9 overall the remodeling in skeletalmuscle and heart is perfectly the same as the phenotypeobserved with exercise and both of them are mediatedthrough activation of calcineurinppar betadelta may act as a key regulator in mediatingpathological angiogenesis for instance ppar betadelta wasshown to regulate retinal angiogenesis in vitro and in vivoand its inhibition reduced preretinal neovascularization possibly via an angiopoietinlike protein angptl4 dependent manner implicating the potential of pparbetadelta in modulating pathological ocular angiogenesisrecently an observation reported that ppar betadeltaknockdown in both retinal pigment epithelial and choroidalendothelial cells caused an antiangiogenic phenotype andppar betadelta promoted laserinduced choroidal neovascular cnv lesions in ppar betadelta mice moreover pharmacological inhibition of ppar betadelta with theantagonist gsk0660 also resulted in a significantly decreasedcnv lesion size in vivo suggesting a functional role of pparbetadelta in the development of cnv lesions this indicates that ppar betadelta has an important association withpathological angiogenesisangiotensin ii ang ii the biologically active peptide ofthe reninangiotensin system ras is a major blood pressure and cardiovascular homeostasis regulator and is alsorecognized as a potent mitogen angiotensinconvertingenzyme inhibitors were introduced approximately yearsago as antihypertensive agents and have since become asuccessful therapeutic approach for high blood pressurecongestive heart failure and postmyocardial infarction inexperimental systemsthe antitumor eï¬ects of diverseace inhibitors show that these inhibit cell proliferationand possessand anti‚ammatory eï¬ects [“] it has been shown recentlythat activation of ppar betadelta inhibits ang iistimulated protein synthesis in a concentrationdependentmanner and suppresses ang iiinduced generation of reactive oxygen species ros in vascular smooth muscle cells ppar betadelta was further shown to inhibit angiimediated atherosclerosis however it is not clearuntil now if ppar betadelta activation can be consideredis foras an ace inhibitormimicking approach as itexample the case for ppar gamma activators antiangiogenicantimetastatic 0cppar researchthe relevance offurthermorethis hypothetical pparbetadelta feature might be limited for tumor angiogenesiswhere vascular smooth muscle hypertrophy and atherosclerosis do not contribute to the major pathologybesides inducing angiogenesis it has been demonstratedthat ppar betadelta directly acts on early epc through activation of the akt pathway and induces an enhanced vasculogenesis similarlythe ppar betadeltamediatedprovasculogenic eï¬ects are also observed on late epc he showed that ppar betadelta activation withgw501516 induced epc proliferation and tube formationwhereas epc treated with an inhibitor of cyclooxygenasecox or pgi2 synthase or with ppar betadeltaspecificsirna also displayed an opposite eï¬ect furthermoreit has been demonstrated that ppar betadelta inducesangiogenesis and skeletal muscle regeneration throughmatrix metalloproteinase mmp 9mediated insulinlikegrowth factor1 paracrine networks upon epc activation han also observed that ppar betadelta activationpromoted a rapid wound healing with enhanced angiogenesis in a mouse model with skin punch wound overallin addition to ec ppar betadelta is also a key regulator ofepc or even may act as an initiator of activation of epc tofurther induce vasculogenesis ppar betadelta and tumor angiogenesislinked to tumor microenvironmentppar betadelta expression is often upregulated and promotes cancer progression in many major human cancerslung breast and gastric cancers [“]such as colonwhich suggests a crucial role of ppar betadelta in cancercells even though there exist some conflicting studies indicating that the functional role of ppar betadelta in tumorigenesis or carcinogenesis still remains highly controversial [“] and dependent on specific tumor or cancer cell typesthus here we discuss the promotion of ppar betadelta intumor progression through facilitating tumor angiogenesisppar betadelta has been suggested as a critical œhubnode transcriptional factor which governs a tumor œangiogenic switch [ “] in the transcriptional networkanalysis it was reported that tumor growth and tumor angiogenesis were markedly inhibited in ppar betadeltanullmice in comparison with wildtype mice moreoverthe elevated ppar betadelta expression level was also considered to be highly correlated to pathologically advancedtumor stage and increased cancer risk for recurrence and distant metastasis in patients with pancreatic cancer indicating the crucial association of ppar betadelta withtumor angiogenesis progression and cancer invasivenessppar betadelta may indirectly facilitate tumor angiogenesis and progression through its function on the tumormicroenvironment tme where tumor angiogenesis is fostered moreover a tumor also releases some extracellular signals to closely communicate and constantly collaborate withtme to facilitate tumor angiogenesis in order to furtherenable tumor growth and progression for instance it wasshown that colon cancer cells with ppar betadelta knockoutfailed to stimulate ec vascularization in response to hypoxicstress whereas wildtype cells exposed to hypoxia were ableto induce angiogenesis [ ] suggesting that ppar betadelta is required for the promotion of angiogenesis in hypoxic stressmediated tme moreover in the tme tumorltrating myeloid cells are considered as the most important cells for fostering tumor angiogenesis among the multiple diï¬erent kinds of stromal cells besides stimulatingtumor angiogenesis tumor myeloid cells also support tumrowth by suppressing tumor immunity and promotingtumor metastasis to distinct sites interestingly it hasbeen demonstrated that ppar betadelta activation intumorltrating myeloid cells stimulates cancer cell invasion and facilitates tumor angiogenesis via an interleukin il10 dependent manner moreover impairedtumor growth and angiogenesis were observed in pparbetadelta ko bmt mice due to ppar betadelta deficiencyin tumor myeloid cells suggesting that ppar betadeltaplays a key role in tumor angiogenesis and progression intumor myeloid cells of tmefurthermore the endoplasmic reticulum er an essential anelle involved in many cellular functions is implicated in tme in cancer stressors like hypoxia nutrientdeprivation and acidosis disrupt er function and lead toaccumulation of unfolded proteins in er a condition knownas er stress cells adapt to er stress by activating an integrated signal transduction pathway called the unfolded protein response upr upr represents a survival response bythe cells to restore er homeostasis and has both survivaland cell death eï¬ects the mechanisms that determine cellfate during er stress are not well understood for instanceshort exposure to er stress initially increases akt signalingbut longterm er stress suppresses akt signaling ppar betadelta activation has been shown to reduce endoplasmic reticulum er stressassociated ‚ammation inskeletal muscle through an ampkdependent mechanism and to reduce ‚ammation in response to chronic erstress in cardiac cells furthermore it has been nicelyshown that ppar betadelta can repress rasoncogeneinduced er stress to promote senescence in tumors thisis mediated through the decrease of pakt activity promoting cellular senescence through upregulation of p53 and p27expression it would be interesting to investigate thedirect eï¬ects of ppar betadelta on senescence of tumorendothelial cells in an in vivo setting we recently showedthat senescent endothelial cells are indispensable for ahealthy lifespan and that removal of senescent endotheliumdisrupts vascular function leading to diminished vessel densities and fibrotic lesions if ppar betadelta mediatessenescence of tumor endothelium thereby protecting vesselintegrity this might explain the enhanced tumor growthand vascularization upon ppar betadelta activationobserved by us and others [ ]most recently zuo demonstrated that pparbetadelta in cancer cells regulates tumor angiogenesisin vivo and in vitro by promoting the secretion of proangiogenic factors including vegf and interleukin il8 most importantly in our recent works it has beenshown that conditionalinducible vascular endotheliumspecific ppar betadelta overexpression in vivo leads to 0cppar researchenhanced tumor angiogenesis tumor growth and metastasis formationfurther indicating a vascular ecspecificppar betadelta action mechanism in tumor progressionindependent of some controversial observations of pparbetadelta in specific tumor or cancer cell types wagner also firstly reported the mouse model in whichrapid induction of cardiac angiogenesis and cardiac hypertrophy were observed [ ] crosstalk between ppar betadelta and signalmolecules ppar betadelta activation or overexpressionmay upregulate the expression of its various downstream signal molecules involved in tumor angiogenesis includingproangiogenic factors such as vegf pdgf and fgfproinvasive matrixdegrading enzymes such as mmp9pro‚ammatory mediators such as cox2 and cytokinesand chemokines such as il1 and cxcl8 even some ofwhich have been further identified as ppar betadelta directtarget genes besides a leading role of ppar betadelta amongthe signal molecules ppar betadelta may function in tmelinked to diverse kinds of cells through direct or indirectmodulation of its downstream molecules interplay between ppar betadelta and ‚ammatoryangiogenesis ‚ammatory angiogenesis is a crucial processin tumor progression for instance the pro‚ammatorymediator cyclooxygenase2 cox2 is considered as a keyregulator of angiogenesis and tumor growth through multiple downstream proangiogenic mechanisms such as production of vegf and induction of mmps moreover selectiveinhibition of cox2 has also been shown to suppress angiogenesis in vivo and in vitro it is well known that vegfaplays a critical role in both angiogenesis and vasculogenesis and it leads the directional migration of tip cells andstalk cell proliferation in microtubule branches [ ] ithas also been demonstrated that mmp9 triggers the œangiogenic switch during carcinogenesis and enhances the availability of vegf to its receptors furthermore it hasbeen reported that ‚ammatory cell mmp9 initiates theonset of tumor neovascularization during which there existsfunctionalincludingmmp9 leptin is shown to mediate angiogenesisin vivo and in vitro through induction of ec proliferationand expression of mmp2 and mmp9 and to furtherpromote ec diï¬erentiation and directional migrationthrough enhancement of cox2 activity leptin couldalso induce angiogenesis via transactivation of vegfr inecs additionally besides inducing angiogenesisppar betadelta also functions in chronic ‚ammationfacilitating tumorigenesis through induction of cox2 andits product prostaglandin e2 pge2 in vivo [ ]interestingly cox2 vegf mmp9 and leptin have beenidentified as ppar betadelta target genes via a direct transcriptional activation mechanism in hepatocellular carcinoma cells colorectal cancer cells [ ] epcs[ ] and liposarcoma cells respectivelylinks between vegf and mmpsin tme tumorltrating ‚ammatory cells also helpto induce and sustain tumor angiogenesis and further tofacilitate tissue invasion and tumor metastatic spread byreleasing some signal molecules such as proinvasive mmp9and ‚ammatory chemokines [“] chemotaxis is alsoa crucial process for inducing angiogenesis in tumors eitherdirectly by attracting ecs towards tumor cells to form newvessels or indirectly by mediating immune ‚ammatorycells to ltrate eventually promoting tumor angiogenesis chemotaxis of tumor cells and stromal cells in tmeis also required for tumor dissemination during tumor progression and metastasis [ ]cxc chemokines such as cxcl8 encoding il8 andcxcl5 are also involved in cox2associated angiogenesisto contribute to nonsmallcelllung cancer progression[ ] it is further shown that il8 directly regulatesangiogenesis via recruitment of neutrophils whichfurther drives vegf activation moreoveril8responding neutrophils are considered as the major sourceof angiogenesisinducing mmp9 [ ] chemokine cc motif ligand ccl2 in addition to the promotionof angiogenesis [ ] also enhances tumor metastasis furthermore myeloid monocytic cellssuch asmyeloidderivedtumormdscsassociated macrophages tams and dendritic cells arerecruited to the tumor site mainly by ccl2 and producemany proangiogenic factorssuch as vegf cxcl8plateletderived growth factor pdgf and transforminggrowth factor beta tgf beta [“] in fact bothtgf beta and hypoxia are potentinducers of vegfexpression in tumor cells and collaborate with tme toprovide the foundation of tumor angiogenesis and cancercell invasion importantly il8 has been reported asa key target gene of ppar betadelta to promote angiogenesis in vivo and in vitro and ccl2 expression isalso significantly upregulated upon vascular ppar betadelta overexpression in vivo suppressorcellscox2 also mediates il1 betainduced angiogenesisin vitro and in vivo [ ] il1 beta supports neovascularization through the regulation of the expression of vegfand its receptor vegfr2 flk1kdr on ecs il1 actsas an upstream pro‚ammatory mediator that initiates anddisseminates the ‚ammatory state by inducing a localinteractive network and increasing adhesion moleculeexpression on ecs and leukocytes which facilitates tumorassociated angiogenesis in tme ‚ammatory il1beta recruits myeloid cells from bone marrow and activatesthem to produce proangiogenic factors such as vegf vegffurther activates ecs and myeloid cells promoting tumorinvasiveness and fostering tumor angiogenesis in addition il6 also stimulates angiogenesis and vasculogenesis[ ] however gopinathan observed an il6induced newly forming vascular structure with defectivepericyte pc coverage ex vivo thus facilitating cancercell ltration and tumor metastasis through vascular leakage interestingly il1 and il6 expression levels are significantly upregulated in the ppar betadelta overexpressionmouse model reported recently in summary ppar betadelta seems to act as a key leaderin ‚ammatory mediatordriven tumor angiogenesis linkedto tme in which many pro‚ammatory mediators chemokines and proangiogenic factors closely communicate with 0cppar researcheach other and also associate with tumorltrating myeloid cells such as neutrophils tams and mdscs other key ppar betadeltamediated proangiogenicfactors it has been demonstrated that wilms™ tumor suppressor wt1 is a major regulator of tumor neovascularization andtumor progression e26 avian leukemia oncogene ets1 also plays a key role in regulating vascular development and haemopoiesis particularly in angiogenesis in addition ets1 promotes cancer cell invasion throughupregulation of mmps consistent with this silencingof ets1 in highly invasive breast cancer cells also reducesthe expression of mmp9 and mmp1 ets1 also acts as a key regulator of mmps such asmmp1 mmp3 and mmp9 in human cancerassociatedfibroblasts cafs [ ] cafs support tumor growthby secreting growth factors such as vegf fgf pdgf andchemokines to stimulate angiogenesis and thereby promotecancer cell invasion and metastasis formation [ ]cafs as metastatic tumor stroma are a crucial componentin tumor progression through the remodeling of the ecmstructure thus helping a tumor to acquire an aggressive phenotype [ ] ppar betadelta in cafs also exhibits aprotumorigenic eï¬ect it was reported that ablation of pparbetadelta in cafs attenuated tumor growth by altering theredox balance in tme suggesting that ppar betadeltain cafs is also an important player in tumor developmentets1 induces the expression of vegf vegfr1 andvegfr2 in ecs [“] in turn vegf is also a majorinducer of ets1 in ecs through the activation of either thepi3kakt pathway or the mekerk12 signal cascade[ ] wt1 is also reported to regulate tumor angiogenesis via direct transactivation of ets1 sryrelated hmgbox sox18 has also beenreported previously to induce angiogenesis during tissuerepair and wound healing and cancer progression and most recently it was further shown that specificecderived endovascular progenitors initiated a vasculogenic process and diï¬erentiated into more mature endothelial phenotypes within the core of the growing tumorsthrough reactivation of sox18 interestingly theseimportant proangiogenic molecules including wt1 ets1and sox18 are also significantly upregulated in the vascularppar betadelta overexpression model in vivo andwt1 is also identified as a target gene of ppar betadeltain melanoma cells ppar betadelta may facilitate cancer progression atdiverse cellular levels in tme ppar betadelta activationis shown to induce colonic cancer stem cell csc expansionand to promote the liver metastasis of colorectal cancerin vivo via direct transactivation of the nanog gene nanog as a key transcriptional factor governs the selfrenewal and pluripotency of stem cells and cancer cellsexpressing nanog also often exhibit stem cell properties protooncogene ckitcd117 is known as the maststem cell factor receptor and receptor tyrosine kinase andits activation in cscs may regulate the stemness to controltumor progression and drug resistance to tyrosine kinaseinhibitors moreover ckit has been identified as a potentialmarker of the cancer stemlike cells in addition ckitnot only functions on ecs [ ] but also belongs to thetumor angiogenesispromoting molecule [“] studiesalso suggested that activation of ckit enhances the expression of vegf that can be suppressed by imatinib an inhibitor of ckit in gastrointestinal stromal tumor cells whichthereby has an impact on tumor angiogenesis [ ] ckit is also involved in pathological ocular neovascularization and is regulated transcriptionally by wt1 and ppar betadelta pdgfb and its receptor pdgfr beta also known asangiogenic factors are suggested to enhance angiogenesisand vasculogenesis via their function in ecs [“] andepcs and to regulate vascular permeability and vesselmaturation through recruitment of pericytes pcs and smooth muscle cells smcs in newly formingvessels moreover pdgfb and pdgfr beta also interactwith other proangiogenic factors such as fgf2 [ ]vegfa and its receptor vegfr2 furthermorepdgfb and pdgfr beta may also aï¬ect cancer growthand progression by directly acting on tme besides thecrosstalk with cafs [“] pdgfr beta in stromalfibroblasts may mediate pdgfbinduced tam recruitment thus implicating a role of pdgfr beta in tumorstroma to facilitate tumor progression most recently it wasfurther shown that specific targeting of pdgfr beta kinaseactivity in tme inhibited cancer growth and vascularizationin cancers with high pdgfb expression such as llc therefore this indicates the diverse role of pdgfb andpdgfr beta in facilitating tumor angiogenesis and progression at diï¬erent cellular levels in tme pdgfr beta is demonstrated as a target of telomeric repeat binding factor trf2 that is further activated transcriptionally by wt1 pdgfb and pdgfr beta have further been identifiedas critical targets of ppar betadelta via a direct transactivation mechanism in vivo in conclusion a variety of key signal molecules involvedin tumor angiogenesis and tumor progression and metastasishave either been identified as ppar betadelta direct targetsor largely upregulated in the vascular ppar betadelta overexpression model in vivo reported recently thus pparbetadelta activation seems to give rise to a highly angiogenicphenotype and even plays a œhallmark role in promotingtumor angiogenesis and progression interestingly it appearsthat there could also exist a widely interactive networkbetween the downstream protumorangiogenic moleculesas described above therefore the crosstalk network is established between ppar betadelta and the various signal molecules and also between those molecules figure 1amoreover in addition to cancer cells ppar betadeltamay also produce pleiotropic eï¬ects in tme by modulatingdownstream key molecules to act on ecs epcs pcs smcscscs cafs and tumorltrating ‚ammatory cells indirectly facilitating tumor angiogenesis and further promotingcancer development figure 1b other ppar betadelta target genes ppar betadeltaregulates the transcription oftarget genes via a direct 0cppar researchil1 betacox2leptinppar betadeltavegfvegfrpdgfr betatrf2wt1ets1mmp9pdgfbckitcxcl8il8il10ccl2cxcl8il8pdgfr betaappar betadeltaets1mmp9pdgfr betananogckittumorassociatedmacrophage tamdendritic cellneutrophilmyeloidderivedsuppressor cell mdscvegfmmp9pdgfbckitsox18pdgfr betaendothelial cell ece

cancer7564

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "peroxisome proliferatoractivated receptorsppars asligandactivated transcription factors belong to the steroidreceptor superfamily which includes three isoforms pparalpha ppar betadelta and ppar gamma ppars formheterodimers with retinoic x receptors and regulate theexpression of various genes upon ligand binding ppars alsointeract with corepressors or coactivators to modulate thetranscription of its downstream target genes ppars asimportant transcriptional regulators have been suggested tobe involved in lipid metabolism and multiple cellular functions for instance ppar alpha also functions in fatty acidbetaoxidation and vascular ‚ammation ppar gammaacts as a regulator in adipocyte diï¬erentiation and type diabetes ppar betadelta is a key player in cardiac energyproduction angiogenesis and particularly in cancer progression ppar alpha and ppar gamma exert predominantly anantiangiogenic eï¬ect [“] but there still exist conflictingstudies showing opposite results [ ] on the contraryppar betadelta produces more obviously proangiogeniceï¬ects [“] in this review we will focus on the promotingrole of ppar betadelta in angiogenesis especially in tumorangiogenesis the network of interplay between ppar betadelta and its various downstream signal molecules and alsobetween those key molecules will be further discussed andestablished remarkably diverse important signal moleculesinvolved in tumor angiogenesis and progression and cancercell metabolism have been identified as direct ppar betadelta target genes angiogenesisangiogenesis is the physiological process through which anew capillary network forms from the preexisting vasculature[ ] whereas vasculogenesis denotes de novo bloodvessel formation mostly during embryogenesis in whichendothelial progenitor cells epc migrate to sites of vascularization then diï¬erentiate into endothelial cells ec andcoalesce into the initial vascular plexus [ ] besides theinteraction between proangiogenic factors and antiangiogenic factors angiogenesis is also a multiple step biologicalprocess during which a variety of molecules cooperateincluding cell adhesion molecules matrix metalloproteinases 0cppar researchmmps extracellular matrix ecm and basement membrane componentsangiogenesis is a physiological and vital process in development and growth an imbalance of proangiogenic andantiangiogenic factors causes angiogenesis in pathologicalconditions such as diabetic retinopathy and tumor growththus when the imbalance comes to a point at which angiogenesis is triggered by tumor cells then an œangiogenicswitch of tumor cells is turned on during tumor progression the œangiogenic switch is often activated and remainson [“] inducing angiogenesis is known as a hallmarkof cancer and angiogenesis is also a fundamental stepby which most benign tumors transition into malignant ones tumor angiogenesis tumor needs to sprout new vesselsand further develop a vascular network in order to supplynutrients and oxygen remove waste products support a continually high proliferative rate and ultimately expand neoplastic growth [ ] hence angiogenesis is essential forhelping sustain tumor growth and facilitate tumor progression besides being a requirement for angiogenesis an abnormal vasculature also helps to promote tumor progression andmetastasis the tumor vascular wall is imperfect and prone toleakage so it is much easier for tumor cells to directly penetrate into the blood vessels or lymphatic vessels and then proliferate at another distant site to form metastasis due to intensive abnormal neovascularization in tumortissues most malignant tumors grow rapidly and acquirethe ability to spread to adjacent and distant ans whichmakes them more malignant and even life threateningtherefore angiogenesis indeed plays an important role intumor progression and metastasis and to intervene with thisprocess would obviously prevent tumor development andspread thus this has been regarded as a critical target forantitumor therapy ppar alpha and angiogenesisit was reported firstly that a selective ppar alpha agonistwy14643 did not show any eï¬ect on angiogenesis or ec proliferation but some subsequent studies showed that theactivation of ppar alpha inhibited angiogenesis in vitro byusing fenofibrate a clinically used ppar alpha agonist moreover fenofibrate suppressed ec proliferation migration and tube formation through inhibition of protein kinaseb akt and disruption of the cytoskeleton furthermore ppar alpha activation was shown to inhibit vascularendothelial growth factor vegf induced ec migrationand basic fibroblast growth factor bfgffgf2 inducedcorneal angiogenesis in vitro and in vivo especiallyin vivo reduced tumor growth and microvessel numberswere observed in mice implanted with melanoma lewis lungcarcinoma llc fibrosarcoma and glioblastoma due to asystemic treatment of ppar alpha ligand and the antiangiogenic state induced through activation of ppar alpha withelevated thrombospondin1 tsp1 and endostatin expression howeverit was demonstrated inanother observation that activation of ppar alpha stimuin that same yearlated neovascularization in vivo with increased phosphorylation of endothelial nitric oxide synthase enos and akt via avegfdependent manner furthermore zhang andward also suggested that ppar alpha activation inducedproangiogenic responses in human ocular cells inanother study it was shown that a new ppar alpha agonistrk13675 had no eï¬ect on angiogenesis recentlyppar alpha activation is further shown to have antineovascularization eï¬ects with downregulation of vegf and angiopoietin expression in a rat alkali burn model in summary the role of ppar alpha in angiogenesis isstill controversial some observations showed that ligandactivation of ppar alpha had antiangiogenic eï¬ects mediated either through upregulation of antiangiogenic factorssuch as tsp1 and endostatin or downregulation of proangiogenic factors including vegf fgf2 akt and angiopoietins others also reported opposite results showing aproangiogenic role upon ppar alpha activation thus thespecific molecular mechanism is still unclear and needs tobe further studied ppar gamma and angiogenesisligand activation of ppar gamma was previously shown toinhibit human umbilical vein endothelial cell huvec tubeformation in collagen gels and vegfinduced choroidalneovascularization in vitro and in vivo another studyalso demonstrated that ec apoptosis was induced throughtreatment with the ppar gamma ligand 15dpgj2 furthermore rosiglitazone a potent ppar gamma agonist wasshown to inhibit primary tumor growth and metastasisthrough both direct and indirect antiangiogenic eï¬ectsin vitro and bfgfinduced corneal neovascularizationin vivo moreover a similar observation also displayedthe inhibition of vegfinduced angiogenesis in a chickchorioallantonic membrane model in a mouse modelwith ischemiainduced retinopathy pioglitazone a ppargamma agonist also showed a protective eï¬ect against pathological neoangiogenesis through upregulation of anti‚ammatory adipokine adiponectin additionally theppar gamma antagonist gw9662 was shown to reverseomega3 polyunsaturated fatty acidinduced reduction ofeselectin angiopoietin2 vascular cell adhesion molecule and intracellular adhesion molecule1 implicatingan antiangiogenic potential of ppar gamma itself howeveropposite results also showed that pioglitazone enhanced neovascularization and inhibited apoptosis of epc in vitro andin vivo via a phosphoinositide3kinase pi3k dependentmanner nadra observed that ppar gammanull embryosdisplayed a vascular structural defect at e95 moreover disanized placental layers and an altered placental microvasculature were observed in pregnant wildtype mice treatedwith the ppar gamma agonist rosiglitazone as well asreduced expression of proangiogenic factorsincludingvegf proliferin and plateletendothelial cell adhesionmolecule1 pecam1cd31 suggesting a crucial roleof ppar gamma in placental vascular development the 0cppar researchmajor antiangiogenic properties on ppar gamma activationwere also reviewed here notablyin most cancersthe canonical wntbetacatenin pathway is upregulated while on the contrary ppargamma is downregulated interestingly in numerous tissuesthe activation of ppar gamma inhibits the betacateninpathway whereascanonicalwntbetacatenin signal cascade also inactivates ppargamma implicating a negative regulatory role of ppargamma in carcinogenesis where tumor angiogenesis mightbe a fundamental stepstimulation ofthethein summary ppar gamma predominantly displays anantiangiogenic eï¬ect that may be mediated through the inhibition of vegf or bfgfinduced neovascularization andreduction of the expression level of some proangiogenicfactors ppar betadelta and angiogenesisunlike ppar alpha and ppar gamma on the contrarymany studies have explicitly shown the proangiogenic eï¬ectsof ppar betadelta on physiological and pathological angiogenesis the first evidence provided in a study is that activation of ppar betadelta with gw501516 a highly selectiveppar betadelta agonistinduces huvec proliferationand an increased expression of vegf and its receptorvegfr1 flt1 besides inducing ec proliferationppar betadelta activation by itsligand prostacyclinpgi2 also stimulates upregulation of alpha expression an antiapoptotic and anti‚ammatory protein whichthereby protects ecs from h2o2induced apoptosis and oxidant injury moreover a subsequent study further provides evidence that activation of ppar betadelta withgw501516 induces angiogenesis during which vegfrelease is considered as a major trigger factor firstlysuggesting the promotion for angiogenesis upon pparbetadelta activationmüllerbrüsselbach show that ppar betadelta mice implanted with llc and b16 melanoma exhibit diminished blood flow and immature microvascular structurescompared with wildtype mice moreover reexpression ofppar betadelta into the matrigelinvading cells triggersmicrovessel maturation and restores normal vascularization indicating a crucial role of ppar betadelta in tumorvascularization additionally another study also observedreduced levels of calcium intracellular channel protein clic4 but it observed enhanced expression of cellular retinol binding protein crbp1 in migrating ecs from pparbetadeltanull mice both of which play a role in tumorvascularization [ ] it was reported that ppar betadeltawas required for placentation and most of the pparbetadeltanull mutant embryos died at e95 to e105 due toabnormal celltocell communication atthe placentaldecidual interface however in these studies [“] adefect in angiogenesis was not observed during normal development in ppar betadeltaknockout micesome observations also show the important role of pparbetadelta in physiological angiogenesis for instance skeletal musclespecific ppar betadelta overexpression leads toppar betadeltaan increase in the number of oxidative muscle fibers andrunning endurance in adult mice [“] moreover pparbetadelta activation promotes a rapid muscle remodelingvia a calcineurindependent manner and induces muscleangiogenesis in highly selective ppar betadelta agonistgw0742treated animals furthermore in the heartpharmacologicalstimulation withgw0742 induces rapid cardiac growth and cardiac angiogenesis through direct transcriptional activation of calcineurin interestingly the same cardiac phenotype wasalso observed after treatment with the ppar betadelta agonist gw501516implicating a response specificity forppar betadelta stimulation calcineurin activationfurther leads to the stimulation of nuclear factoractivatedt cell c3 nfatc3 and an enhanced expression of hypoxiainducible factor alpha hif1alpha and cyclindependentkinase cdk9 overall the remodeling in skeletalmuscle and heart is perfectly the same as the phenotypeobserved with exercise and both of them are mediatedthrough activation of calcineurinppar betadelta may act as a key regulator in mediatingpathological angiogenesis for instance ppar betadelta wasshown to regulate retinal angiogenesis in vitro and in vivoand its inhibition reduced preretinal neovascularization possibly via an angiopoietinlike protein angptl4 dependent manner implicating the potential of pparbetadelta in modulating pathological ocular angiogenesisrecently an observation reported that ppar betadeltaknockdown in both retinal pigment epithelial and choroidalendothelial cells caused an antiangiogenic phenotype andppar betadelta promoted laserinduced choroidal neovascular cnv lesions in ppar betadelta mice moreover pharmacological inhibition of ppar betadelta with theantagonist gsk0660 also resulted in a significantly decreasedcnv lesion size in vivo suggesting a functional role of pparbetadelta in the development of cnv lesions this indicates that ppar betadelta has an important association withpathological angiogenesisangiotensin ii ang ii the biologically active peptide ofthe reninangiotensin system ras is a major blood pressure and cardiovascular homeostasis regulator and is alsorecognized as a potent mitogen angiotensinconvertingenzyme inhibitors were introduced approximately yearsago as antihypertensive agents and have since become asuccessful therapeutic approach for high blood pressurecongestive heart failure and postmyocardial infarction inexperimental systemsthe antitumor eï¬ects of diverseace inhibitors show that these inhibit cell proliferationand possessand anti‚ammatory eï¬ects [“] it has been shown recentlythat activation of ppar betadelta inhibits ang iistimulated protein synthesis in a concentrationdependentmanner and suppresses ang iiinduced generation of reactive oxygen species ros in vascular smooth muscle cells ppar betadelta was further shown to inhibit angiimediated atherosclerosis however it is not clearuntil now if ppar betadelta activation can be consideredis foras an ace inhibitormimicking approach as itexample the case for ppar gamma activators antiangiogenicantimetastatic 0cppar researchthe relevance offurthermorethis hypothetical pparbetadelta feature might be limited for tumor angiogenesiswhere vascular smooth muscle hypertrophy and atherosclerosis do not contribute to the major pathologybesides inducing angiogenesis it has been demonstratedthat ppar betadelta directly acts on early epc through activation of the akt pathway and induces an enhanced vasculogenesis similarlythe ppar betadeltamediatedprovasculogenic eï¬ects are also observed on late epc he showed that ppar betadelta activation withgw501516 induced epc proliferation and tube formationwhereas epc treated with an inhibitor of cyclooxygenasecox or pgi2 synthase or with ppar betadeltaspecificsirna also displayed an opposite eï¬ect furthermoreit has been demonstrated that ppar betadelta inducesangiogenesis and skeletal muscle regeneration throughmatrix metalloproteinase mmp 9mediated insulinlikegrowth factor1 paracrine networks upon epc activation han also observed that ppar betadelta activationpromoted a rapid wound healing with enhanced angiogenesis in a mouse model with skin punch wound overallin addition to ec ppar betadelta is also a key regulator ofepc or even may act as an initiator of activation of epc tofurther induce vasculogenesis ppar betadelta and tumor angiogenesislinked to tumor microenvironmentppar betadelta expression is often upregulated and promotes cancer progression in many major human cancerslung breast and gastric cancers [“]such as colonwhich suggests a crucial role of ppar betadelta in cancercells even though there exist some conflicting studies indicating that the functional role of ppar betadelta in tumorigenesis or carcinogenesis still remains highly controversial [“] and dependent on specific tumor or cancer cell typesthus here we discuss the promotion of ppar betadelta intumor progression through facilitating tumor angiogenesisppar betadelta has been suggested as a critical œhubnode transcriptional factor which governs a tumor œangiogenic switch [ “] in the transcriptional networkanalysis it was reported that tumor growth and tumor angiogenesis were markedly inhibited in ppar betadeltanullmice in comparison with wildtype mice moreoverthe elevated ppar betadelta expression level was also considered to be highly correlated to pathologically advancedtumor stage and increased cancer risk for recurrence and distant metastasis in patients with pancreatic cancer indicating the crucial association of ppar betadelta withtumor angiogenesis progression and cancer invasivenessppar betadelta may indirectly facilitate tumor angiogenesis and progression through its function on the tumormicroenvironment tme where tumor angiogenesis is fostered moreover a tumor also releases some extracellular signals to closely communicate and constantly collaborate withtme to facilitate tumor angiogenesis in order to furtherenable tumor growth and progression for instance it wasshown that colon cancer cells with ppar betadelta knockoutfailed to stimulate ec vascularization in response to hypoxicstress whereas wildtype cells exposed to hypoxia were ableto induce angiogenesis [ ] suggesting that ppar betadelta is required for the promotion of angiogenesis in hypoxic stressmediated tme moreover in the tme tumorltrating myeloid cells are considered as the most important cells for fostering tumor angiogenesis among the multiple diï¬erent kinds of stromal cells besides stimulatingtumor angiogenesis tumor myeloid cells also support tumrowth by suppressing tumor immunity and promotingtumor metastasis to distinct sites interestingly it hasbeen demonstrated that ppar betadelta activation intumorltrating myeloid cells stimulates cancer cell invasion and facilitates tumor angiogenesis via an interleukin il10 dependent manner moreover impairedtumor growth and angiogenesis were observed in pparbetadelta ko bmt mice due to ppar betadelta deficiencyin tumor myeloid cells suggesting that ppar betadeltaplays a key role in tumor angiogenesis and progression intumor myeloid cells of tmefurthermore the endoplasmic reticulum er an essential anelle involved in many cellular functions is implicated in tme in cancer stressors like hypoxia nutrientdeprivation and acidosis disrupt er function and lead toaccumulation of unfolded proteins in er a condition knownas er stress cells adapt to er stress by activating an integrated signal transduction pathway called the unfolded protein response upr upr represents a survival response bythe cells to restore er homeostasis and has both survivaland cell death eï¬ects the mechanisms that determine cellfate during er stress are not well understood for instanceshort exposure to er stress initially increases akt signalingbut longterm er stress suppresses akt signaling ppar betadelta activation has been shown to reduce endoplasmic reticulum er stressassociated ‚ammation inskeletal muscle through an ampkdependent mechanism and to reduce ‚ammation in response to chronic erstress in cardiac cells furthermore it has been nicelyshown that ppar betadelta can repress rasoncogeneinduced er stress to promote senescence in tumors thisis mediated through the decrease of pakt activity promoting cellular senescence through upregulation of p53 and p27expression it would be interesting to investigate thedirect eï¬ects of ppar betadelta on senescence of tumorendothelial cells in an in vivo setting we recently showedthat senescent endothelial cells are indispensable for ahealthy lifespan and that removal of senescent endotheliumdisrupts vascular function leading to diminished vessel densities and fibrotic lesions if ppar betadelta mediatessenescence of tumor endothelium thereby protecting vesselintegrity this might explain the enhanced tumor growthand vascularization upon ppar betadelta activationobserved by us and others [ ]most recently zuo demonstrated that pparbetadelta in cancer cells regulates tumor angiogenesisin vivo and in vitro by promoting the secretion of proangiogenic factors including vegf and interleukin il8 most importantly in our recent works it has beenshown that conditionalinducible vascular endotheliumspecific ppar betadelta overexpression in vivo leads to 0cppar researchenhanced tumor angiogenesis tumor growth and metastasis formationfurther indicating a vascular ecspecificppar betadelta action mechanism in tumor progressionindependent of some controversial observations of pparbetadelta in specific tumor or cancer cell types wagner also firstly reported the mouse model in whichrapid induction of cardiac angiogenesis and cardiac hypertrophy were observed [ ] crosstalk between ppar betadelta and signalmolecules ppar betadelta activation or overexpressionmay upregulate the expression of its various downstream signal molecules involved in tumor angiogenesis includingproangiogenic factors such as vegf pdgf and fgfproinvasive matrixdegrading enzymes such as mmp9pro‚ammatory mediators such as cox2 and cytokinesand chemokines such as il1 and cxcl8 even some ofwhich have been further identified as ppar betadelta directtarget genes besides a leading role of ppar betadelta amongthe signal molecules ppar betadelta may function in tmelinked to diverse kinds of cells through direct or indirectmodulation of its downstream molecules interplay between ppar betadelta and ‚ammatoryangiogenesis ‚ammatory angiogenesis is a crucial processin tumor progression for instance the pro‚ammatorymediator cyclooxygenase2 cox2 is considered as a keyregulator of angiogenesis and tumor growth through multiple downstream proangiogenic mechanisms such as production of vegf and induction of mmps moreover selectiveinhibition of cox2 has also been shown to suppress angiogenesis in vivo and in vitro it is well known that vegfaplays a critical role in both angiogenesis and vasculogenesis and it leads the directional migration of tip cells andstalk cell proliferation in microtubule branches [ ] ithas also been demonstrated that mmp9 triggers the œangiogenic switch during carcinogenesis and enhances the availability of vegf to its receptors furthermore it hasbeen reported that ‚ammatory cell mmp9 initiates theonset of tumor neovascularization during which there existsfunctionalincludingmmp9 leptin is shown to mediate angiogenesisin vivo and in vitro through induction of ec proliferationand expression of mmp2 and mmp9 and to furtherpromote ec diï¬erentiation and directional migrationthrough enhancement of cox2 activity leptin couldalso induce angiogenesis via transactivation of vegfr inecs additionally besides inducing angiogenesisppar betadelta also functions in chronic ‚ammationfacilitating tumorigenesis through induction of cox2 andits product prostaglandin e2 pge2 in vivo [ ]interestingly cox2 vegf mmp9 and leptin have beenidentified as ppar betadelta target genes via a direct transcriptional activation mechanism in hepatocellular carcinoma cells colorectal cancer cells [ ] epcs[ ] and liposarcoma cells respectivelylinks between vegf and mmpsin tme tumorltrating ‚ammatory cells also helpto induce and sustain tumor angiogenesis and further tofacilitate tissue invasion and tumor metastatic spread byreleasing some signal molecules such as proinvasive mmp9and ‚ammatory chemokines [“] chemotaxis is alsoa crucial process for inducing angiogenesis in tumors eitherdirectly by attracting ecs towards tumor cells to form newvessels or indirectly by mediating immune ‚ammatorycells to ltrate eventually promoting tumor angiogenesis chemotaxis of tumor cells and stromal cells in tmeis also required for tumor dissemination during tumor progression and metastasis [ ]cxc chemokines such as cxcl8 encoding il8 andcxcl5 are also involved in cox2associated angiogenesisto contribute to nonsmallcelllung cancer progression[ ] it is further shown that il8 directly regulatesangiogenesis via recruitment of neutrophils whichfurther drives vegf activation moreoveril8responding neutrophils are considered as the major sourceof angiogenesisinducing mmp9 [ ] chemokine cc motif ligand ccl2 in addition to the promotionof angiogenesis [ ] also enhances tumor metastasis furthermore myeloid monocytic cellssuch asmyeloidderivedtumormdscsassociated macrophages tams and dendritic cells arerecruited to the tumor site mainly by ccl2 and producemany proangiogenic factorssuch as vegf cxcl8plateletderived growth factor pdgf and transforminggrowth factor beta tgf beta [“] in fact bothtgf beta and hypoxia are potentinducers of vegfexpression in tumor cells and collaborate with tme toprovide the foundation of tumor angiogenesis and cancercell invasion importantly il8 has been reported asa key target gene of ppar betadelta to promote angiogenesis in vivo and in vitro and ccl2 expression isalso significantly upregulated upon vascular ppar betadelta overexpression in vivo suppressorcellscox2 also mediates il1 betainduced angiogenesisin vitro and in vivo [ ] il1 beta supports neovascularization through the regulation of the expression of vegfand its receptor vegfr2 flk1kdr on ecs il1 actsas an upstream pro‚ammatory mediator that initiates anddisseminates the ‚ammatory state by inducing a localinteractive network and increasing adhesion moleculeexpression on ecs and leukocytes which facilitates tumorassociated angiogenesis in tme ‚ammatory il1beta recruits myeloid cells from bone marrow and activatesthem to produce proangiogenic factors such as vegf vegffurther activates ecs and myeloid cells promoting tumorinvasiveness and fostering tumor angiogenesis in addition il6 also stimulates angiogenesis and vasculogenesis[ ] however gopinathan observed an il6induced newly forming vascular structure with defectivepericyte pc coverage ex vivo thus facilitating cancercell ltration and tumor metastasis through vascular leakage interestingly il1 and il6 expression levels are significantly upregulated in the ppar betadelta overexpressionmouse model reported recently in summary ppar betadelta seems to act as a key leaderin ‚ammatory mediatordriven tumor angiogenesis linkedto tme in which many pro‚ammatory mediators chemokines and proangiogenic factors closely communicate with 0cppar researcheach other and also associate with tumorltrating myeloid cells such as neutrophils tams and mdscs other key ppar betadeltamediated proangiogenicfactors it has been demonstrated that wilms™ tumor suppressor wt1 is a major regulator of tumor neovascularization andtumor progression e26 avian leukemia oncogene ets1 also plays a key role in regulating vascular development and haemopoiesis particularly in angiogenesis in addition ets1 promotes cancer cell invasion throughupregulation of mmps consistent with this silencingof ets1 in highly invasive breast cancer cells also reducesthe expression of mmp9 and mmp1 ets1 also acts as a key regulator of mmps such asmmp1 mmp3 and mmp9 in human cancerassociatedfibroblasts cafs [ ] cafs support tumor growthby secreting growth factors such as vegf fgf pdgf andchemokines to stimulate angiogenesis and thereby promotecancer cell invasion and metastasis formation [ ]cafs as metastatic tumor stroma are a crucial componentin tumor progression through the remodeling of the ecmstructure thus helping a tumor to acquire an aggressive phenotype [ ] ppar betadelta in cafs also exhibits aprotumorigenic eï¬ect it was reported that ablation of pparbetadelta in cafs attenuated tumor growth by altering theredox balance in tme suggesting that ppar betadeltain cafs is also an important player in tumor developmentets1 induces the expression of vegf vegfr1 andvegfr2 in ecs [“] in turn vegf is also a majorinducer of ets1 in ecs through the activation of either thepi3kakt pathway or the mekerk12 signal cascade[ ] wt1 is also reported to regulate tumor angiogenesis via direct transactivation of ets1 sryrelated hmgbox sox18 has also beenreported previously to induce angiogenesis during tissuerepair and wound healing and cancer progression and most recently it was further shown that specificecderived endovascular progenitors initiated a vasculogenic process and diï¬erentiated into more mature endothelial phenotypes within the core of the growing tumorsthrough reactivation of sox18 interestingly theseimportant proangiogenic molecules including wt1 ets1and sox18 are also significantly upregulated in the vascularppar betadelta overexpression model in vivo andwt1 is also identified as a target gene of ppar betadeltain melanoma cells ppar betadelta may facilitate cancer progression atdiverse cellular levels in tme ppar betadelta activationis shown to induce colonic cancer stem cell csc expansionand to promote the liver metastasis of colorectal cancerin vivo via direct transactivation of the nanog gene nanog as a key transcriptional factor governs the selfrenewal and pluripotency of stem cells and cancer cellsexpressing nanog also often exhibit stem cell properties protooncogene ckitcd117 is known as the maststem cell factor receptor and receptor tyrosine kinase andits activation in cscs may regulate the stemness to controltumor progression and drug resistance to tyrosine kinaseinhibitors moreover ckit has been identified as a potentialmarker of the cancer stemlike cells in addition ckitnot only functions on ecs [ ] but also belongs to thetumor angiogenesispromoting molecule [“] studiesalso suggested that activation of ckit enhances the expression of vegf that can be suppressed by imatinib an inhibitor of ckit in gastrointestinal stromal tumor cells whichthereby has an impact on tumor angiogenesis [ ] ckit is also involved in pathological ocular neovascularization and is regulated transcriptionally by wt1 and ppar betadelta pdgfb and its receptor pdgfr beta also known asangiogenic factors are suggested to enhance angiogenesisand vasculogenesis via their function in ecs [“] andepcs and to regulate vascular permeability and vesselmaturation through recruitment of pericytes pcs and smooth muscle cells smcs in newly formingvessels moreover pdgfb and pdgfr beta also interactwith other proangiogenic factors such as fgf2 [ ]vegfa and its receptor vegfr2 furthermorepdgfb and pdgfr beta may also aï¬ect cancer growthand progression by directly acting on tme besides thecrosstalk with cafs [“] pdgfr beta in stromalfibroblasts may mediate pdgfbinduced tam recruitment thus implicating a role of pdgfr beta in tumorstroma to facilitate tumor progression most recently it wasfurther shown that specific targeting of pdgfr beta kinaseactivity in tme inhibited cancer growth and vascularizationin cancers with high pdgfb expression such as llc therefore this indicates the diverse role of pdgfb andpdgfr beta in facilitating tumor angiogenesis and progression at diï¬erent cellular levels in tme pdgfr beta is demonstrated as a target of telomeric repeat binding factor trf2 that is further activated transcriptionally by wt1 pdgfb and pdgfr beta have further been identifiedas critical targets of ppar betadelta via a direct transactivation mechanism in vivo in conclusion a variety of key signal molecules involvedin tumor angiogenesis and tumor progression and metastasishave either been identified as ppar betadelta direct targetsor largely upregulated in the vascular ppar betadelta overexpression model in vivo reported recently thus pparbetadelta activation seems to give rise to a highly angiogenicphenotype and even plays a œhallmark role in promotingtumor angiogenesis and progression interestingly it appearsthat there could also exist a widely interactive networkbetween the downstream protumorangiogenic moleculesas described above therefore the crosstalk network is established between ppar betadelta and the various signal molecules and also between those molecules figure 1amoreover in addition to cancer cells ppar betadeltamay also produce pleiotropic eï¬ects in tme by modulatingdownstream key molecules to act on ecs epcs pcs smcscscs cafs and tumorltrating ‚ammatory cells indirectly facilitating tumor angiogenesis and further promotingcancer development figure 1b other ppar betadelta target genes ppar betadeltaregulates the transcription oftarget genes via a direct 0cppar researchil1 betacox2leptinppar betadeltavegfvegfrpdgfr betatrf2wt1ets1mmp9pdgfbckitcxcl8il8il10ccl2cxcl8il8pdgfr betaappar betadeltaets1mmp9pdgfr betananogckittumorassociatedmacrophage tamdendritic cellneutrophilmyeloidderivedsuppressor cell mdscvegfmmp9pdgfbckitsox18pdgfr betaendothelial cell ece Answer:

Colon_Cancer

we report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus cryosurgical excisionwas performed tissue pathology confirmed the diagnosis of metastatic embryonal carcinoma the patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin bep chemotherapy regimenkeywordscryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourcorrespondencewenchien cheng division of pulmonary and critical care medicine department of internal medicinechina medical university hospital no yuderoad north dis taichung city taiwanemail wcchengdrgmailcomreceived july revised july accepted july associate editor james horespirology case reports e00644 101002rcr2644introductionlung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases ebms from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [“] primary lung cancer is the most common cause of endobronchialtumours extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [“] although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma ebms from embryonal carcinomas are extremely rare in this report we present acase of ebm from a primary embryonal carcinomacase reporta 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days he reported no fever chills coldsweats weight loss or decreased appetite a chest radiograph at admission revealed complete collapse of the leftlung fig 1a computed tomography ct was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumbronchoscopytumourobstructing thefig 1b theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 ttf1negative sallike protein sall4positive and cluster of differentiation cd30positive these findings were consistentwith a final diagnosis of metastatic embryonal carcinomafig we checked the levels of tumour markers in thepatient including those of alphafetoprotein afp betahuman chorionic gonadotropin bhcg and lactate dehydrogenase ldh each tumour marker was found to be the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian pacific society of respirologythis is an open access under the terms of the creative commons attributionnoncommercialnoderivs license which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modifications or adaptations are made vol iss e00644page 0cebm from embryonal carcinomack teng figure chestradiograph and bronchoscopic view of the endobronchial metastasesebm a complete collapse of the left lungon chest radiograph b bronchoscopic viewof the endobronchial tumour within the leftmain bronchusfigure tumour pathology of metastatic embryonal carcinoma a embryonal carcinoma with a complex glandular growth pattern the characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magnification — b immunohistochemical staining with antithyroid transcription factor1 ttf1 highlighting cells in the alveolar space original magnification — c immunohistochemical staining with antisallikeprotein sall4 revealed diffuse nuclear staining original magnification — d immunohistochemical staining with anticluster of differentiation cd30 highlighting diffuse membranous staining original magnification —presentin high levels afp ngml bhcg miuml and ldh iul the patientunderwent a right orchiectomy followed by a bepbleomycin etoposide cisplatin chemotherapy regimendiscussionwe report here the case of a young man with an ebmfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumourslikewise manykindslung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverebms from extrapulmonary malignancies are rare [“] primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith ebm primarily breast colon and renal carcinomas[“] ebms from testicular seminomas are also extremelyrare the majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian pacific society of respirology 0cck teng ebm from embryonal carcinomatable reports of previous cases of ebmslocationdiagnostic methodpathology¶zt¼rk moreirameyer case case the orifice of right upper loberight main bronchusleft main bronchus main carina andright main bronchus fibreoptic bronchoscopy mixed gctfibreoptic bronchoscopy mixed gctvideobronchoscopyembryonal carcinoma¶zsu the orifice of the right upper lobefibreoptic bronchoscopytesticular seminomaturan varkey our caseand right intermediary loberight intermediate bronchusleft main bronchusleft main bronchusebm endobronchial metastases gct germ cell tumourembryonic carcinomas or seminomas “there are only a few published reports of primary testicularembryonic carcinomas resulting in ebms [“]the mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently howeversome patients are asymptomatic in our patient symptoms on presentation included haemoptysis and shortnessof breathresults from chest radiography in patients with ebmcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyhowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difficult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these findings alone toconfirm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue the flexible bronchoscopy fibreoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy the former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure the patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the final pathology reportconfirmed the diagnosis of metastatic embryonal carcinomawe had evaluated the presence of afp bhcg andldh tumour markers elevated afp levels can be secretedby germ cell tumours gcts including embryonal carcinoma yolk sac tumour or teratoma in gcts detectablerigid bronchoscopybronchoscopyfibreoptic bronchoscopyand cryosurgerysomatictype gctembryonal carcinomaembryonal carcinomabhcg elevation is observed in both seminomas and nonseminomas the serum level of ldh was directly correlated with tumour burden in nonseminomatous gctswhich is also useful for the surveillance of patients withadvanced seminoma the tumour markers in ourpatient showed elevated levels of afp bhcg and ldhthis was compatible with the diagnosis of embryonal carcinoma moreirameyer also evaluated the patienttumour markers and found elevated levels of afp ngml and bhcg miuml the elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma ¶zsu onlyevaluated the patient™s bhcg level which was found to beelevated miuml and the final diagnosis wasmetastatic testicular seminoma on comparison with previous case reports table ours was the first case in which the tissue was obtainedusing cryosurgery other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation apc to control bleedingcryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree sampleshaemorrhage was observed to be similar during both procedures further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesin conclusion ebms from primary gcts notably thoseassociated with total or partial collapse are extremely rarewe have presented this case to emphasize the importanceof distinguishing ebm from primary lung carcinoma andto report the first case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian pacific society of respirology 0cebm from embryonal carcinomadisclosure statementappropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisreferences ¶zt¼rk a aktas¸ z and yılmaz a endobronchialmetastasis of mixed germ cell tumors two cases tuberktoraks “ lee sh jung jy kim dh endobronchialmetastases from extrathoracic malignancy yonsei med j“ ikemura k lin dm martyn cp endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing lung “ moreirameyer a bautistaherrera d hern¡ndezembryonal endobronchialgonz¡lez mck teng carcinoma“j bronchologyinterv pulmonol ¶zsu s erol mm oztuna f endobronchial metastasis from testicular seminoma med princ pract “tumoraltesticular germ cell turan d akif ¶zg¼l m kirkil gendobronchial metastasis ofeurasian j pulmonol “et varkey b and heckman mg diagnosis of a case ofembryonal carcinoma by bronchial biopsy chest “ paradis tj dixon j and tieu bh the role of bronchoscopy in the diagnosis of airway disease j thorac dis“ aktas z gunay e hoca nt endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisann thorac med “ barlow lj badalato gm and mckiernan jm serumtumor markers in the evaluation of male germ cell tumorsnat rev urol “ the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian pacific society of respirology 0c'

cancer7565

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: we report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus cryosurgical excisionwas performed tissue pathology confirmed the diagnosis of metastatic embryonal carcinoma the patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin bep chemotherapy regimenkeywordscryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourcorrespondencewenchien cheng division of pulmonary and critical care medicine department of internal medicinechina medical university hospital no yuderoad north dis taichung city taiwanemail wcchengdrgmailcomreceived july revised july accepted july associate editor james horespirology case reports e00644 101002rcr2644introductionlung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases ebms from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [“] primary lung cancer is the most common cause of endobronchialtumours extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [“] although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma ebms from embryonal carcinomas are extremely rare in this report we present acase of ebm from a primary embryonal carcinomacase reporta 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days he reported no fever chills coldsweats weight loss or decreased appetite a chest radiograph at admission revealed complete collapse of the leftlung fig 1a computed tomography ct was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumbronchoscopytumourobstructing thefig 1b theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 ttf1negative sallike protein sall4positive and cluster of differentiation cd30positive these findings were consistentwith a final diagnosis of metastatic embryonal carcinomafig we checked the levels of tumour markers in thepatient including those of alphafetoprotein afp betahuman chorionic gonadotropin bhcg and lactate dehydrogenase ldh each tumour marker was found to be the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian pacific society of respirologythis is an open access under the terms of the creative commons attributionnoncommercialnoderivs license which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modifications or adaptations are made vol iss e00644page 0cebm from embryonal carcinomack teng figure chestradiograph and bronchoscopic view of the endobronchial metastasesebm a complete collapse of the left lungon chest radiograph b bronchoscopic viewof the endobronchial tumour within the leftmain bronchusfigure tumour pathology of metastatic embryonal carcinoma a embryonal carcinoma with a complex glandular growth pattern the characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magnification — b immunohistochemical staining with antithyroid transcription factor1 ttf1 highlighting cells in the alveolar space original magnification — c immunohistochemical staining with antisallikeprotein sall4 revealed diffuse nuclear staining original magnification — d immunohistochemical staining with anticluster of differentiation cd30 highlighting diffuse membranous staining original magnification —presentin high levels afp ngml bhcg miuml and ldh iul the patientunderwent a right orchiectomy followed by a bepbleomycin etoposide cisplatin chemotherapy regimendiscussionwe report here the case of a young man with an ebmfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumourslikewise manykindslung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverebms from extrapulmonary malignancies are rare [“] primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith ebm primarily breast colon and renal carcinomas[“] ebms from testicular seminomas are also extremelyrare the majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian pacific society of respirology 0cck teng ebm from embryonal carcinomatable reports of previous cases of ebmslocationdiagnostic methodpathology¶zt¼rk moreirameyer case case the orifice of right upper loberight main bronchusleft main bronchus main carina andright main bronchus fibreoptic bronchoscopy mixed gctfibreoptic bronchoscopy mixed gctvideobronchoscopyembryonal carcinoma¶zsu the orifice of the right upper lobefibreoptic bronchoscopytesticular seminomaturan varkey our caseand right intermediary loberight intermediate bronchusleft main bronchusleft main bronchusebm endobronchial metastases gct germ cell tumourembryonic carcinomas or seminomas “there are only a few published reports of primary testicularembryonic carcinomas resulting in ebms [“]the mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently howeversome patients are asymptomatic in our patient symptoms on presentation included haemoptysis and shortnessof breathresults from chest radiography in patients with ebmcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyhowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difficult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these findings alone toconfirm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue the flexible bronchoscopy fibreoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy the former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure the patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the final pathology reportconfirmed the diagnosis of metastatic embryonal carcinomawe had evaluated the presence of afp bhcg andldh tumour markers elevated afp levels can be secretedby germ cell tumours gcts including embryonal carcinoma yolk sac tumour or teratoma in gcts detectablerigid bronchoscopybronchoscopyfibreoptic bronchoscopyand cryosurgerysomatictype gctembryonal carcinomaembryonal carcinomabhcg elevation is observed in both seminomas and nonseminomas the serum level of ldh was directly correlated with tumour burden in nonseminomatous gctswhich is also useful for the surveillance of patients withadvanced seminoma the tumour markers in ourpatient showed elevated levels of afp bhcg and ldhthis was compatible with the diagnosis of embryonal carcinoma moreirameyer also evaluated the patienttumour markers and found elevated levels of afp ngml and bhcg miuml the elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma ¶zsu onlyevaluated the patient™s bhcg level which was found to beelevated miuml and the final diagnosis wasmetastatic testicular seminoma on comparison with previous case reports table ours was the first case in which the tissue was obtainedusing cryosurgery other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation apc to control bleedingcryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree sampleshaemorrhage was observed to be similar during both procedures further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesin conclusion ebms from primary gcts notably thoseassociated with total or partial collapse are extremely rarewe have presented this case to emphasize the importanceof distinguishing ebm from primary lung carcinoma andto report the first case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian pacific society of respirology 0cebm from embryonal carcinomadisclosure statementappropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisreferences ¶zt¼rk a aktas¸ z and yılmaz a endobronchialmetastasis of mixed germ cell tumors two cases tuberktoraks “ lee sh jung jy kim dh endobronchialmetastases from extrathoracic malignancy yonsei med j“ ikemura k lin dm martyn cp endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing lung “ moreirameyer a bautistaherrera d hern¡ndezembryonal endobronchialgonz¡lez mck teng carcinoma“j bronchologyinterv pulmonol ¶zsu s erol mm oztuna f endobronchial metastasis from testicular seminoma med princ pract “tumoraltesticular germ cell turan d akif ¶zg¼l m kirkil gendobronchial metastasis ofeurasian j pulmonol “et varkey b and heckman mg diagnosis of a case ofembryonal carcinoma by bronchial biopsy chest “ paradis tj dixon j and tieu bh the role of bronchoscopy in the diagnosis of airway disease j thorac dis“ aktas z gunay e hoca nt endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisann thorac med “ barlow lj badalato gm and mckiernan jm serumtumor markers in the evaluation of male germ cell tumorsnat rev urol “ the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian pacific society of respirology 0c' Answer:

Colon_Cancer

among synchronous colorectal cancers scrcs reported previously the incidence of quadruple advanced scrcs is very rarewe present the case who underwent laparoscopic twosegment resection of the colon requiring two anastomoses that wasperformed for quadruple advanced cancers and four tumors were curatively removed there were no signs of recurrence at months after surgery laparoscopic surgery provided less invasiveness even for quadruple advanced scrcs in terms of earlyrecovery with an acceptable longterm outcomeintroductionsynchronous colorectal cancers scrcs are characterized bythe simultaneous occurrence of multiple primary tumors inthe same patient synchronous malignancies most commonlyoccur in the colon among other ans [“] the occurrence ofadvanced scrcs is rare and may be identified at any locationwithin the large intestine the prevalence of scrcs is reportedto range from to among these however the incidence of quadruple advanced scrcs is extremely rare accounting for of all scrcs surgical resection is considered thestandard treatment for scrcs as a surgical approach laparoscopic surgery has significant advantages in terms of shortterm outcomes including early recovery and no disadvantageouslongterm outcomes according to recent reports laparoscopicsurgery has been used in scrcs but these reports noted thatcontroversy remains concerning operative procedures for multiple segmental resections and for total or subtotal colectomywe report the case who presented with quadruple synchronousadvanced cancers arising from the colon which were successfully treated with laparoscopic twosegment colectomyreceived may accepted june published by oxford university press and jscr publishing ltd all rights reserved the authors this is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0cj ma figure colonoscopy images showing four tumors a one cauliflowerlike tumor with lumen stenosis is located in the ascending colon b another cauliflowerliketumor is located in the descending colon the third c and fourth d tumors are located in the sigmoid coloncase presentationa 70yearold man who was positive for a blood stool testvisited our hospital colonoscopy computed tomography ctand barium enema indicated quadruple concurrent locallyadvanced cancers the firsttumor with observed lumenstenosis was located in the ascending colon the secondtumor was located in the descending colon and the third andfourth tumors were located in the sigmoid colon fig ctrevealed marked intestinal wall thickness in the ascendingdescending and sigmoid colon fig preoperative precisesimulation using 3d angiography was performed to determineadequate lymph node dissection along the arteries feedingthe tumors and appropriate resection to avoid anastomoticleakageswe planned the appropriate placement of trocars as shownin figure because we wanted to create a single minilaparotomy for specimen retrieval and extracorporeal reconstruction after lymph node dissection and mobilization of thecolonduring the operation laparoscopic exploration confirmed thepresence of known four tumors with no invasion of the serosasubsequently a right hemicolectomy and sigmoid colectomywere performed laparoscopically the right half of the colon wasseparated and a sidetoside anastomosis between the jejunumand transverse colon was performed followed by the sigmoidcolon and a colorectal anastomosis between the descendingcolon and rectum was performed the resected tissue specimensrevealed four tumors fig histological examination showedthat the first tumor in the ascending colon the second tumor inthe descending colon and the third tumor in the sigmoid colonhad invaded up to the subserosa whereas the fourth tumor inthe sigmoid colon had invaded up to the muscularis propriafig according to the american joint committee on cancertumornodemetastasis staging system the pstage was iiia t3n1m0the patient was discharged days after surgery for adjuvantchemotherapy the patient chose to take an oral fluoropyrimidine agent for months fortunately there have been no signsof metastasis or recurrence after the operation at months offollowupdiscussionwe reported a rare case of quadruple scrcs all four tumorswere removed curatively by laparoscopic surgery with d3 lymphnode dissection we planned a strategy for quadruple scrcsbased on preserving the remnant large intestine and sufficientd3 lymph node dissection through a laparoscopic approachwe believe that laparoscopic surgery can be a safe even forquadruple scrcs this is the first case report of laparoscopicsurgery with d3 lymph node dissection for quadruple advancedscrcsthe incidence of malignant scrc with four or five synchronous lesions is extremely rare with a rate of beingreported this is a quite rare case of quadruple synchronous 0cquadruple advanced synchronous colorectal cancersfigure placement of trocars and miniincision in the present casethan the index cancer however all of the scrcs in our patienthad the same histological grade and t staging pstage iiiawith the tumor locations being in the ascending descending andsigmoid colonsurgical management of scrcs needs to be tailored tothe individual based on tumor location invasion status andthe patient™s health condition some studies have suggestedtotal or subtotal colectomy to remove any potential existingsynchronous tumors or polyps that have not been detected however other studies recommend a more conservativesurgical approach it is thought that the removal of the entirecolon will prevent the development of metachronous tumorsand a previous study indicated that subtotal colectomy mayincrease defecation frequency as the normal colon cannot bepreserved we successfully performed laparoscopic surgerycombining twosegment resection of a right hemicolectomyand sigmoid colectomy with no intra or postoperative adverseevents in our patient we tried preserving as much colonas possible considering the patient™s quality oflife aftersurgery in addition to performing sufficient d3 dissection ofcourse the meaning of preserving colon in terms of patientpostoperative quality of life needs to be more clearly assessed infuturewe encountered a rare case of advanced quadruple scrcsfor which we achieved a curative resection that required twoanastomoses through a laparoscopic approach we suggest thatlaparoscopic surgery that requires multiple anastomoses foradvanced scrcs can be a safe procedure even if the number ofcolorectal cancers is multiplefigure abdominal ct scan revealing a tumor of the ascending colon aarrowhead a tumor in the descending colon b arrowhead and two tumorsin the sigmoid colon are also visible c d arrowheadadvanced cancer arising from the ascending descending andthe sigmoid colon it was reported that scrcs often occur inthe same or adjacent segment of the large intestine and thatother smaller colorectal cancers in the patients with scrcs wereusually smaller and of lower pathological grade and t stagingconflict of interest statementnone declared 0cj ma figure the surgical specimens of the ascending colon cancer a descending colon cancer b and the two sigmoid colon cancers c and dfigure histopathological examination of the tissue specimens revealed four tumors showing cancerous cells arranged in a tubular pattern 0cfundingnonereferencesjiang x xu c tang d wang d laparoscopic subtotal colectomy for synchronous triple colorectal cancer a case reportoncol lett “ yang j peng jy chen w synchronous colorectal cancersa review of clinical features diagnosis treatment and prognosis dig surg “ aky l synchronous colorectal cancer clinical pathological and molecular implications world j gastroenterol“ fukatsu h kato j nasu ji kawamoto h okada h yamamotoh clinical characteristics of synchronous colorectalcancer are different according to tumour location dig liverdis “ holubar sd wolff bg poola vp soop m multiple synchronous colonic anastomoses are they safe colorectal dis“quadruple advanced synchronous colorectal cancers li z wang d wei y liu p xu j clinical outcomes oflaparoscopicassisted synchronous bowel anastomoses forsynchronous colorectal cancer initial clinical experienceoncotarget “ nosho k kure sirahara n shima k baba yspiegleman d a prospective cohort study showsunique epigenetic genetic and prognostic features ofsynchronous colorectal cancers gastroenterology “20e1“ lam ak carmichael r gertraud buettner p gopalan dho yh siu s clinicopathological significance of synchronous carcinoma in colorectal cancer am j surg “ easson am cotterchio m crosby ja sutherland h dale daronson m a populationbased study of the extent ofsurgical resection of potentially curable colon cancer annsurg oncol “ tsantilas d ntinas apetrasp zambas n aimogrambi s frangandreas g metachronouscolorectals202“adenocarcinomastech coloproctol 0c'

cancer7566

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: among synchronous colorectal cancers scrcs reported previously the incidence of quadruple advanced scrcs is very rarewe present the case who underwent laparoscopic twosegment resection of the colon requiring two anastomoses that wasperformed for quadruple advanced cancers and four tumors were curatively removed there were no signs of recurrence at months after surgery laparoscopic surgery provided less invasiveness even for quadruple advanced scrcs in terms of earlyrecovery with an acceptable longterm outcomeintroductionsynchronous colorectal cancers scrcs are characterized bythe simultaneous occurrence of multiple primary tumors inthe same patient synchronous malignancies most commonlyoccur in the colon among other ans [“] the occurrence ofadvanced scrcs is rare and may be identified at any locationwithin the large intestine the prevalence of scrcs is reportedto range from to among these however the incidence of quadruple advanced scrcs is extremely rare accounting for of all scrcs surgical resection is considered thestandard treatment for scrcs as a surgical approach laparoscopic surgery has significant advantages in terms of shortterm outcomes including early recovery and no disadvantageouslongterm outcomes according to recent reports laparoscopicsurgery has been used in scrcs but these reports noted thatcontroversy remains concerning operative procedures for multiple segmental resections and for total or subtotal colectomywe report the case who presented with quadruple synchronousadvanced cancers arising from the colon which were successfully treated with laparoscopic twosegment colectomyreceived may accepted june published by oxford university press and jscr publishing ltd all rights reserved the authors this is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0cj ma figure colonoscopy images showing four tumors a one cauliflowerlike tumor with lumen stenosis is located in the ascending colon b another cauliflowerliketumor is located in the descending colon the third c and fourth d tumors are located in the sigmoid coloncase presentationa 70yearold man who was positive for a blood stool testvisited our hospital colonoscopy computed tomography ctand barium enema indicated quadruple concurrent locallyadvanced cancers the firsttumor with observed lumenstenosis was located in the ascending colon the secondtumor was located in the descending colon and the third andfourth tumors were located in the sigmoid colon fig ctrevealed marked intestinal wall thickness in the ascendingdescending and sigmoid colon fig preoperative precisesimulation using 3d angiography was performed to determineadequate lymph node dissection along the arteries feedingthe tumors and appropriate resection to avoid anastomoticleakageswe planned the appropriate placement of trocars as shownin figure because we wanted to create a single minilaparotomy for specimen retrieval and extracorporeal reconstruction after lymph node dissection and mobilization of thecolonduring the operation laparoscopic exploration confirmed thepresence of known four tumors with no invasion of the serosasubsequently a right hemicolectomy and sigmoid colectomywere performed laparoscopically the right half of the colon wasseparated and a sidetoside anastomosis between the jejunumand transverse colon was performed followed by the sigmoidcolon and a colorectal anastomosis between the descendingcolon and rectum was performed the resected tissue specimensrevealed four tumors fig histological examination showedthat the first tumor in the ascending colon the second tumor inthe descending colon and the third tumor in the sigmoid colonhad invaded up to the subserosa whereas the fourth tumor inthe sigmoid colon had invaded up to the muscularis propriafig according to the american joint committee on cancertumornodemetastasis staging system the pstage was iiia t3n1m0the patient was discharged days after surgery for adjuvantchemotherapy the patient chose to take an oral fluoropyrimidine agent for months fortunately there have been no signsof metastasis or recurrence after the operation at months offollowupdiscussionwe reported a rare case of quadruple scrcs all four tumorswere removed curatively by laparoscopic surgery with d3 lymphnode dissection we planned a strategy for quadruple scrcsbased on preserving the remnant large intestine and sufficientd3 lymph node dissection through a laparoscopic approachwe believe that laparoscopic surgery can be a safe even forquadruple scrcs this is the first case report of laparoscopicsurgery with d3 lymph node dissection for quadruple advancedscrcsthe incidence of malignant scrc with four or five synchronous lesions is extremely rare with a rate of beingreported this is a quite rare case of quadruple synchronous 0cquadruple advanced synchronous colorectal cancersfigure placement of trocars and miniincision in the present casethan the index cancer however all of the scrcs in our patienthad the same histological grade and t staging pstage iiiawith the tumor locations being in the ascending descending andsigmoid colonsurgical management of scrcs needs to be tailored tothe individual based on tumor location invasion status andthe patient™s health condition some studies have suggestedtotal or subtotal colectomy to remove any potential existingsynchronous tumors or polyps that have not been detected however other studies recommend a more conservativesurgical approach it is thought that the removal of the entirecolon will prevent the development of metachronous tumorsand a previous study indicated that subtotal colectomy mayincrease defecation frequency as the normal colon cannot bepreserved we successfully performed laparoscopic surgerycombining twosegment resection of a right hemicolectomyand sigmoid colectomy with no intra or postoperative adverseevents in our patient we tried preserving as much colonas possible considering the patient™s quality oflife aftersurgery in addition to performing sufficient d3 dissection ofcourse the meaning of preserving colon in terms of patientpostoperative quality of life needs to be more clearly assessed infuturewe encountered a rare case of advanced quadruple scrcsfor which we achieved a curative resection that required twoanastomoses through a laparoscopic approach we suggest thatlaparoscopic surgery that requires multiple anastomoses foradvanced scrcs can be a safe procedure even if the number ofcolorectal cancers is multiplefigure abdominal ct scan revealing a tumor of the ascending colon aarrowhead a tumor in the descending colon b arrowhead and two tumorsin the sigmoid colon are also visible c d arrowheadadvanced cancer arising from the ascending descending andthe sigmoid colon it was reported that scrcs often occur inthe same or adjacent segment of the large intestine and thatother smaller colorectal cancers in the patients with scrcs wereusually smaller and of lower pathological grade and t stagingconflict of interest statementnone declared 0cj ma figure the surgical specimens of the ascending colon cancer a descending colon cancer b and the two sigmoid colon cancers c and dfigure histopathological examination of the tissue specimens revealed four tumors showing cancerous cells arranged in a tubular pattern 0cfundingnonereferencesjiang x xu c tang d wang d laparoscopic subtotal colectomy for synchronous triple colorectal cancer a case reportoncol lett “ yang j peng jy chen w synchronous colorectal cancersa review of clinical features diagnosis treatment and prognosis dig surg “ aky l synchronous colorectal cancer clinical pathological and molecular implications world j gastroenterol“ fukatsu h kato j nasu ji kawamoto h okada h yamamotoh clinical characteristics of synchronous colorectalcancer are different according to tumour location dig liverdis “ holubar sd wolff bg poola vp soop m multiple synchronous colonic anastomoses are they safe colorectal dis“quadruple advanced synchronous colorectal cancers li z wang d wei y liu p xu j clinical outcomes oflaparoscopicassisted synchronous bowel anastomoses forsynchronous colorectal cancer initial clinical experienceoncotarget “ nosho k kure sirahara n shima k baba yspiegleman d a prospective cohort study showsunique epigenetic genetic and prognostic features ofsynchronous colorectal cancers gastroenterology “20e1“ lam ak carmichael r gertraud buettner p gopalan dho yh siu s clinicopathological significance of synchronous carcinoma in colorectal cancer am j surg “ easson am cotterchio m crosby ja sutherland h dale daronson m a populationbased study of the extent ofsurgical resection of potentially curable colon cancer annsurg oncol “ tsantilas d ntinas apetrasp zambas n aimogrambi s frangandreas g metachronouscolorectals202“adenocarcinomastech coloproctol 0c' Answer:

Colon_Cancer

the heterogeneity of cancer cells is generally accepted and astem celllike subpopulation that is called œcancer stem cellscscs has been identified in various types of malignanttumors although the lack of consensus on the definitioncscs are widely recognized as a small subpopulation amongcancer cells with the properties of selfrenewal and tumor initiation as cscs play a critical role in the recurrence andmetastasis of cancer targeting the cscs is thought to bea promising approach for curing cancera large number of past studies have tried to identify andcharacterize the cscs as normal tissuespecific stem cellsare considered as the main origin of cancer the cscsare also thought to be inherited at least partially the characterization of normal tissuespecific stem cells thereforemany studies on the identificationpurification of cscs havesimply shared markers of hematopoietic stem cells includingthe most popularly used cell surface markers of cd44 andcd133 [ ] cd44 is a type i transmembrane glycoproteinthat is expressed on hematopoietic fibroblastic and glial cellsand functionally known to mediate cellcell and cellmatrixinteractions previous studies have demonstrated that thecd44 is not only a biomarker but also plays critical roles inthe maintenance of cscs the resistance to various therapiesstresses and the metastasis of cancer cells [“]cd133 is originally identified as protein expressing on thecell surface of hematopoietic stem cells and has subsequently been found to be critical in the maintenance ofœstemness of stem cells in various tissues [“] cd133has also been found in some csc [“] which contributesto therapeutic resistance through the activation of akt bcl2and mapkpi3k signaling pathways [“] although theexpressions of cd44 and cd133 in cancer cells likely associate with the resistances to radiotherapy chemotherapy andvarious stresses the diï¬erent significance between cd44and cd133 has not yet been well understoodin this study we investigated whether the expression ofcd44 and cd133 in human colorectal cancer cells hct8diï¬erently contributed to drug resistance our data indicated 0cstem cells internationalthat the expression of cd133 rather than cd44 closely associated with doxorubicin dxr resistance at least partiallythrough drug excretion and redox regulation materials and methods cell culture human colorectal cancer hct8 cells werecultured in rpmi medium fujifilm wako purechemical japan supplemented with fbs gibco°thermo fisher scientific ma usa at c in a humidifiedatmosphere of air and co2 separation of cd44 and cd133positive cells fromhct8 cells we separated the parent hct8 cells intocd44positive cd44 and cd133positive cd133 cellsby a twostep magnetic cell sorting method as described previously [ ] briefly hct8 cells were collected as a singlecell suspension by trypsinization and then incubated withmagnetic microbeadconjugated antihuman cd44 antibodymiltenyi biotec germany for min after washing cellswere separated into cd44 and cd44 subpopulations byusing the automacs„¢ pro separator miltenyi biotecaccording to the manufacturer™s instruction the purifiedcd44 cells were further expanded and then harvested as asinglecellsuspension to be incubated with magneticmicrobeadconjugated antihuman cd133 antibody miltenyibiotec for min after washing the cd44cd133 andcd44cd133 subpopulations were separated as describedabove this twostep isolation enabled us to obtain a sufficientnumber of cd44 cd44 cd44cd133 and cd44cd133 cells for our experimentsto verify the purity of each subpopulation isolated cellswere stained with pelabelled mouse antihuman cd133clone ac133 miltenyi biotec and fitclabelled mouseantihuman cd44 clone db105 miltenyi biotec according to the supplied protocols flow cytometry analysis wasperformed using a facscalibur becton dickinson asdescribed previously mouse igg1pe miltenyi biotecand mouse igg1fitc miltenyi biotec were used as a negative control cytotoxicity assays cells were seeded in 96well cultureplates at a density of — cells per well and cultured overnight the cells were then treated with various concentrationsof dxr fujifilm wako pure chemical in the absence orpresence of verapamil fujifilm wako pure chemicalcytotoxicity assays were performed using the cell proliferation kit i mtt roche applied science germany asdescribed previously the absorbance was measured at nm using a microplate reader multiskan fc thermofisher scientific analysis on the expression of abc transporters theexpressions of the atpbinding cassette subfamilies of bmember abcb1 or g member abcg2 were analyzedby flow cytometry briefly cells were incubated with mouseprimary antibodies against human abcb1 and abcg2bd biosciences ca usa and then labeled by fitcconjugated antimouse igg bd biosciences according tothe manufacturer™s instruction respective isotype controlswere used as a negative control after washing flow cytometry analysis was performed using a facscalibur analysis of cellular accumulation of dxr the intracellular accumulation of dxr was analyzed by flow cytometrybriefly cells were treated by μm dxr for hr in theabsence or presence of μm verapamil or μm buthionine sulfoximine bso sigmaaldrich mo usa cellswere then collected as a singlecell suspension and washedtwice with icecold phosphatebuï¬ered saline the accumulation of dxr within cells was evaluated by the intracellularfluorescence intensity using a facscalibur the nucleusaccumulation of dxr was analyzed by using cell pelletstreated with triton x100pbs as assay material asdescribed previously expressionlevelsanalysisimmunoblot detection of intracellular ros the intracellular roslevel based on the oxidation of ²²dichlorodihydrofluorescein diacetate h2dcfda molecular probes thermofisher scientific was measured to form the fluorescent compound ²²dichlorofluorescein dcf using a facscaliburofphosphorylatedp38 map kinase phosphop38mapk totalp38mapk and nuclear factor erythroid 2related factor nrf2 in the cells were estimated by immunoblotting brieflycell lysate μg of total protein was separated by sodiumdodecyl sulfate“polyacrylamide gel electrophoresis sdspage gel transferred to pvdf membranes biorad causa and then incubated with primary antibodies cell signaling technology ma usafollowed by appropriatehrplabeled secondary antibodies dako agilent pathologysolutions ca usa blots were developed by enhancedchemiluminescence using an ecl kit ge healthcare lifesciences pa usa semiquantitation was done by measuringthe density of bands using the image quant las minibiomolecular imager ge healthcare life sciences asdescribed previously sirna treatment smallinterfering rna sirnaspecific targeting to cd133 on targetplus sirnaand a scramble sirna on targetplus sirna negativecontrol were obtained from dharmacon horizon discovery cambridge uk cells were seeded in 6well plates — cellswell and incubated for hr transfectionswere performed using dharmafect sirna transfectionreagents dharmacon according to the manufacturer™sinstructions analyses were done at hr after sirnatransfection statistical analysis all of the results are presented as themeans ± sd statistical significance was determined by oneway analysis of variance anova followed by tukey™s testdr spss ii chicago il diï¬erences were considered significant when p results hct8 cells were separated into various subpopulationsbased on their expressions of cd44 and cd133 first we 0cstem cells internationalparent cellshlffl1hdccd44hlfhlfcd44ˆ’fl1hcd44fl1hahlfhlfcd44cd133ˆ’fl1hcd44cd133fl1hparent cd44ˆ’cd44 cd44133ˆ’ cd44133enilesab fo noitarefilorp llecparent cd44ˆ’ cd44 cd44ˆ’cd44bcfigure continued 0cstem cells internationalenilesabstnuo0ccd44cd133ˆ’ ± stnuo0ccd44cd133 ± fl2hfl2hsyad stnuo0c ± fl2hstnuo0cd ± fl2hfigure the separation of hct8 colorectal cancer cells into diï¬erent subpopulations based on the expression of cd44 and cd133 arepresentative dot plots of flow cytometry analysis show the purities of each subpopulation of isolated cells quantitative data in the dotplots are presented as the percentages of positive cells from three independent experiments b representative photos of morphologicalproperties upper and mtt assay on cell growth lower at hr after the initiation of culture data are presented as the mean ± sd fromthree independent experiments c d representative histograms of flow cytometry analysis showed the expressions of cd44 c andcd133 d at baseline and days after cell culture the dotted vertical lines through histograms indicate the diï¬erence in the expressionpeaks between the baseline and at days after culture quantitative data in the histograms are presented as the mean fluorescentintensity from three independent experimentsseparated the hct8 cells into cd44 and cd44 subpopulations and compared their sensitivity to anticancer drugs ofdxr and cisplatin cisdiaminedichloroplatine cddphowever no diï¬erence in the sensitivity to the two drugswas observed between cd44 and cd44 cells data notshown we further tried to purify a small population ofcd133 cells from these cd44 cells cd44 cells almostnegatively expressed with cd133 figure 1a as a resultwe separated hct cells into diï¬erent subpopulationsincluding cd44 cd44 cd44cd133 and cd44cd133 cells the purities of isolated cells in each subpopulation were confirmed to be around by flow cytometryfigure 1aandphenotype changein diï¬erent growthsubpopulations of cells the morphology and proliferationof these cells could not be found obviously diï¬erent amongsubpopulations figure 1b the expression of cd44 in allsubpopulations kept stable within days of reculturingfrom the frozen cells that stocked immediately after isolationinterestingly the expression of cd44 was a tendency todecrease with culture time in cd44 fluorescence intensity ± at baseline vs ± at days p figure 1c and cd44cd133 cells fluorescence intensity ± at baseline vs ± at days p figure 1c but still kept stable in cd44cd133 cells at days after reculturing fluorescence intensity ± at baseline vs ± at days p figure 1cthe expression of cd133 in cd44cd133 cells kept verystable fluorescence intensity ± at baseline vs ± at days p figure 1d and cd44cd133cells did not turn to express cd133 within days of reculturing fluorescence intensity ± at baseline vs ± at days p figure 1d therefore we usedthe cells within days after reculturing from the frozenstocked cells in subsequent experiments dxr resistance of cd44cd133 cells next we evaluated the sensitivity of cells to dxr by mtt assay withthe addition of μm of dxr in medium we foundthat the survival of cd44cd133 cells was significantlyhigher than all other subpopulations of cells after hr of 0cstem cells international ytilibaiv llecŽŽŽŽŽðœ‡mparent cellscd44ˆ’ cellscd44 cellscd44133ˆ’ cellscd44133 cellsdxrbso003hct8doxb24 hfl3h006hct8 cd44ˆ’doxb24 hfl3h009hct8 cd44doxb24 hsllec tnerapstnuocsllec ˆ’dcstnuocsllec dcstnuocdxr001hct8dox24 hfl3hstnuocadxrverapamil002hct8doxv24 hstnuocfl3h004hct8 cd44ˆ’dox24 h005hct8 cd44ˆ’doxv24 hstnuocstnuocfl3hfl3h007hct8 cd44dox24 h006hct8 cd44doxv24 hstnuocstnuocfl3hfl3hsllec ˆ’dcsllec dc006hct8 cd44cd133ˆ’dox24 h006hct8 cd44cd133ˆ’doxv24 hstnuocstnuocstnuocfl3hfl3h00hct8 cd44cd133dox24 h00hct8 cd44cd133doxv24 hstnuocfl3hstnuocstnuocfl3hbfigure continuedfl3h012hct8 cd133ˆ’doxb24 hfl3h015hct8 cd44cd133doxb24 hfl3h 0cstnuocstem cells internationalabcg2abcb1stnuocfl1hparent cellscd44ˆ’cellscd44cellscd44133ˆ’ cellscd44133 cells ± ± ± ± ± parent cellscd44ˆ’cellscd44cellscd44133ˆ’ cellscd44133 cellscfl1h ± ± ± ± ± figure dxr resistance of diï¬erent subpopulations of cells a mtt assay was done to evaluate the cytotoxicity of dxr data are expressedas the percentile of baseline before dxr treatment from three independent experiments ˆ—p vs all other subpopulations brepresentative histograms of flow cytometry analysis show the accumulation of dxr in cells hr after the treatment with μm dxrin the absence or presence of μm verapamil and μm bso the dotted vertical lines through histograms indicated the mean levels ofdxr accumulation in cd44cd133 cells for comparing with other subpopulations of cells the results were reproducible in threeindependent experiments c representative histograms of flow cytometry analysis show the expression of the abcb1 or abcg2 indiï¬erent subpopulations of cells quantitative data in the histograms are presented as the mean fluorescent intensity from threeindependent experimentsculture p vs other groups at diï¬erent dxr concentrations figure2ato understand the relevant mechanism we measured theintracellular accumulation of dxr in cells by flow cytometrythe accumulation of dxr in cd44cd133 cells wasdetected as the lowest among these subpopulations at hrafter the exposure to μm dxr figure 2b we furtherfound that the intracellular accumulation of dxr in cd44cd133 cells was obviously increased by the treatment withverapamil an inhibitor for drug efflux cell membrane transporters of abcb1 and abcg2 figure 2b however theintracellular accumulation of dxr in cd44cd133 cellsdid not change by the treatment with bso a glutathione synthesis inhibitor that indirectly regulates drug efflux throughabcc1 figure 2b we also confirmed that the expressionof abcb1 p vs other groups but not abcg2 wasenhanced in cd44cd133 cells figure 2c suggestingthe probable role of abcb1 on dxr resistance in cd44cd133 cellsto further confirm the causal relationship between theenhanced drug efflux and dxr resistance we evaluatedthe cytotoxicity of dxr in the presence or absence of verapamil unexpectedly verapamil only partially enhanced thecytotoxicity of dxr in either cd44cd133 or cd44cd133 cells figure 3ait is well known that dxr interacts with nuclear dna toinhibit macromolecular biosynthesis therefore we alsoestimated the eï¬ect of verapamil on the nuclear accumulation of dxr the nuclear accumulation of dxr wasobserved obviously less in cd44cd133 than cd44cd133 cells but tended to have comparable levels withverapamil treatment figure 3b cd44cd133 cells showed better stress tolerance thancd44cd133 cells it is well known that the stress responsekinase p38mapk can be activated by various extracellularstresses and plays critical roles in cell survival and apoptosis although the basal level of phosphorylated p38mapkwas detected very similar between cd44cd133 andcd44cd133 cells p figure lower expressionwas observed in cd44cd133 than cd44cd133 cellsafter dxr exposure even under verapamiltreatmentp figure this suggests a better tolerance tostress of cd44cd133 cells independent on the accumulation of dxr 0cstem cells internationalˆ’ limaparev ytilibaiv llec limaparev ytilibaiv llec hoursŽŽŽŽ 𝜇mŽŽŽŽðœ‡mcd44cd133 cellscd44cd133ˆ’ cellsa hoursŽŽŽ ˆ’ limaparevstnuoccd44133ˆ’ cd44133𝜇mfl3hŽŽŽŽðœ‡m limaparevstnuocfl3hbfigure dxr resistance and nuclear dxr accumulation in cd44cd133 and cd44cd133 cells in the absence or presence of drug effluxinhibitor a mtt assay was done to compare the cytotoxicity of dxr in cd44cd133 and cd44cd133 cells with or without theaddition of μm verapamil data were expressed as a percent of baseline before dxr treatment from three independent experiments ˆ—p vs cd44cd133 cells b representative histograms of flow cytometry analysis showed the nuclear accumulation of dxr incells hr after the treatment by μm dxr with or without the addition of μm verapamil the results were reproducible in threeindependent experimentsverapamil ˆ’verapamil phosphop38 mapk totalp38 mapk noisserpxe evitalercd133 ˆ’p p p ˆ’ ˆ’ ˆ’ ˆ’ control hours dxr treatmentp p p ˆ’ˆ’control ˆ’ ˆ’ ˆ’ hours dxr treatmentfigure diï¬erent expression of phosphorylated p38mapk between cd44cd133 and cd44cd133 cells representative blots andsemiquantitative data on the expression of phosphorylated p38mapk and total p38mapk in cells treated with μm dxr in theabsence or presence of μm verapamil the quantitative data are normalized to total p38mapk data are expressed as relative values tocd44cd133 cells without dxr treatment and presented as the mean ± sd from three independent experiments 0cstem cells internationaldxr ˆ’dxr ˆ’ limaparevstnuoc limaparevstnuoccd44133cd44133ˆ’stnuocfl1hfl1hstnuocfl1hafl1hverapamil ˆ’verapamil nrf2𝛽tubulin noisserpxe evitalercd133 ˆ’p p p ˆ’ ˆ’ ˆ’ ˆ’ controldxr treatment hours bp p ˆ’ˆ’control ˆ’ ˆ’ ˆ’ dxr treatment hours figure diï¬erent antioxidant capacity between cd44cd133 and cd44cd133 cells a representative histograms of flow cytometryanalysis show the intracellular ros levels hr after the treatment by μm dxr in the absence or presence of μm verapamil theresults were reproducible in three independent experiments b representative blots and semiquantitative data on the expression of nrf2in cells treated with μm dxr in the absence or presence of μm verapamil the quantitative data are normalized to βtubulin dataare expressed as relative values to cd44cd133 cells without dxr treatment and presented as the mean ± sd from three independentexperiments cd44cd133 cells showed higher antioxidantcapacity than cd44cd133 cells it is also well known thatdxr generates ros and oxidative stress due to ros generation may induce the activation of p38mapk therefore weestimated the ros levels in cells with or without dxr exposure we observed a lower level of ros in cd44cd133 0cstem cells internationalstnuocstnuocstnuocstnuocstnuoc0nm ± stnuocfl2h5nm ± stnuocfl2h10nm ± stnuocfl2h15nm ± stnuocfl2h25nm ± stnuocfl2h0nm ± fl2h25nm ± ytilibaiv llecfl2h50nm ± fl2h75nm ± fl2h100nm ± fl2hŽŽŽŽðœ‡m cd44133ˆ’ cd44133 cd44133 control sirna cd44133 cd133 sirna abfigure continued 0cstnuo0cstnuo0cexpression of abcb10nm ± 0nmstnuo0c ± fl1h5nm ± fl1h50nmstnuo0c ± fl1hfl1h25nmstnuo0c ± stnuo0cfl1h100nm ± fl1hstem cells internationalaccumulation of dxrcontrol sirna 5nm ± fl3hcd133 sirna 5nm ± stnuo0cstnuo0cfl3hcdfigure the eï¬ect of silencing cd133 expression on dxr resistance of cd44cd133 cells a representative histograms of flowcytometry analysis on the expression of cd133 in cd44cd133 cells after silencing by diï¬erent dosages of targeted sirna quantitativedata in the histograms are presented as the mean fluorescent intensity from three independent experiments b mtt assay was done toevaluate the cytotoxicity to dxr cells were treated with nm sirna for hr followed by dxr treatment for another hr data areexpressed as a percent of baseline before dxr treatment from three independent experiments ˆ—p vs cd44cd133 cells crepresentative histograms of flow cytometry analysis on the expression of abcb1 in cells after silencing by diï¬erent dosages of targetedsirna quantitative data in the histograms are presented as the mean fluorescent intensity from three independent experiments drepresentative histograms of flow cytometry analysis on the accumulation of dxr quantitative data in the histograms are presented asthe mean fluorescent intensity from three independent experimentsthan cd44cd133 cells especially under dxr exposurebut verapamil did not obviously change the ros levelsfigure 5a based on these findings we speculated thatthe enhanced antioxidant capacity in cd44cd133 cellsmight help to maintain a lower level of phosphorylatedp38mapknrf2 a transcription factor that is well known to beactivated by oxidative stress such as ros and electrophilicsubstances can protect cells against various stresses we alsocompared the expression level of nrf2 between cd44cd133 and cd44cd133 cells western blotting showeda higher expression of nrf2 in cd44cd133 than cd44cd133 cells especially under dxr exposure p figure 5b and the enhanced expression of nrf2 in cd44cd133 cells was not cancelled by verapamil treatmentp figure 5b sirna treatment to further confirm the regulatory roleof cd133 in drug resistance we tried to silence cd133expression in cd44cd133 cells by sirna and then estimated cytotoxicity of dxr although the decrease ofcd133 expression was clearly observed by targeted sirnap vs nm figure 6a dxr resistance of cd44cd133 cells only partially improved figure 6b unexpectedlythe silencing of cd133 did not change theexpression of abcb1 in cd44cd133 cells even using 0cstem cells internationalexcessive concentrations of cd133 sirna p vs nmfigure 6c we also confirmed that the silencing of cd133did not aï¬ect the accumulation of dxr in cd44cd133cells p vs control sirna figure 6dthis suggests that beyond the drug excretion and redoxregulation other complex mechanisms are also likelyinvolved in the dxr resistance in cd44cd133 cells discussionby using the wellrecognized cell surface markers of cd44and cd133 for csc identification we tried to separate thehct8 human colon cancer cells into cd44 cd44 cd44cd133 and cd44cd133 subpopulations and then investigated how the expressions of cd44 and cd133 associatedwith drug resistance actually we checked several cancer celllines on the expression of cd44 and cd133 including helacells and a549 cells however both hela cells and a549 cellsshowed almost expression of cd44 only the hct8cells showed a partial expression of cd44 about anda rare expression of cd133 therefore we only isolateddiï¬erent subpopulations from hct8 cells for this studyfirst we found that the expression level of cd44 keptvery stable in the cd44cd133 cells but gradually declinedin cd44cd133 cells during a cell passaging process on theother hand some of cd44 cells shifted to express cd44 during a cell passaging process figure 1c these findings suggested the plasticity of cd44 expression in hct8 cellsactually ohata et al reported that cd44 highexpressedcells from human intractable colon cancer patients can diï¬erentiate into cd44 lowexpressed cells and a fraction of cd44lowexpressed cells can also generate cd44 highexpressedcells in a xenograft mouse model however it is unclearwhy the cd44cd133 cells but not cd44cd133 cellsstably maintain the expression level of cd44 unlike theextensive expression of cd44 with high plasticity the expression of cd133 was only observed in very few of the hct8cells with poor plasticitya number of previous studies have demonstrated thatcscs are likely resistant to chemotherapeutic drugs thecd44cd133 cells but not the cd44 and cd44cd133cells showed dxr resistance figure 2a according to thisdata the expression of cd133 but not cd44 seems to beclosely associated with drug resistance actuallythesecd44cd133 cells showed the enhanced expression ofabcb1 and the decreased intracellular accumulation ofdxr figures 2b and 2c liu et al reported that nonsmallcell lung cancer cells treated with lowdose cddp aresufficient to enrich cd133 cells and upregulate abcb1expression through notch signaling which thereforeincreases the crossresistance to dxr however theinhibition of abcb1 by verapamil only partially improvedthe dxr resistance of cd44cd133 cells in this studyto find other potential mechanisms involving in thedxr resistance of cd44cd133 cells we investigatedseveral interesting aspects including the stress protectionand redox regulation we found that p38mapk one of themost popular protein kinases known to be activated byinflammatory cytokines lipopolysaccharide osmotic shockultraviolet light and other stresses was more obviouslyinduced by dxr in cd44cd133 cells than cd44cd133cells figure moreover the activation of p38 mapk wasnot dependent on the intracellular accumulation of dxrfigure dxr is known to insert between the base pairs of dna oftumor cells and exhibits antitumor eï¬ects by suppressing thebiosynthesis of both dna and rna through the inhibitionof dna polymerase rna polymerase and topoisomeraseii reactions furthermore it is believed that dxr has theability to generate sufficient ros to raise oxidative stressindeed we observed dxrinduced ros generation in bothcd44cd133 and cd44cd133 cells butthe dxrinduced ros generation was detected even higher in cd44cd133 than cd44cd133 cells independent on the intracellular accumulation of dxr figures 5a 2b and 3bsuggesting the enhanced antioxidant capacity in cd44cd133 cellsthe keap1nrf2 control system plays a central role in theantioxidant defense mechanisms nrf2 is known as a transcription factor to activate various genes involving in biological defense mechanisms it has been reported that nrf2 isconstantly expressed in many cancer cells [“] moreover the enhanced expression of nrf2 has been confirmedto associate with poor prognosis of cancer patients [“]in our study nrf2 expression was detected higher in cd44cd133 than cd44cd133 cells and the diï¬erence in nrf2expression was observed even clearer between cells with dxradministrationindependent on the dxr accumulationfigure 5b these findings also clearly indicate theenhanced antioxidant capacity in cd44cd133 cellsalthough the absence of direct evidence by interferenceexperiment pathways involving in the stress protection andredox regulation might at least partially contributed to thedxr resistance of cd44cd133 cellsvery strangely our data showed that the silencing ofcd133 expression in cd44cd133 cells by sirna couldonly partially increase the cytotoxicity of dxr figure 6bbut did not change the expression of abcb1 and the intracellular accumulation of dxr figure 6c other unknownmechanisms beyond the drug excretion and redox regulationare asked to be defined on the dxr resistance of cd44cd133 cellsbased on data from the present study the expression ofcd133 rather than cd44 more closely associated with theresistance of cancer cells to anticancer drug as complexmechanisms including the drug excretion and redox regulation are likely involved in the drug resistance of cscs multiple approaches may be needed to overcome the big problemof drug resistance in cancer patientsabbreviationsabcb1abcg2atpbinding cassette subfamily b member 1pglycoproteinmultidrug resistance protein1mdr1atpbinding cassette subfamily g member2breast cancer resistanceproteinbcrpcd388 0cabcc1bsocscsdxrmttatpbinding cassette subfamily c member1multidrug resistanceassociated protein1mrp1buthionine sulfoximinecancer stem cellsdoxorubicinadriamycin345dimethylthiazol2yl25diphenyltetrazolium bromidenuclear factor erythroid 2related factor nrf2p38mapk p38 map kinaserossirnareactive oxygen speciessmall interfering rnadata availabilitythe data that support the findings of this study are availablefrom the corresponding author upon reasonable requestdisclosurethe funder played no role in the study design data collectionand analysis decision to publish or preparation of themanuscriptconflicts of interestthe authors indicate no potential conflicts of interestacknowledgmentsthis work was supported by a grantinaid for the ministryof education science sports culture and technology ofjapan grant numbers and 16k15622 and thecollaborative research program of the atomic bomb diseaseinstitute of nagasaki universityreferences r c elble œthe role of cancer stem cells in relapse of solidtumors frontiers in bioscience vol e4 no pp “ j e visvader œcells of origin in cancer nature vol no pp “ h clevers œthe cancer stem cell premises promises andchallenges nature medicine vol no pp “ y kinugasa t matsui and n takakura œcd44 expressed oncancerassociated fibroblasts is a functional molecule supporting the stemness and drug resistance of malignant cancer cellsin the tumor microenvironment stem cells vol no pp “ t ishimoto o nagano t yae et al œcd44 variant regulatesredox status in cancer cells by stabilizing the xct subunit ofsystem xc and thereby promotes tumor growth cancer cellvol no pp “ m tamada o nagano s tateyama et al œmodulation ofglucose metabolism by cd44 contributes to antioxidant statusand drug resistance in cancer cells cancer research vol no pp “ r c bates n s edwards g f burns and d e fisher œacd44 survival pathway triggers chemoresistance via lyn kinasestem cells internationaland phosphoinositide 3kinaseakt in colon carcinoma cellscancer research vol no pp “ l y w bourguignon k peyrollier w xia and e giladœhyaluronancd44 interaction activates stem cell markernanogandankyrinregulated multidrug efflux in breast and ovariantumor cells the journal of biological chemistry vol no pp “ stat3mediated mdr1expressiongene l y w bourguignon c earle g wong c c spevak andk krueger œstem cell marker nanog and stat3 signalingpromote microrna21 expression and chemoresistance inhyaluronancd44activated head and neck squamous cellcarcinoma cells oncogene vol no pp “ k tajima r ohashi y sekido et al œosteopontinmediatedenhanced hyaluronan binding induces multidrug resistance inmesothelioma cells oncogene vol no pp “ j ni p j cozzi j l hao et al œcd44 variant is associatedwith prostate cancer metastasis and chemoradioresistanceprostate vol no pp “ u carling l barkhatov h m reims et al œcan we ablateliver lesions close to large portal and hepatic veins with mrguided hifu an experimental study in a porcine modelblood vol no pp “ y wu and p y wu œcd133 as a marker for cancer stem cellsprogresses and concerns stem cells and development vol no pp “ u m gehling s erg¼n u schumacher et al œin vitro diï¬erentiation of endothelial cells from ac133positive progenitorcells blood vol no pp “ m peichev a j naiyer d pereira et al œexpression ofvegfr2 and ac133 by circulating human cd34 cellsidentifies a population of functional endothelial precursorsblood vol no pp “ n uchida d w buck d he et al œdirect isolation of humancentral nervous system stem cells proceedings of the nationalacademy of sciences of the united states of america vol no pp “ b j cummings n uchida s j tamaki et al œhuman neuralstem cells diï¬erentiate and promote locomotor recovery inspinal cordinjured mice proceedings of the national academy of sciences of the united states of america vol no pp “ b bussolati s bruno c grange et al œisolation of renal progenitor cells from adult human kidney the american journalof pathology vol no pp “ l riccivitiani d g lombardi e pilozzi et al œidentification and expansion of human coloncancerinitiating cellsnature vol no pp “ c a o™brien a pollett s gallinger and j e dick œa humancolon cancer cell capable of initiating tumour growth inimmunodeficient mice nature vol no pp “ l lin a liu z peng et al œstat3 is necessary for proliferation and survival in colon cancerinitiating cells cancerresearch vol no pp “ n haraguchi m ohkuma h sakashita et al œcd133cd44 population efficiently enriches colon cancer initiating cellsannals of surgical oncology vol no pp “ 0cstem cells international d inoue t suzuki y mitsuishi et al œaccumulation ofp62sqstm1 is associated with poor prognosis in patientswith lung adenocarcinoma cancer science vol no pp “ j q ma h tuersun s j jiao j h zheng j b xiao anda hasim œfunctional role of nrf2 in cervical carcinogenesis plos one vol no article e0133876 q yang h deng h xia et al œhigh nfe2related factor expression predicts poor prognosis in patients with lung cancer a metaanalysis of cohort studies free radical researchvol pp “ s sarvi a c mackinnon n avlonitis et al œcd133 cancerstemlike cells in small cell lung cancer are highly tumorigenicand chemoresistant but sensitive to a novel neuropeptideantagonist cancer research vol no pp “ q zhang s shi y yen j brown j q ta and a d le œasubpopulation of cd133 cancer stemlike cells characterized in human oral squamous cell carcinoma confer resistanceto chemotherapy cancer letters vol no pp “ s ma t k lee b j zheng k w chan and x y guanœcd133 hcc cancer stem cells confer chemoresistance bypreferential expression of the aktpkb survival pathwayoncogene vol no pp “ s bao q wu r e mclendon et al œglioma stem cells promote radioresistance by preferential activation of the dnadamage response nature vol no pp “ c yan l luo c y guo et al œdoxorubicininduced mitophagy contributes to drug resistance in cancer stem cells fromhct8 human colorectal cancer cells cancer letters vol pp “ s goto y ihara y urata et al œdoxorubicininduced dnaintercalation and scavenging by nuclear glutathionestransferase ϝ the faseb journal vol no pp “ h ohata t ishiguro y aihara et al œinduction of the stemlike cell regulator cd44 by rho kinase inhibition cont

cancer7567

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: the heterogeneity of cancer cells is generally accepted and astem celllike subpopulation that is called œcancer stem cellscscs has been identified in various types of malignanttumors although the lack of consensus on the definitioncscs are widely recognized as a small subpopulation amongcancer cells with the properties of selfrenewal and tumor initiation as cscs play a critical role in the recurrence andmetastasis of cancer targeting the cscs is thought to bea promising approach for curing cancera large number of past studies have tried to identify andcharacterize the cscs as normal tissuespecific stem cellsare considered as the main origin of cancer the cscsare also thought to be inherited at least partially the characterization of normal tissuespecific stem cells thereforemany studies on the identificationpurification of cscs havesimply shared markers of hematopoietic stem cells includingthe most popularly used cell surface markers of cd44 andcd133 [ ] cd44 is a type i transmembrane glycoproteinthat is expressed on hematopoietic fibroblastic and glial cellsand functionally known to mediate cellcell and cellmatrixinteractions previous studies have demonstrated that thecd44 is not only a biomarker but also plays critical roles inthe maintenance of cscs the resistance to various therapiesstresses and the metastasis of cancer cells [“]cd133 is originally identified as protein expressing on thecell surface of hematopoietic stem cells and has subsequently been found to be critical in the maintenance ofœstemness of stem cells in various tissues [“] cd133has also been found in some csc [“] which contributesto therapeutic resistance through the activation of akt bcl2and mapkpi3k signaling pathways [“] although theexpressions of cd44 and cd133 in cancer cells likely associate with the resistances to radiotherapy chemotherapy andvarious stresses the diï¬erent significance between cd44and cd133 has not yet been well understoodin this study we investigated whether the expression ofcd44 and cd133 in human colorectal cancer cells hct8diï¬erently contributed to drug resistance our data indicated 0cstem cells internationalthat the expression of cd133 rather than cd44 closely associated with doxorubicin dxr resistance at least partiallythrough drug excretion and redox regulation materials and methods cell culture human colorectal cancer hct8 cells werecultured in rpmi medium fujifilm wako purechemical japan supplemented with fbs gibco°thermo fisher scientific ma usa at c in a humidifiedatmosphere of air and co2 separation of cd44 and cd133positive cells fromhct8 cells we separated the parent hct8 cells intocd44positive cd44 and cd133positive cd133 cellsby a twostep magnetic cell sorting method as described previously [ ] briefly hct8 cells were collected as a singlecell suspension by trypsinization and then incubated withmagnetic microbeadconjugated antihuman cd44 antibodymiltenyi biotec germany for min after washing cellswere separated into cd44 and cd44 subpopulations byusing the automacs„¢ pro separator miltenyi biotecaccording to the manufacturer™s instruction the purifiedcd44 cells were further expanded and then harvested as asinglecellsuspension to be incubated with magneticmicrobeadconjugated antihuman cd133 antibody miltenyibiotec for min after washing the cd44cd133 andcd44cd133 subpopulations were separated as describedabove this twostep isolation enabled us to obtain a sufficientnumber of cd44 cd44 cd44cd133 and cd44cd133 cells for our experimentsto verify the purity of each subpopulation isolated cellswere stained with pelabelled mouse antihuman cd133clone ac133 miltenyi biotec and fitclabelled mouseantihuman cd44 clone db105 miltenyi biotec according to the supplied protocols flow cytometry analysis wasperformed using a facscalibur becton dickinson asdescribed previously mouse igg1pe miltenyi biotecand mouse igg1fitc miltenyi biotec were used as a negative control cytotoxicity assays cells were seeded in 96well cultureplates at a density of — cells per well and cultured overnight the cells were then treated with various concentrationsof dxr fujifilm wako pure chemical in the absence orpresence of verapamil fujifilm wako pure chemicalcytotoxicity assays were performed using the cell proliferation kit i mtt roche applied science germany asdescribed previously the absorbance was measured at nm using a microplate reader multiskan fc thermofisher scientific analysis on the expression of abc transporters theexpressions of the atpbinding cassette subfamilies of bmember abcb1 or g member abcg2 were analyzedby flow cytometry briefly cells were incubated with mouseprimary antibodies against human abcb1 and abcg2bd biosciences ca usa and then labeled by fitcconjugated antimouse igg bd biosciences according tothe manufacturer™s instruction respective isotype controlswere used as a negative control after washing flow cytometry analysis was performed using a facscalibur analysis of cellular accumulation of dxr the intracellular accumulation of dxr was analyzed by flow cytometrybriefly cells were treated by μm dxr for hr in theabsence or presence of μm verapamil or μm buthionine sulfoximine bso sigmaaldrich mo usa cellswere then collected as a singlecell suspension and washedtwice with icecold phosphatebuï¬ered saline the accumulation of dxr within cells was evaluated by the intracellularfluorescence intensity using a facscalibur the nucleusaccumulation of dxr was analyzed by using cell pelletstreated with triton x100pbs as assay material asdescribed previously expressionlevelsanalysisimmunoblot detection of intracellular ros the intracellular roslevel based on the oxidation of ²²dichlorodihydrofluorescein diacetate h2dcfda molecular probes thermofisher scientific was measured to form the fluorescent compound ²²dichlorofluorescein dcf using a facscaliburofphosphorylatedp38 map kinase phosphop38mapk totalp38mapk and nuclear factor erythroid 2related factor nrf2 in the cells were estimated by immunoblotting brieflycell lysate μg of total protein was separated by sodiumdodecyl sulfate“polyacrylamide gel electrophoresis sdspage gel transferred to pvdf membranes biorad causa and then incubated with primary antibodies cell signaling technology ma usafollowed by appropriatehrplabeled secondary antibodies dako agilent pathologysolutions ca usa blots were developed by enhancedchemiluminescence using an ecl kit ge healthcare lifesciences pa usa semiquantitation was done by measuringthe density of bands using the image quant las minibiomolecular imager ge healthcare life sciences asdescribed previously sirna treatment smallinterfering rna sirnaspecific targeting to cd133 on targetplus sirnaand a scramble sirna on targetplus sirna negativecontrol were obtained from dharmacon horizon discovery cambridge uk cells were seeded in 6well plates — cellswell and incubated for hr transfectionswere performed using dharmafect sirna transfectionreagents dharmacon according to the manufacturer™sinstructions analyses were done at hr after sirnatransfection statistical analysis all of the results are presented as themeans ± sd statistical significance was determined by oneway analysis of variance anova followed by tukey™s testdr spss ii chicago il diï¬erences were considered significant when p results hct8 cells were separated into various subpopulationsbased on their expressions of cd44 and cd133 first we 0cstem cells internationalparent cellshlffl1hdccd44hlfhlfcd44ˆ’fl1hcd44fl1hahlfhlfcd44cd133ˆ’fl1hcd44cd133fl1hparent cd44ˆ’cd44 cd44133ˆ’ cd44133enilesab fo noitarefilorp llecparent cd44ˆ’ cd44 cd44ˆ’cd44bcfigure continued 0cstem cells internationalenilesabstnuo0ccd44cd133ˆ’ ± stnuo0ccd44cd133 ± fl2hfl2hsyad stnuo0c ± fl2hstnuo0cd ± fl2hfigure the separation of hct8 colorectal cancer cells into diï¬erent subpopulations based on the expression of cd44 and cd133 arepresentative dot plots of flow cytometry analysis show the purities of each subpopulation of isolated cells quantitative data in the dotplots are presented as the percentages of positive cells from three independent experiments b representative photos of morphologicalproperties upper and mtt assay on cell growth lower at hr after the initiation of culture data are presented as the mean ± sd fromthree independent experiments c d representative histograms of flow cytometry analysis showed the expressions of cd44 c andcd133 d at baseline and days after cell culture the dotted vertical lines through histograms indicate the diï¬erence in the expressionpeaks between the baseline and at days after culture quantitative data in the histograms are presented as the mean fluorescentintensity from three independent experimentsseparated the hct8 cells into cd44 and cd44 subpopulations and compared their sensitivity to anticancer drugs ofdxr and cisplatin cisdiaminedichloroplatine cddphowever no diï¬erence in the sensitivity to the two drugswas observed between cd44 and cd44 cells data notshown we further tried to purify a small population ofcd133 cells from these cd44 cells cd44 cells almostnegatively expressed with cd133 figure 1a as a resultwe separated hct cells into diï¬erent subpopulationsincluding cd44 cd44 cd44cd133 and cd44cd133 cells the purities of isolated cells in each subpopulation were confirmed to be around by flow cytometryfigure 1aandphenotype changein diï¬erent growthsubpopulations of cells the morphology and proliferationof these cells could not be found obviously diï¬erent amongsubpopulations figure 1b the expression of cd44 in allsubpopulations kept stable within days of reculturingfrom the frozen cells that stocked immediately after isolationinterestingly the expression of cd44 was a tendency todecrease with culture time in cd44 fluorescence intensity ± at baseline vs ± at days p figure 1c and cd44cd133 cells fluorescence intensity ± at baseline vs ± at days p figure 1c but still kept stable in cd44cd133 cells at days after reculturing fluorescence intensity ± at baseline vs ± at days p figure 1cthe expression of cd133 in cd44cd133 cells kept verystable fluorescence intensity ± at baseline vs ± at days p figure 1d and cd44cd133cells did not turn to express cd133 within days of reculturing fluorescence intensity ± at baseline vs ± at days p figure 1d therefore we usedthe cells within days after reculturing from the frozenstocked cells in subsequent experiments dxr resistance of cd44cd133 cells next we evaluated the sensitivity of cells to dxr by mtt assay withthe addition of μm of dxr in medium we foundthat the survival of cd44cd133 cells was significantlyhigher than all other subpopulations of cells after hr of 0cstem cells international ytilibaiv llecŽŽŽŽŽðœ‡mparent cellscd44ˆ’ cellscd44 cellscd44133ˆ’ cellscd44133 cellsdxrbso003hct8doxb24 hfl3h006hct8 cd44ˆ’doxb24 hfl3h009hct8 cd44doxb24 hsllec tnerapstnuocsllec ˆ’dcstnuocsllec dcstnuocdxr001hct8dox24 hfl3hstnuocadxrverapamil002hct8doxv24 hstnuocfl3h004hct8 cd44ˆ’dox24 h005hct8 cd44ˆ’doxv24 hstnuocstnuocfl3hfl3h007hct8 cd44dox24 h006hct8 cd44doxv24 hstnuocstnuocfl3hfl3hsllec ˆ’dcsllec dc006hct8 cd44cd133ˆ’dox24 h006hct8 cd44cd133ˆ’doxv24 hstnuocstnuocstnuocfl3hfl3h00hct8 cd44cd133dox24 h00hct8 cd44cd133doxv24 hstnuocfl3hstnuocstnuocfl3hbfigure continuedfl3h012hct8 cd133ˆ’doxb24 hfl3h015hct8 cd44cd133doxb24 hfl3h 0cstnuocstem cells internationalabcg2abcb1stnuocfl1hparent cellscd44ˆ’cellscd44cellscd44133ˆ’ cellscd44133 cells ± ± ± ± ± parent cellscd44ˆ’cellscd44cellscd44133ˆ’ cellscd44133 cellscfl1h ± ± ± ± ± figure dxr resistance of diï¬erent subpopulations of cells a mtt assay was done to evaluate the cytotoxicity of dxr data are expressedas the percentile of baseline before dxr treatment from three independent experiments ˆ—p vs all other subpopulations brepresentative histograms of flow cytometry analysis show the accumulation of dxr in cells hr after the treatment with μm dxrin the absence or presence of μm verapamil and μm bso the dotted vertical lines through histograms indicated the mean levels ofdxr accumulation in cd44cd133 cells for comparing with other subpopulations of cells the results were reproducible in threeindependent experiments c representative histograms of flow cytometry analysis show the expression of the abcb1 or abcg2 indiï¬erent subpopulations of cells quantitative data in the histograms are presented as the mean fluorescent intensity from threeindependent experimentsculture p vs other groups at diï¬erent dxr concentrations figure2ato understand the relevant mechanism we measured theintracellular accumulation of dxr in cells by flow cytometrythe accumulation of dxr in cd44cd133 cells wasdetected as the lowest among these subpopulations at hrafter the exposure to μm dxr figure 2b we furtherfound that the intracellular accumulation of dxr in cd44cd133 cells was obviously increased by the treatment withverapamil an inhibitor for drug efflux cell membrane transporters of abcb1 and abcg2 figure 2b however theintracellular accumulation of dxr in cd44cd133 cellsdid not change by the treatment with bso a glutathione synthesis inhibitor that indirectly regulates drug efflux throughabcc1 figure 2b we also confirmed that the expressionof abcb1 p vs other groups but not abcg2 wasenhanced in cd44cd133 cells figure 2c suggestingthe probable role of abcb1 on dxr resistance in cd44cd133 cellsto further confirm the causal relationship between theenhanced drug efflux and dxr resistance we evaluatedthe cytotoxicity of dxr in the presence or absence of verapamil unexpectedly verapamil only partially enhanced thecytotoxicity of dxr in either cd44cd133 or cd44cd133 cells figure 3ait is well known that dxr interacts with nuclear dna toinhibit macromolecular biosynthesis therefore we alsoestimated the eï¬ect of verapamil on the nuclear accumulation of dxr the nuclear accumulation of dxr wasobserved obviously less in cd44cd133 than cd44cd133 cells but tended to have comparable levels withverapamil treatment figure 3b cd44cd133 cells showed better stress tolerance thancd44cd133 cells it is well known that the stress responsekinase p38mapk can be activated by various extracellularstresses and plays critical roles in cell survival and apoptosis although the basal level of phosphorylated p38mapkwas detected very similar between cd44cd133 andcd44cd133 cells p figure lower expressionwas observed in cd44cd133 than cd44cd133 cellsafter dxr exposure even under verapamiltreatmentp figure this suggests a better tolerance tostress of cd44cd133 cells independent on the accumulation of dxr 0cstem cells internationalˆ’ limaparev ytilibaiv llec limaparev ytilibaiv llec hoursŽŽŽŽ 𝜇mŽŽŽŽðœ‡mcd44cd133 cellscd44cd133ˆ’ cellsa hoursŽŽŽ ˆ’ limaparevstnuoccd44133ˆ’ cd44133𝜇mfl3hŽŽŽŽðœ‡m limaparevstnuocfl3hbfigure dxr resistance and nuclear dxr accumulation in cd44cd133 and cd44cd133 cells in the absence or presence of drug effluxinhibitor a mtt assay was done to compare the cytotoxicity of dxr in cd44cd133 and cd44cd133 cells with or without theaddition of μm verapamil data were expressed as a percent of baseline before dxr treatment from three independent experiments ˆ—p vs cd44cd133 cells b representative histograms of flow cytometry analysis showed the nuclear accumulation of dxr incells hr after the treatment by μm dxr with or without the addition of μm verapamil the results were reproducible in threeindependent experimentsverapamil ˆ’verapamil phosphop38 mapk totalp38 mapk noisserpxe evitalercd133 ˆ’p p p ˆ’ ˆ’ ˆ’ ˆ’ control hours dxr treatmentp p p ˆ’ˆ’control ˆ’ ˆ’ ˆ’ hours dxr treatmentfigure diï¬erent expression of phosphorylated p38mapk between cd44cd133 and cd44cd133 cells representative blots andsemiquantitative data on the expression of phosphorylated p38mapk and total p38mapk in cells treated with μm dxr in theabsence or presence of μm verapamil the quantitative data are normalized to total p38mapk data are expressed as relative values tocd44cd133 cells without dxr treatment and presented as the mean ± sd from three independent experiments 0cstem cells internationaldxr ˆ’dxr ˆ’ limaparevstnuoc limaparevstnuoccd44133cd44133ˆ’stnuocfl1hfl1hstnuocfl1hafl1hverapamil ˆ’verapamil nrf2𝛽tubulin noisserpxe evitalercd133 ˆ’p p p ˆ’ ˆ’ ˆ’ ˆ’ controldxr treatment hours bp p ˆ’ˆ’control ˆ’ ˆ’ ˆ’ dxr treatment hours figure diï¬erent antioxidant capacity between cd44cd133 and cd44cd133 cells a representative histograms of flow cytometryanalysis show the intracellular ros levels hr after the treatment by μm dxr in the absence or presence of μm verapamil theresults were reproducible in three independent experiments b representative blots and semiquantitative data on the expression of nrf2in cells treated with μm dxr in the absence or presence of μm verapamil the quantitative data are normalized to βtubulin dataare expressed as relative values to cd44cd133 cells without dxr treatment and presented as the mean ± sd from three independentexperiments cd44cd133 cells showed higher antioxidantcapacity than cd44cd133 cells it is also well known thatdxr generates ros and oxidative stress due to ros generation may induce the activation of p38mapk therefore weestimated the ros levels in cells with or without dxr exposure we observed a lower level of ros in cd44cd133 0cstem cells internationalstnuocstnuocstnuocstnuocstnuoc0nm ± stnuocfl2h5nm ± stnuocfl2h10nm ± stnuocfl2h15nm ± stnuocfl2h25nm ± stnuocfl2h0nm ± fl2h25nm ± ytilibaiv llecfl2h50nm ± fl2h75nm ± fl2h100nm ± fl2hŽŽŽŽðœ‡m cd44133ˆ’ cd44133 cd44133 control sirna cd44133 cd133 sirna abfigure continued 0cstnuo0cstnuo0cexpression of abcb10nm ± 0nmstnuo0c ± fl1h5nm ± fl1h50nmstnuo0c ± fl1hfl1h25nmstnuo0c ± stnuo0cfl1h100nm ± fl1hstem cells internationalaccumulation of dxrcontrol sirna 5nm ± fl3hcd133 sirna 5nm ± stnuo0cstnuo0cfl3hcdfigure the eï¬ect of silencing cd133 expression on dxr resistance of cd44cd133 cells a representative histograms of flowcytometry analysis on the expression of cd133 in cd44cd133 cells after silencing by diï¬erent dosages of targeted sirna quantitativedata in the histograms are presented as the mean fluorescent intensity from three independent experiments b mtt assay was done toevaluate the cytotoxicity to dxr cells were treated with nm sirna for hr followed by dxr treatment for another hr data areexpressed as a percent of baseline before dxr treatment from three independent experiments ˆ—p vs cd44cd133 cells crepresentative histograms of flow cytometry analysis on the expression of abcb1 in cells after silencing by diï¬erent dosages of targetedsirna quantitative data in the histograms are presented as the mean fluorescent intensity from three independent experiments drepresentative histograms of flow cytometry analysis on the accumulation of dxr quantitative data in the histograms are presented asthe mean fluorescent intensity from three independent experimentsthan cd44cd133 cells especially under dxr exposurebut verapamil did not obviously change the ros levelsfigure 5a based on these findings we speculated thatthe enhanced antioxidant capacity in cd44cd133 cellsmight help to maintain a lower level of phosphorylatedp38mapknrf2 a transcription factor that is well known to beactivated by oxidative stress such as ros and electrophilicsubstances can protect cells against various stresses we alsocompared the expression level of nrf2 between cd44cd133 and cd44cd133 cells western blotting showeda higher expression of nrf2 in cd44cd133 than cd44cd133 cells especially under dxr exposure p figure 5b and the enhanced expression of nrf2 in cd44cd133 cells was not cancelled by verapamil treatmentp figure 5b sirna treatment to further confirm the regulatory roleof cd133 in drug resistance we tried to silence cd133expression in cd44cd133 cells by sirna and then estimated cytotoxicity of dxr although the decrease ofcd133 expression was clearly observed by targeted sirnap vs nm figure 6a dxr resistance of cd44cd133 cells only partially improved figure 6b unexpectedlythe silencing of cd133 did not change theexpression of abcb1 in cd44cd133 cells even using 0cstem cells internationalexcessive concentrations of cd133 sirna p vs nmfigure 6c we also confirmed that the silencing of cd133did not aï¬ect the accumulation of dxr in cd44cd133cells p vs control sirna figure 6dthis suggests that beyond the drug excretion and redoxregulation other complex mechanisms are also likelyinvolved in the dxr resistance in cd44cd133 cells discussionby using the wellrecognized cell surface markers of cd44and cd133 for csc identification we tried to separate thehct8 human colon cancer cells into cd44 cd44 cd44cd133 and cd44cd133 subpopulations and then investigated how the expressions of cd44 and cd133 associatedwith drug resistance actually we checked several cancer celllines on the expression of cd44 and cd133 including helacells and a549 cells however both hela cells and a549 cellsshowed almost expression of cd44 only the hct8cells showed a partial expression of cd44 about anda rare expression of cd133 therefore we only isolateddiï¬erent subpopulations from hct8 cells for this studyfirst we found that the expression level of cd44 keptvery stable in the cd44cd133 cells but gradually declinedin cd44cd133 cells during a cell passaging process on theother hand some of cd44 cells shifted to express cd44 during a cell passaging process figure 1c these findings suggested the plasticity of cd44 expression in hct8 cellsactually ohata et al reported that cd44 highexpressedcells from human intractable colon cancer patients can diï¬erentiate into cd44 lowexpressed cells and a fraction of cd44lowexpressed cells can also generate cd44 highexpressedcells in a xenograft mouse model however it is unclearwhy the cd44cd133 cells but not cd44cd133 cellsstably maintain the expression level of cd44 unlike theextensive expression of cd44 with high plasticity the expression of cd133 was only observed in very few of the hct8cells with poor plasticitya number of previous studies have demonstrated thatcscs are likely resistant to chemotherapeutic drugs thecd44cd133 cells but not the cd44 and cd44cd133cells showed dxr resistance figure 2a according to thisdata the expression of cd133 but not cd44 seems to beclosely associated with drug resistance actuallythesecd44cd133 cells showed the enhanced expression ofabcb1 and the decreased intracellular accumulation ofdxr figures 2b and 2c liu et al reported that nonsmallcell lung cancer cells treated with lowdose cddp aresufficient to enrich cd133 cells and upregulate abcb1expression through notch signaling which thereforeincreases the crossresistance to dxr however theinhibition of abcb1 by verapamil only partially improvedthe dxr resistance of cd44cd133 cells in this studyto find other potential mechanisms involving in thedxr resistance of cd44cd133 cells we investigatedseveral interesting aspects including the stress protectionand redox regulation we found that p38mapk one of themost popular protein kinases known to be activated byinflammatory cytokines lipopolysaccharide osmotic shockultraviolet light and other stresses was more obviouslyinduced by dxr in cd44cd133 cells than cd44cd133cells figure moreover the activation of p38 mapk wasnot dependent on the intracellular accumulation of dxrfigure dxr is known to insert between the base pairs of dna oftumor cells and exhibits antitumor eï¬ects by suppressing thebiosynthesis of both dna and rna through the inhibitionof dna polymerase rna polymerase and topoisomeraseii reactions furthermore it is believed that dxr has theability to generate sufficient ros to raise oxidative stressindeed we observed dxrinduced ros generation in bothcd44cd133 and cd44cd133 cells butthe dxrinduced ros generation was detected even higher in cd44cd133 than cd44cd133 cells independent on the intracellular accumulation of dxr figures 5a 2b and 3bsuggesting the enhanced antioxidant capacity in cd44cd133 cellsthe keap1nrf2 control system plays a central role in theantioxidant defense mechanisms nrf2 is known as a transcription factor to activate various genes involving in biological defense mechanisms it has been reported that nrf2 isconstantly expressed in many cancer cells [“] moreover the enhanced expression of nrf2 has been confirmedto associate with poor prognosis of cancer patients [“]in our study nrf2 expression was detected higher in cd44cd133 than cd44cd133 cells and the diï¬erence in nrf2expression was observed even clearer between cells with dxradministrationindependent on the dxr accumulationfigure 5b these findings also clearly indicate theenhanced antioxidant capacity in cd44cd133 cellsalthough the absence of direct evidence by interferenceexperiment pathways involving in the stress protection andredox regulation might at least partially contributed to thedxr resistance of cd44cd133 cellsvery strangely our data showed that the silencing ofcd133 expression in cd44cd133 cells by sirna couldonly partially increase the cytotoxicity of dxr figure 6bbut did not change the expression of abcb1 and the intracellular accumulation of dxr figure 6c other unknownmechanisms beyond the drug excretion and redox regulationare asked to be defined on the dxr resistance of cd44cd133 cellsbased on data from the present study the expression ofcd133 rather than cd44 more closely associated with theresistance of cancer cells to anticancer drug as complexmechanisms including the drug excretion and redox regulation are likely involved in the drug resistance of cscs multiple approaches may be needed to overcome the big problemof drug resistance in cancer patientsabbreviationsabcb1abcg2atpbinding cassette subfamily b member 1pglycoproteinmultidrug resistance protein1mdr1atpbinding cassette subfamily g member2breast cancer resistanceproteinbcrpcd388 0cabcc1bsocscsdxrmttatpbinding cassette subfamily c member1multidrug resistanceassociated protein1mrp1buthionine sulfoximinecancer stem cellsdoxorubicinadriamycin345dimethylthiazol2yl25diphenyltetrazolium bromidenuclear factor erythroid 2related factor nrf2p38mapk p38 map kinaserossirnareactive oxygen speciessmall interfering rnadata availabilitythe data that support the findings of this study are availablefrom the corresponding author upon reasonable requestdisclosurethe funder played no role in the study design data collectionand analysis decision to publish or preparation of themanuscriptconflicts of interestthe authors indicate no potential conflicts of interestacknowledgmentsthis work was supported by a grantinaid for the ministryof education science sports culture and technology ofjapan grant numbers and 16k15622 and thecollaborative research program of the atomic bomb diseaseinstitute of nagasaki universityreferences r c elble œthe role of cancer stem cells in relapse of solidtumors frontiers in bioscience vol e4 no pp “ j e visvader œcells of origin in cancer nature vol no pp “ h clevers œthe cancer stem cell premises promises andchallenges nature medicine vol no pp “ y kinugasa t matsui and n takakura œcd44 expressed oncancerassociated fibroblasts is a functional molecule supporting the stemness and drug resistance of malignant cancer cellsin the tumor microenvironment stem cells vol no pp “ t ishimoto o nagano t yae et al œcd44 variant regulatesredox status in cancer cells by stabilizing the xct subunit ofsystem xc and thereby promotes tumor growth cancer cellvol no pp “ m tamada o nagano s tateyama et al œmodulation ofglucose metabolism by cd44 contributes to antioxidant statusand drug resistance in cancer cells cancer research vol no pp “ r c bates n s edwards g f burns and d e fisher œacd44 survival pathway triggers chemoresistance via lyn kinasestem cells internationaland phosphoinositide 3kinaseakt in colon carcinoma cellscancer research vol no pp “ l y w bourguignon k peyrollier w xia and e giladœhyaluronancd44 interaction activates stem cell markernanogandankyrinregulated multidrug efflux in breast and ovariantumor cells the journal of biological chemistry vol no pp “ stat3mediated mdr1expressiongene l y w bourguignon c earle g wong c c spevak andk krueger œstem cell marker nanog and stat3 signalingpromote microrna21 expression and chemoresistance inhyaluronancd44activated head and neck squamous cellcarcinoma cells oncogene vol no pp “ k tajima r ohashi y sekido et al œosteopontinmediatedenhanced hyaluronan binding induces multidrug resistance inmesothelioma cells oncogene vol no pp “ j ni p j cozzi j l hao et al œcd44 variant is associatedwith prostate cancer metastasis and chemoradioresistanceprostate vol no pp “ u carling l barkhatov h m reims et al œcan we ablateliver lesions close to large portal and hepatic veins with mrguided hifu an experimental study in a porcine modelblood vol no pp “ y wu and p y wu œcd133 as a marker for cancer stem cellsprogresses and concerns stem cells and development vol no pp “ u m gehling s erg¼n u schumacher et al œin vitro diï¬erentiation of endothelial cells from ac133positive progenitorcells blood vol no pp “ m peichev a j naiyer d pereira et al œexpression ofvegfr2 and ac133 by circulating human cd34 cellsidentifies a population of functional endothelial precursorsblood vol no pp “ n uchida d w buck d he et al œdirect isolation of humancentral nervous system stem cells proceedings of the nationalacademy of sciences of the united states of america vol no pp “ b j cummings n uchida s j tamaki et al œhuman neuralstem cells diï¬erentiate and promote locomotor recovery inspinal cordinjured mice proceedings of the national academy of sciences of the united states of america vol no pp “ b bussolati s bruno c grange et al œisolation of renal progenitor cells from adult human kidney the american journalof pathology vol no pp “ l riccivitiani d g lombardi e pilozzi et al œidentification and expansion of human coloncancerinitiating cellsnature vol no pp “ c a o™brien a pollett s gallinger and j e dick œa humancolon cancer cell capable of initiating tumour growth inimmunodeficient mice nature vol no pp “ l lin a liu z peng et al œstat3 is necessary for proliferation and survival in colon cancerinitiating cells cancerresearch vol no pp “ n haraguchi m ohkuma h sakashita et al œcd133cd44 population efficiently enriches colon cancer initiating cellsannals of surgical oncology vol no pp “ 0cstem cells international d inoue t suzuki y mitsuishi et al œaccumulation ofp62sqstm1 is associated with poor prognosis in patientswith lung adenocarcinoma cancer science vol no pp “ j q ma h tuersun s j jiao j h zheng j b xiao anda hasim œfunctional role of nrf2 in cervical carcinogenesis plos one vol no article e0133876 q yang h deng h xia et al œhigh nfe2related factor expression predicts poor prognosis in patients with lung cancer a metaanalysis of cohort studies free radical researchvol pp “ s sarvi a c mackinnon n avlonitis et al œcd133 cancerstemlike cells in small cell lung cancer are highly tumorigenicand chemoresistant but sensitive to a novel neuropeptideantagonist cancer research vol no pp “ q zhang s shi y yen j brown j q ta and a d le œasubpopulation of cd133 cancer stemlike cells characterized in human oral squamous cell carcinoma confer resistanceto chemotherapy cancer letters vol no pp “ s ma t k lee b j zheng k w chan and x y guanœcd133 hcc cancer stem cells confer chemoresistance bypreferential expression of the aktpkb survival pathwayoncogene vol no pp “ s bao q wu r e mclendon et al œglioma stem cells promote radioresistance by preferential activation of the dnadamage response nature vol no pp “ c yan l luo c y guo et al œdoxorubicininduced mitophagy contributes to drug resistance in cancer stem cells fromhct8 human colorectal cancer cells cancer letters vol pp “ s goto y ihara y urata et al œdoxorubicininduced dnaintercalation and scavenging by nuclear glutathionestransferase ϝ the faseb journal vol no pp “ h ohata t ishiguro y aihara et al œinduction of the stemlike cell regulator cd44 by rho kinase inhibition cont Answer:

Colon_Cancer

"adipogenesis is the process through which mesenchymalstem cells mscs commit to the adipose lineage and diï¬erentiate into adipocytes during this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro one isfibroid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells there are two kinds of fibroid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells anothergroup is fibroblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3t3l1 and 3t3f422a cells cancer cells with tumorinitiation ability designated as cancer stem cells cscshave the characteristics of tumorigenesis and the expressionof specific stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential in addition to cscs cancer cells undergoing epithelialmesenchymaltransformation emt havebeen reported to be induced to diï¬erentiate into adipocytes[“] lung cancer ncih446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro the adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of fibroblastderived cancer cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes brca mutations can be inducedto diï¬erentiate by poly adpribose polymerase parp 0cstem cells internationalinhibitors the nuclear receptor peroxisome proliferatoractivated receptor Î pparÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells thyroid cancer cells expressing the pparÎ fusion proteinppfp can be induced to diï¬erentiate into adipocytes bypioglitazone adipogenesis can be induced in welldiï¬erentiated liposarcoma wdlps and dediï¬erentiatedliposarcoma ddlps cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine ibmx in this review we highlight some of the crucial transcription factors that induce adipogenesis both in mscs and inincluding the wellstudied pparÎ and ccaatcscsenhancerbinding proteins cebps as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation we focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes adipocyte differentiationcell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype adipogenesishas diï¬erent stages each stage has a specific gene expression pattern in general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases the firstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes the preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types in the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespecificproteins the diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation after the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge after reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes adipocyte precursors exhibit transient mitosiscalled œclonal expansion a process that relies on the actionof induced diï¬erentiation factors some preadipocyte cellsmouse cell lines 3t3l1 3t3f442a undergo one or tworounds of cell division prior to diï¬erentiation whereasother cell lines mouse c3h10t12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion whether œmitotic clonal expansion is required for adiposediï¬erentiation remains controversial however it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] when cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets regulatory pathways inpreadipocytes commitmentadipocyte diï¬erentiation is a complex process in which geneexpression is finely regulated the most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process figure wnt signal pathway in adipogenesis wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo the wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to ‚uence cell fate and development wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of pparÎ and cebpα constitutive wnt10b expression inhibits adipogenesis wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes in vivo transgenic expression of wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development wnt10a and wnt6 have also been identified as determinantsof brown adipocyte development [ ] wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation indicating that preadipocytes integrate inputs fromseveral competing wnt signals the hedgehog hh signaling pathway mechanismthree vertebrate hh ligands including sonic hedgehogshhindian hedgehog ihh and desert hedgehogdhh have been identified and initiated a signaling cascademediated by patched ptch1 and ptch2 receptors [ ]hh signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as c3h10t12 ks483 calvaria mscslines and mouse adiposederived stromal cells thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after hhsignaling was inhibited although it is generally agreedthat hh expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking hh signaling andadipogenesis remain poorly defined erkmapkppar signal pathway extracellularregulated protein kinase erk is required in the proliferativephase of diï¬erentiation erk activity blockade in 3t3l1 0cstem cells internationaldex insulin demxwnt 10band othersshhpbc smotgf𝛽p smad3 smad3testosterone𝛽catentinarirspi3kaktcrebpkapcrebfoxo1a2tcflef gata23cebp𝛽mapkg3k3𝛽p2cebp𝛽cebpαppará½»bmpssmad1srebpadipocytegenesfigure regulation pathways in preadipocytes commitment bmp and wnt families are mediators of mscs commitment to producepreadipocytes exposure of growtharrested preadipocytes to diï¬erentiation inducers igf1 glucocorticoid and camp triggers dnareplication leading to adipocyte gene expression due to a transcription factor cascade the dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis inthe terminal diï¬erentiation phase erk1 activity leads topparÎ phosphorylation which inhibits adipocyte diï¬erentiation this implies that erk1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedthis reduction is mediated in part by mitogenactivatedprotein kinase mapk phosphatase1 mkp1 [ ]these extracellular and intracellular regulation factors causeadipocytespecific gene expression and eventually lead toadipocyte formation adipocyte differentiationregulatory proteins pparÎ and adipocyte diï¬erentiation pparÎ is a member of the nuclearreceptor superfamily and is both necessaryand sufficient for adipogenesis forced expression ofpparÎ is sufficient to induce adipocyte diï¬erentiation broblasts indeedthe proadipogenic cebps andkrüppellike factors klfs have all been shown to induceat least one of the two pparÎ promoters in contrast antiadipogenic transcription factor gata functioned in part byrepressing pparÎ expression pparÎ itself has twoisomers the relative roles of pparÎ1 and pparÎ2 in adipogenesis remain an open question pparÎ2 is mainlyexpressed in adipose tissue while pparÎ1 is expressed inmany other tissues although both can promote adipocytediï¬erentiation pparÎ2 could do so eï¬ectively at very lowligand concentration compared with pparÎ1 the twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesispparÎ1 can also be expressed in cell types other than adipocytes ren et al used engineered zincfinger proteins tothe expression ofthe endogenous pparÎ1 andinhibitpparÎ2 promoters in 3t3l1 cells ectopic expression ofpparÎ2 promotes adipogenesis whereas that of pparÎ1does not zhang et al reported that pparÎ2 deficiencyimpairs the development of adipose tissue and insulin sensitivity there are transcriptional cascades between adipocytesgenes including pparÎ and cebpα which are the coreadipocyte diï¬erentiation regulators in the early stage of adipocyte diï¬erentiation the expression of cebp and cebpδincrease which upregulates cebpα expressionfurtheractivate pparÎ pparÎ activating cebpα in turn resultsin a positive feedback pparÎ binding with retinoic acid xreceptor rxr forms diï¬erent heterodimers the variousdimmers can combine with the pparÎ response elementppre and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes cebps participate in adipogenesis and several cebpfamily members are expressed in adipocytesincludingcebpα cebp cebpÎ cebpδ and cebphomologous protein chop the temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of cebp and cebpδ leads tocebpα expression this notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationcebp is crucial for adipogenesis in immortalized preadipocyte lines cebp and cebpδ promote adipogenesis atleast in part by inducing cebpα and pparÎ cebpαinduces many adipocyte genes directly and plays an important role in adipose tissue development once cebpα isexpressed its expression is maintained through autoactivation despite the importance of cebps in adipogenesis 0cstem cells internationalthese transcription factors clearly cannot function efficientlyin the absence of pparÎ cebp cannot induce cebpαexpression in the absence of pparÎ which is required torelease histone deacetylase1 hdac1 from the cebpαpromoter furthermore ectopic cebpα expressioncannot induce adipogenesis in pparΓ“ï¬broblasts however cebpα also plays an important role in diï¬erentiated adipocytes overexpression of exogenous pparÎ incebpαdeficient cells showed that although cebpα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] figure human fibroblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning amplification however pparÎ exogenousligands need to be added to promote adipocyte diï¬erentiation therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of pparÎ bmp and transforming growth factor tgf inadipocyte diï¬erentiation a variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein bmp transforminggrowth factor tgf insulininsulinlike growthfactor igf1 tumor necrosis factor α and interleukin matrix metalloproteinase fibroblast growthfactor fgf and fgf2 bmp and tgf have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells the tgf superfamily members bmps and myostatinregulate the diï¬erentiation of many cell types includingadipocytes tgf inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of tgf impairsadipocyte development inhibition of adipogenesis couldbe obtained through blocking of endogenous tgf with adominantnegative tgf receptor or drosophila mothersagainst decapentaplegic protein smad inhibitionsmad3 binds to cebps and inhibits their transcriptionalactivity including their ability to transactivate the pparÎ2promoter [ ] exposure of multipotent mesenchymalcells to bmp4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion theeï¬ects of bmp2 are more complex and depend on the presence of other signaling molecules bmp2 alone has little eï¬ecton adipogenesis and it interacts with other factors such astgf and insulin to stimulate adipogenesis of embryonicstem cells bmp2 stimulates adipogenesis of multipotentc3h10t12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations klfs in adipocyte diï¬erentiation during adipocyte differentiation some klf family members are overexpressedsuch as klf4 klf5 klf9 and klf15 while klf16 expression is reduced [ ] klf15 is the first klf family members which were identified to be involved in adipocytediï¬erentiation its expression increased significantly on thesixth day of 3t3l1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in mscs andmouse embryonic fibroblasts inhibition of klf15 by sirnaor mutation led to a decrease in pparÎ cebpα fatty acidbinding protein fabp4 and glucose transporter glut4 however overexpression of klf15 in nih3t3cells was found to be associated with lipid accumulation aswell as increases in pparÎ and fabp4 mice with complete absence of klf5 showed embryonal lethality and micewith singlechromosome klf5 knockout showed a significant reduction in white fat in adulthood suggesting thatklf5 plays an important role in adipocyte diï¬erentiationklf5 can be activated by cebp or cebpδ which isinvolved in early adipocyte diï¬erentiation klf5 can beactivated by cebp or cebpδ which is involved in earlyadipocyte diï¬erentiation direct binding of klf5 to thepparÎ2 promoter in combination with cebps inducespparÎ2 expression transfection of klf5 dominantnegative mutants in 3t3l1 cells reduced lipid droplet accumulation and inhibited pparÎ and cebpα expressionwhereas overexpression of wild klf5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation similar to klf5 klf9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as pparÎ cebpα and fabp4 hence inhibitingadipocyte diï¬erentiation however klf9 overexpressiondid not upregulate the expression of pparÎ and cebpα in addition klf4 can transactivate cebp by bindingto the region of kb upstream of the cebp promoter and promote lipid diï¬erentiation klf6 can forma complex with histone deacetylase3 hdac3 inhibitingpreadipocyte factor1 pref1 expression and promotinglipid diï¬erentiation klf2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation overexpressed klf2 can bind to thecaccc region of pparÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of pparÎcebpα and sterolregulated elementbinding proteinssrebp by inhibiting the promoter activity rnasequence analysisshowed that klfl6 expression wasdecreased on the first day of adipocyte diï¬erentiation of3t3l1 cells adipocyte diï¬erentiation was promoted byklf16 knockdown but was inhibited by klf16 overexpression via inhibition of pparÎ promoter activity in addition klf3 and klf7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] signal transducers and activators of transcriptionstats and adipocyte diï¬erentiation the activated statprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription in the adipocyte diï¬erentiation of mouse 3t3l1 cells the expression of stat1 andstat5 was significantly increased while that of stat3and stat6 was not significantly changed in the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells stat1 expression was significantly decreased while the expression of stat3 and stat5 wasincreased and stat6 expression was unchanged therole of stat1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process early adipocytediï¬erentiation of 3t3l1 cells was inhibited by stat1 0cstem cells internationalklf5srebp1cklf15klf2chopcebpá½»krox20ligandcebp𝛽cebp𝛿gata23ppará½»cebp𝛼proadipogenicantiadipogenicgenes of terminaladipocytedifferentiationfigure a cascade of transcription factors that regulate adipogenesis pparÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis pparÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors cebpα is regulated through a series of inhibitory protein“protein interactions some transcription factorfamilies include several members that participate in adipogenesis such as the klfs black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î loss of stat1 in 3t3l1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α other studies have found thatstat1 is required for adipose diï¬erentiation and stat1overexpression in c3h10t12 cells can prevent the inhibition of lipid diï¬erentiation caused by bcell lymphoma6knockdown there was no abnormal adipose tissuein stat1 knockout mice stat3 not only aï¬ectsthe proliferation of 3t3l1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein the fabp4 promoter was used to specificallyknock out stat3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice stat5a and stat5b have diï¬erenteï¬ects on adipocyte diï¬erentiation abnormal adipose tissuewas found in the mice with stat5a or stat5b knockout ordouble knockout and the amount of adipose tissue was onlyonefifth of the original adipose tissue in mice withoutknockdown histone modification in adipocyte diï¬erentiation histone deacetylase sirtuin sirt plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation during the adipocyte diï¬erentiation of c3h1012 cells sirt1 expressiondecreased overexpression of sirt1 activated thewnt signal which caused the deacetylation of cateninthe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation sirt1 knockdown resulted inincreased acetylation of the histones h3k9 and h4k16 inthe secreted frizzledrelated protein sfrp and sfrp2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation forkhead box proteino foxo is a member of the transcription factor foxofamily it can recruit cyclic amp response elementbindingprotein cbphistone acetyltransferase p300 to initiate anacetylation the acetylated foxo1 can be phosphorylatedby phosphorylated protein kinase b pkbakt the phosphorylation of foxo1 by akt inhibits the transcriptionalactivation of foxo1 the acetylation of foxo1 lost the ability of dnabinding affinity and promoted its shuttling fromnuclei to cytoplasm sirt1 and sirt2 can deacetylateand active foxo1 activated foxo1 nonphosphorylatednuclear foxo1 in the nucleus binds to the promoters of target genes encoding p21 p27 and pparÎ and initiates subsequent transcriptions sirt2 inhibits the acetylation andphosphorylation of foxo1 thereby induces the accumulation of activated foxo1 in the nucleus activated foxo1could inhibit adipogenesis via pparÎ [“] lysinespecific histone demethylase lsd1 expression increasedduring the adipocyte diï¬erentiation of 3t3l1 cells lsd1could reduce the dimethylation levels of histone h3k9 andh3k4 in the cebpα promoter region thereby promotingadipocyte diï¬erentiation set domaincontaining setd8 catalyzed the monomethylation of h4k20 andpromoted pparÎ expression the activation of pparÎ transcriptional activity leads to the induction of monomethylatedh4k20 and modification of pparÎ and its targets therebypromoting adipogenesis enhancer of zeste homolog ezh2 is a methyltransferase and can bind methyl groupsto histone h3k27 which is also necessary for lipid diï¬erentiation the absence of ezh2 in brown fat precursors results inreduced levels of the wnt promoter histone h3k27me3which is also saved by the ectopic ezh2 expression or theuse of a wntcatenin signal inhibitor in addition histone demethylases such as lysinespecific histone demethylase lsdkdm kdm6 and histone lysine demethylasephf2 are also involved in adipose diï¬erentiation andkdm2b inhibits transcription factor activator protein 2αpromoter via h3k4me3 and h3k36me2 role of microrna and long noncodingrna in adipogenesismicrorna mir can bind and cut target genes or inhibittarget gene translation endogenous sirna can be producedby the action of dicer enzyme and bind to a specific proteinto change its cellular location many kinds of mirsare involved in regulating adipocyte diï¬erentiation the 0cstem cells internationalexpression of mir143 increased during the diï¬erentiationof adipose progenitor cells overexpression of mir143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation inhibition of mir143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] additionally mir8 promotes adipocyte diï¬erentiation by inhibiting wnt signaling moreover mir mir103 mir21 mir519d mir210 mir30mir204211 and mir375 also play a certain role in promoting adipocyte diï¬erentiation while mir130 mir448and let7y inhibit lipid diï¬erentiation [ ] in additionto mirs long noncoding rna lncrna is a type of noncoding rna and is important during epigenetic regulationand can form a doublestranded rna complex with mrnacauses protein transcription lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of pparÎ2 as a novel lncrna hoxaas3 expression increasedduring the adipose diï¬erentiation of mscs and hoxaas3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation zhu et al reported that hoxaas3 interacted with ezh2 toregulate lineage commitment of mscs hoxa as3 canregulate the trimethylation level of h3k27 in the runx2promoter region by binding to ezh2 therefore hoxaas3 is considered to be an epigenetic switch regulating mscslineage specificity adipocyte diï¬erentiationassociatedlncrna can act as a competitive endogenous rna of mir in the process of lipid diï¬erentiation thereby promotingthe expression of sirt1 the target gene of mir204 and thusinhibiting lipid diï¬erentiation the lncrna neat1can also regulate adipocyte diï¬erentiation under the ‚uence of mirna140 other lncrna including lncrnablnc1 and plnc are also involved in regulating adipocytediï¬erentiation [ ] other biochemical response involved inadipocyte differentiation unfolded protein responses in adipocyte diï¬erentiationin the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded rnadependent proteinkinaselike er kinase and activating transcription factoratf 6α knockdown of atf6α aï¬ects the expressionof adipocytes genes and inhibits c3h10t12 adipocyte differentiation the inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3t3l1 cells is also due to inducedchop and decorin expressions and this inhibitory eï¬ectis ameliorated by chop knockout in the adipocytediï¬erentiation process of 3t3l1 cells increases in pparÎand cebpα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated eukaryotic translation initiaeif 2α phosphorylated endoribonucleasetion factorire1α atf4 chop and other unfolded protein responsesendoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationadditionally eif2α mutation results in continuous activation or overexpression of chop which also inhibits adipocyte diï¬erentiation after the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationfigure role of oxidative stress in adipogenesis during thedirectional diï¬erentiation of mscs mitochondrial complexi and iii and nadph oxidase nox4 are the main sourcesof oxygen species ros production currently it is believedthat ros aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through ‚uencing the signaling pathwaysincluding the wnt hh and foxo signaling cascade duringmscs diï¬erentiation the diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which specifically manifests as low atpcellcontents and a high rate of oxygen consumption the lackof these characteristics indicates stem cell diï¬erentiation adipocyte diï¬erentiation is a highly dependent rosactivation factor related to mitosis and cell maturation schroder et al found that exogenous h2o2 could stimulate adipocyte diï¬erentiation of mouse 3t3l1 cells andhuman adipocyte progenitor cells in the absence of insulinh2o2 regulates adipocyte diï¬erentiation of 3t3l1 cells ina dosedependent manner high doses of h2o2 and μm promote adipocyte diï¬erentiation [ ] tormos et al found that ros synthesis increased in humanmscs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation byknocking down rieske ironsulfur protein and ubiquinonebinding protein ros produced by mitochondrial complexiii was found to be necessary in initiating adipose diï¬erentiation however other studies have shown that theexpression levels of adiponectin and pparÎ were decreasedby using h2o2 “ mm in 3t3l1 cells free radical nitric oxide no also promotes lipid diï¬erentiationbecause treatment with no inducer hydroxylamine or nosynthase nos substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells nosinduced adipose diï¬erentiation mainly via enos rather thaninos ros can induce adipose diï¬erentiation primarily by inhibiting wnt foxo and hh signaling pathwaysthat inhibit lipid diï¬erentiation autophagy in adipocyte diï¬erentiation the increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation baerga et al confirmed that the adipocyte diï¬erentiation efficiency was significantly inhibited in mouse embryonic fibroblasts lacking autophagyrelated gene atg agene encoding an essential protein required for autophagy knockdown of atg5 in 3t3l1 cells promotesproteasomedependent degradation of pparÎ2therebyinhibiting adipocyte diï¬erentiation zhang reportedthat autophagyrelated gene 7atg7 is also crucial for adipose development atg7deficient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cstem cells internationalcebp𝛽 geneebf1 geneklf4egr2cebp𝛽cytosolcebp𝛿 genecebp𝛿klf5geneppará½» geneklf5nr2f2nfkb11433relasrebf1a2rxrappará½»ppará½»rxra heterodimerppará½»rxracorepressor complexfabp4ligands of ppará½»fam120bthrap3ep300ncoa2ncoa3helz2ncoa1crebbpebf1adipoq geneaidrfcebp𝛼 geneznf638znf467cebp𝛼ncor1hdac3ncor2 slc2a4 geneglut4 genelep genefabp4 genecdk4ccnd3plin1 genepck1 genefabp4cd36 geneppararxracoactivator complexppará½»fatty acidrxramediatorcoactivator complexangptlgeneppargc1amediator complex consensuslpl genenucleoplasmproteins bind to gene promoterstranscription of genes into proteinsacting on proteins compoundingtgf𝛽1wnt1wnt10btnf77233adipoqglut4slc2a4 tetramerlepfabp4lipid dropletplin1pck1papa pa4xpalmccd36paangptl4lplfigure regulation of adipocyte diï¬erentiation a regulatory loop exists between pparÎ and cebp activation transcription factor coeebf activates cebpα cebpα activates ebf1 and ebf1 activates pparÎ cebp and cebpδ act directly on the pparÎ gene bybinding its promoter and activating transcription cebpα cebp and cebpδ can activate the ebf1 gene and klf5 the ebf1 and klf5proteins in turn bind the promoter of pparÎ which becomes activated other hormones such as insulin can aï¬ect the expression ofpparÎ and other transcription factors such as srebp1c pparÎ can form a heterodimer with the rxrα in the absence of activatingligands the pparÎrxrα complex recruits transcription repressors such as nuclear receptor corepressor ncor ncor1 andhdac3 upon binding with activating ligands pparÎ causes a rearrangement of adjacent factors corepressors such as ncor2 are lostand coactivators such as transcription intermediary factor tif2 cbp and p300 are recruited which can result in the expression of cyclicampresponsive elementbinding protein creb followed by pparÎ pparÎ expression initiates the expression of downstream genesincluding angiopoietinrelated protein pgar perilipin fabp4 cebpα fatty acid transportrelated proteins carbohydrate metabolismrelated proteins and energy homeostasisrelated proteinslipid metabolism and hormoneinduced lipolysis in the adipocytes were altered autophagy related gene atg4b isactivated by cebp in the process of lipid diï¬erentiationand autophagy activation is necessary for the degradationof klf2 and klf3 two negative regulators of lipid diï¬erentiation these results showed that adipose diï¬erentiation andautophagy are mutually complementary in 3t3l1cells autophagy was inhibited by aspartate ammonia or 0cstem cells internationalmethyladenine at diï¬erent lipid induction periods “ ““ and “ days and only autophagy inhibition at “days hindered the formation of lipid droplets and the expression of lipid marker genes indicating that autophagy wasvery important in the early stage of lipid diï¬erentiation recent studies showed that lc3 is overexpressed in3t3l1 cells further demonstrating the important role ofautophagy in lipid diï¬erentiation role of alternative splicing in adipogenesis selectivesplicing is ‚uenced by splicing regulators which regulateadipocyte diï¬erentiation by regulating the selective splicingof genes specific to this process lipin1 is an important regulator in the process of adipocyte diï¬erentiation and includestwo isomers lipin1α and lipin1 which have diï¬erenteï¬ects high expression of lipin1α promotes adipocyte differentiation while that of lipin1 promotes lipid droplet formation in sam68deficient mice the fifth intron ofserinethreonineprotein kinase mtor was retained resulting in unstable and rapid mtor degradation and inhibitionof adipocyte diï¬erentiation furthermore there arefour isomers of pref1 pref1a and pr

cancer7568

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: "adipogenesis is the process through which mesenchymalstem cells mscs commit to the adipose lineage and diï¬erentiate into adipocytes during this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro one isfibroid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells there are two kinds of fibroid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells anothergroup is fibroblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3t3l1 and 3t3f422a cells cancer cells with tumorinitiation ability designated as cancer stem cells cscshave the characteristics of tumorigenesis and the expressionof specific stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential in addition to cscs cancer cells undergoing epithelialmesenchymaltransformation emt havebeen reported to be induced to diï¬erentiate into adipocytes[“] lung cancer ncih446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro the adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of fibroblastderived cancer cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes brca mutations can be inducedto diï¬erentiate by poly adpribose polymerase parp 0cstem cells internationalinhibitors the nuclear receptor peroxisome proliferatoractivated receptor Î pparÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells thyroid cancer cells expressing the pparÎ fusion proteinppfp can be induced to diï¬erentiate into adipocytes bypioglitazone adipogenesis can be induced in welldiï¬erentiated liposarcoma wdlps and dediï¬erentiatedliposarcoma ddlps cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine ibmx in this review we highlight some of the crucial transcription factors that induce adipogenesis both in mscs and inincluding the wellstudied pparÎ and ccaatcscsenhancerbinding proteins cebps as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation we focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes adipocyte differentiationcell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype adipogenesishas diï¬erent stages each stage has a specific gene expression pattern in general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases the firstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes the preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types in the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespecificproteins the diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation after the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge after reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes adipocyte precursors exhibit transient mitosiscalled œclonal expansion a process that relies on the actionof induced diï¬erentiation factors some preadipocyte cellsmouse cell lines 3t3l1 3t3f442a undergo one or tworounds of cell division prior to diï¬erentiation whereasother cell lines mouse c3h10t12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion whether œmitotic clonal expansion is required for adiposediï¬erentiation remains controversial however it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] when cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets regulatory pathways inpreadipocytes commitmentadipocyte diï¬erentiation is a complex process in which geneexpression is finely regulated the most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process figure wnt signal pathway in adipogenesis wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo the wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to ‚uence cell fate and development wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of pparÎ and cebpα constitutive wnt10b expression inhibits adipogenesis wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes in vivo transgenic expression of wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development wnt10a and wnt6 have also been identified as determinantsof brown adipocyte development [ ] wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation indicating that preadipocytes integrate inputs fromseveral competing wnt signals the hedgehog hh signaling pathway mechanismthree vertebrate hh ligands including sonic hedgehogshhindian hedgehog ihh and desert hedgehogdhh have been identified and initiated a signaling cascademediated by patched ptch1 and ptch2 receptors [ ]hh signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as c3h10t12 ks483 calvaria mscslines and mouse adiposederived stromal cells thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after hhsignaling was inhibited although it is generally agreedthat hh expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking hh signaling andadipogenesis remain poorly defined erkmapkppar signal pathway extracellularregulated protein kinase erk is required in the proliferativephase of diï¬erentiation erk activity blockade in 3t3l1 0cstem cells internationaldex insulin demxwnt 10band othersshhpbc smotgf𝛽p smad3 smad3testosterone𝛽catentinarirspi3kaktcrebpkapcrebfoxo1a2tcflef gata23cebp𝛽mapkg3k3𝛽p2cebp𝛽cebpαppará½»bmpssmad1srebpadipocytegenesfigure regulation pathways in preadipocytes commitment bmp and wnt families are mediators of mscs commitment to producepreadipocytes exposure of growtharrested preadipocytes to diï¬erentiation inducers igf1 glucocorticoid and camp triggers dnareplication leading to adipocyte gene expression due to a transcription factor cascade the dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis inthe terminal diï¬erentiation phase erk1 activity leads topparÎ phosphorylation which inhibits adipocyte diï¬erentiation this implies that erk1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedthis reduction is mediated in part by mitogenactivatedprotein kinase mapk phosphatase1 mkp1 [ ]these extracellular and intracellular regulation factors causeadipocytespecific gene expression and eventually lead toadipocyte formation adipocyte differentiationregulatory proteins pparÎ and adipocyte diï¬erentiation pparÎ is a member of the nuclearreceptor superfamily and is both necessaryand sufficient for adipogenesis forced expression ofpparÎ is sufficient to induce adipocyte diï¬erentiation broblasts indeedthe proadipogenic cebps andkrüppellike factors klfs have all been shown to induceat least one of the two pparÎ promoters in contrast antiadipogenic transcription factor gata functioned in part byrepressing pparÎ expression pparÎ itself has twoisomers the relative roles of pparÎ1 and pparÎ2 in adipogenesis remain an open question pparÎ2 is mainlyexpressed in adipose tissue while pparÎ1 is expressed inmany other tissues although both can promote adipocytediï¬erentiation pparÎ2 could do so eï¬ectively at very lowligand concentration compared with pparÎ1 the twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesispparÎ1 can also be expressed in cell types other than adipocytes ren et al used engineered zincfinger proteins tothe expression ofthe endogenous pparÎ1 andinhibitpparÎ2 promoters in 3t3l1 cells ectopic expression ofpparÎ2 promotes adipogenesis whereas that of pparÎ1does not zhang et al reported that pparÎ2 deficiencyimpairs the development of adipose tissue and insulin sensitivity there are transcriptional cascades between adipocytesgenes including pparÎ and cebpα which are the coreadipocyte diï¬erentiation regulators in the early stage of adipocyte diï¬erentiation the expression of cebp and cebpδincrease which upregulates cebpα expressionfurtheractivate pparÎ pparÎ activating cebpα in turn resultsin a positive feedback pparÎ binding with retinoic acid xreceptor rxr forms diï¬erent heterodimers the variousdimmers can combine with the pparÎ response elementppre and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes cebps participate in adipogenesis and several cebpfamily members are expressed in adipocytesincludingcebpα cebp cebpÎ cebpδ and cebphomologous protein chop the temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of cebp and cebpδ leads tocebpα expression this notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationcebp is crucial for adipogenesis in immortalized preadipocyte lines cebp and cebpδ promote adipogenesis atleast in part by inducing cebpα and pparÎ cebpαinduces many adipocyte genes directly and plays an important role in adipose tissue development once cebpα isexpressed its expression is maintained through autoactivation despite the importance of cebps in adipogenesis 0cstem cells internationalthese transcription factors clearly cannot function efficientlyin the absence of pparÎ cebp cannot induce cebpαexpression in the absence of pparÎ which is required torelease histone deacetylase1 hdac1 from the cebpαpromoter furthermore ectopic cebpα expressioncannot induce adipogenesis in pparΓ“ï¬broblasts however cebpα also plays an important role in diï¬erentiated adipocytes overexpression of exogenous pparÎ incebpαdeficient cells showed that although cebpα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] figure human fibroblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning amplification however pparÎ exogenousligands need to be added to promote adipocyte diï¬erentiation therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of pparÎ bmp and transforming growth factor tgf inadipocyte diï¬erentiation a variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein bmp transforminggrowth factor tgf insulininsulinlike growthfactor igf1 tumor necrosis factor α and interleukin matrix metalloproteinase fibroblast growthfactor fgf and fgf2 bmp and tgf have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells the tgf superfamily members bmps and myostatinregulate the diï¬erentiation of many cell types includingadipocytes tgf inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of tgf impairsadipocyte development inhibition of adipogenesis couldbe obtained through blocking of endogenous tgf with adominantnegative tgf receptor or drosophila mothersagainst decapentaplegic protein smad inhibitionsmad3 binds to cebps and inhibits their transcriptionalactivity including their ability to transactivate the pparÎ2promoter [ ] exposure of multipotent mesenchymalcells to bmp4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion theeï¬ects of bmp2 are more complex and depend on the presence of other signaling molecules bmp2 alone has little eï¬ecton adipogenesis and it interacts with other factors such astgf and insulin to stimulate adipogenesis of embryonicstem cells bmp2 stimulates adipogenesis of multipotentc3h10t12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations klfs in adipocyte diï¬erentiation during adipocyte differentiation some klf family members are overexpressedsuch as klf4 klf5 klf9 and klf15 while klf16 expression is reduced [ ] klf15 is the first klf family members which were identified to be involved in adipocytediï¬erentiation its expression increased significantly on thesixth day of 3t3l1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in mscs andmouse embryonic fibroblasts inhibition of klf15 by sirnaor mutation led to a decrease in pparÎ cebpα fatty acidbinding protein fabp4 and glucose transporter glut4 however overexpression of klf15 in nih3t3cells was found to be associated with lipid accumulation aswell as increases in pparÎ and fabp4 mice with complete absence of klf5 showed embryonal lethality and micewith singlechromosome klf5 knockout showed a significant reduction in white fat in adulthood suggesting thatklf5 plays an important role in adipocyte diï¬erentiationklf5 can be activated by cebp or cebpδ which isinvolved in early adipocyte diï¬erentiation klf5 can beactivated by cebp or cebpδ which is involved in earlyadipocyte diï¬erentiation direct binding of klf5 to thepparÎ2 promoter in combination with cebps inducespparÎ2 expression transfection of klf5 dominantnegative mutants in 3t3l1 cells reduced lipid droplet accumulation and inhibited pparÎ and cebpα expressionwhereas overexpression of wild klf5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation similar to klf5 klf9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as pparÎ cebpα and fabp4 hence inhibitingadipocyte diï¬erentiation however klf9 overexpressiondid not upregulate the expression of pparÎ and cebpα in addition klf4 can transactivate cebp by bindingto the region of kb upstream of the cebp promoter and promote lipid diï¬erentiation klf6 can forma complex with histone deacetylase3 hdac3 inhibitingpreadipocyte factor1 pref1 expression and promotinglipid diï¬erentiation klf2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation overexpressed klf2 can bind to thecaccc region of pparÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of pparÎcebpα and sterolregulated elementbinding proteinssrebp by inhibiting the promoter activity rnasequence analysisshowed that klfl6 expression wasdecreased on the first day of adipocyte diï¬erentiation of3t3l1 cells adipocyte diï¬erentiation was promoted byklf16 knockdown but was inhibited by klf16 overexpression via inhibition of pparÎ promoter activity in addition klf3 and klf7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] signal transducers and activators of transcriptionstats and adipocyte diï¬erentiation the activated statprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription in the adipocyte diï¬erentiation of mouse 3t3l1 cells the expression of stat1 andstat5 was significantly increased while that of stat3and stat6 was not significantly changed in the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells stat1 expression was significantly decreased while the expression of stat3 and stat5 wasincreased and stat6 expression was unchanged therole of stat1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process early adipocytediï¬erentiation of 3t3l1 cells was inhibited by stat1 0cstem cells internationalklf5srebp1cklf15klf2chopcebpá½»krox20ligandcebp𝛽cebp𝛿gata23ppará½»cebp𝛼proadipogenicantiadipogenicgenes of terminaladipocytedifferentiationfigure a cascade of transcription factors that regulate adipogenesis pparÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis pparÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors cebpα is regulated through a series of inhibitory protein“protein interactions some transcription factorfamilies include several members that participate in adipogenesis such as the klfs black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î loss of stat1 in 3t3l1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α other studies have found thatstat1 is required for adipose diï¬erentiation and stat1overexpression in c3h10t12 cells can prevent the inhibition of lipid diï¬erentiation caused by bcell lymphoma6knockdown there was no abnormal adipose tissuein stat1 knockout mice stat3 not only aï¬ectsthe proliferation of 3t3l1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein the fabp4 promoter was used to specificallyknock out stat3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice stat5a and stat5b have diï¬erenteï¬ects on adipocyte diï¬erentiation abnormal adipose tissuewas found in the mice with stat5a or stat5b knockout ordouble knockout and the amount of adipose tissue was onlyonefifth of the original adipose tissue in mice withoutknockdown histone modification in adipocyte diï¬erentiation histone deacetylase sirtuin sirt plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation during the adipocyte diï¬erentiation of c3h1012 cells sirt1 expressiondecreased overexpression of sirt1 activated thewnt signal which caused the deacetylation of cateninthe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation sirt1 knockdown resulted inincreased acetylation of the histones h3k9 and h4k16 inthe secreted frizzledrelated protein sfrp and sfrp2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation forkhead box proteino foxo is a member of the transcription factor foxofamily it can recruit cyclic amp response elementbindingprotein cbphistone acetyltransferase p300 to initiate anacetylation the acetylated foxo1 can be phosphorylatedby phosphorylated protein kinase b pkbakt the phosphorylation of foxo1 by akt inhibits the transcriptionalactivation of foxo1 the acetylation of foxo1 lost the ability of dnabinding affinity and promoted its shuttling fromnuclei to cytoplasm sirt1 and sirt2 can deacetylateand active foxo1 activated foxo1 nonphosphorylatednuclear foxo1 in the nucleus binds to the promoters of target genes encoding p21 p27 and pparÎ and initiates subsequent transcriptions sirt2 inhibits the acetylation andphosphorylation of foxo1 thereby induces the accumulation of activated foxo1 in the nucleus activated foxo1could inhibit adipogenesis via pparÎ [“] lysinespecific histone demethylase lsd1 expression increasedduring the adipocyte diï¬erentiation of 3t3l1 cells lsd1could reduce the dimethylation levels of histone h3k9 andh3k4 in the cebpα promoter region thereby promotingadipocyte diï¬erentiation set domaincontaining setd8 catalyzed the monomethylation of h4k20 andpromoted pparÎ expression the activation of pparÎ transcriptional activity leads to the induction of monomethylatedh4k20 and modification of pparÎ and its targets therebypromoting adipogenesis enhancer of zeste homolog ezh2 is a methyltransferase and can bind methyl groupsto histone h3k27 which is also necessary for lipid diï¬erentiation the absence of ezh2 in brown fat precursors results inreduced levels of the wnt promoter histone h3k27me3which is also saved by the ectopic ezh2 expression or theuse of a wntcatenin signal inhibitor in addition histone demethylases such as lysinespecific histone demethylase lsdkdm kdm6 and histone lysine demethylasephf2 are also involved in adipose diï¬erentiation andkdm2b inhibits transcription factor activator protein 2αpromoter via h3k4me3 and h3k36me2 role of microrna and long noncodingrna in adipogenesismicrorna mir can bind and cut target genes or inhibittarget gene translation endogenous sirna can be producedby the action of dicer enzyme and bind to a specific proteinto change its cellular location many kinds of mirsare involved in regulating adipocyte diï¬erentiation the 0cstem cells internationalexpression of mir143 increased during the diï¬erentiationof adipose progenitor cells overexpression of mir143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation inhibition of mir143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] additionally mir8 promotes adipocyte diï¬erentiation by inhibiting wnt signaling moreover mir mir103 mir21 mir519d mir210 mir30mir204211 and mir375 also play a certain role in promoting adipocyte diï¬erentiation while mir130 mir448and let7y inhibit lipid diï¬erentiation [ ] in additionto mirs long noncoding rna lncrna is a type of noncoding rna and is important during epigenetic regulationand can form a doublestranded rna complex with mrnacauses protein transcription lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of pparÎ2 as a novel lncrna hoxaas3 expression increasedduring the adipose diï¬erentiation of mscs and hoxaas3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation zhu et al reported that hoxaas3 interacted with ezh2 toregulate lineage commitment of mscs hoxa as3 canregulate the trimethylation level of h3k27 in the runx2promoter region by binding to ezh2 therefore hoxaas3 is considered to be an epigenetic switch regulating mscslineage specificity adipocyte diï¬erentiationassociatedlncrna can act as a competitive endogenous rna of mir in the process of lipid diï¬erentiation thereby promotingthe expression of sirt1 the target gene of mir204 and thusinhibiting lipid diï¬erentiation the lncrna neat1can also regulate adipocyte diï¬erentiation under the ‚uence of mirna140 other lncrna including lncrnablnc1 and plnc are also involved in regulating adipocytediï¬erentiation [ ] other biochemical response involved inadipocyte differentiation unfolded protein responses in adipocyte diï¬erentiationin the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded rnadependent proteinkinaselike er kinase and activating transcription factoratf 6α knockdown of atf6α aï¬ects the expressionof adipocytes genes and inhibits c3h10t12 adipocyte differentiation the inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3t3l1 cells is also due to inducedchop and decorin expressions and this inhibitory eï¬ectis ameliorated by chop knockout in the adipocytediï¬erentiation process of 3t3l1 cells increases in pparÎand cebpα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated eukaryotic translation initiaeif 2α phosphorylated endoribonucleasetion factorire1α atf4 chop and other unfolded protein responsesendoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationadditionally eif2α mutation results in continuous activation or overexpression of chop which also inhibits adipocyte diï¬erentiation after the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationfigure role of oxidative stress in adipogenesis during thedirectional diï¬erentiation of mscs mitochondrial complexi and iii and nadph oxidase nox4 are the main sourcesof oxygen species ros production currently it is believedthat ros aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through ‚uencing the signaling pathwaysincluding the wnt hh and foxo signaling cascade duringmscs diï¬erentiation the diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which specifically manifests as low atpcellcontents and a high rate of oxygen consumption the lackof these characteristics indicates stem cell diï¬erentiation adipocyte diï¬erentiation is a highly dependent rosactivation factor related to mitosis and cell maturation schroder et al found that exogenous h2o2 could stimulate adipocyte diï¬erentiation of mouse 3t3l1 cells andhuman adipocyte progenitor cells in the absence of insulinh2o2 regulates adipocyte diï¬erentiation of 3t3l1 cells ina dosedependent manner high doses of h2o2 and μm promote adipocyte diï¬erentiation [ ] tormos et al found that ros synthesis increased in humanmscs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation byknocking down rieske ironsulfur protein and ubiquinonebinding protein ros produced by mitochondrial complexiii was found to be necessary in initiating adipose diï¬erentiation however other studies have shown that theexpression levels of adiponectin and pparÎ were decreasedby using h2o2 “ mm in 3t3l1 cells free radical nitric oxide no also promotes lipid diï¬erentiationbecause treatment with no inducer hydroxylamine or nosynthase nos substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells nosinduced adipose diï¬erentiation mainly via enos rather thaninos ros can induce adipose diï¬erentiation primarily by inhibiting wnt foxo and hh signaling pathwaysthat inhibit lipid diï¬erentiation autophagy in adipocyte diï¬erentiation the increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation baerga et al confirmed that the adipocyte diï¬erentiation efficiency was significantly inhibited in mouse embryonic fibroblasts lacking autophagyrelated gene atg agene encoding an essential protein required for autophagy knockdown of atg5 in 3t3l1 cells promotesproteasomedependent degradation of pparÎ2therebyinhibiting adipocyte diï¬erentiation zhang reportedthat autophagyrelated gene 7atg7 is also crucial for adipose development atg7deficient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cstem cells internationalcebp𝛽 geneebf1 geneklf4egr2cebp𝛽cytosolcebp𝛿 genecebp𝛿klf5geneppará½» geneklf5nr2f2nfkb11433relasrebf1a2rxrappará½»ppará½»rxra heterodimerppará½»rxracorepressor complexfabp4ligands of ppará½»fam120bthrap3ep300ncoa2ncoa3helz2ncoa1crebbpebf1adipoq geneaidrfcebp𝛼 geneznf638znf467cebp𝛼ncor1hdac3ncor2 slc2a4 geneglut4 genelep genefabp4 genecdk4ccnd3plin1 genepck1 genefabp4cd36 geneppararxracoactivator complexppará½»fatty acidrxramediatorcoactivator complexangptlgeneppargc1amediator complex consensuslpl genenucleoplasmproteins bind to gene promoterstranscription of genes into proteinsacting on proteins compoundingtgf𝛽1wnt1wnt10btnf77233adipoqglut4slc2a4 tetramerlepfabp4lipid dropletplin1pck1papa pa4xpalmccd36paangptl4lplfigure regulation of adipocyte diï¬erentiation a regulatory loop exists between pparÎ and cebp activation transcription factor coeebf activates cebpα cebpα activates ebf1 and ebf1 activates pparÎ cebp and cebpδ act directly on the pparÎ gene bybinding its promoter and activating transcription cebpα cebp and cebpδ can activate the ebf1 gene and klf5 the ebf1 and klf5proteins in turn bind the promoter of pparÎ which becomes activated other hormones such as insulin can aï¬ect the expression ofpparÎ and other transcription factors such as srebp1c pparÎ can form a heterodimer with the rxrα in the absence of activatingligands the pparÎrxrα complex recruits transcription repressors such as nuclear receptor corepressor ncor ncor1 andhdac3 upon binding with activating ligands pparÎ causes a rearrangement of adjacent factors corepressors such as ncor2 are lostand coactivators such as transcription intermediary factor tif2 cbp and p300 are recruited which can result in the expression of cyclicampresponsive elementbinding protein creb followed by pparÎ pparÎ expression initiates the expression of downstream genesincluding angiopoietinrelated protein pgar perilipin fabp4 cebpα fatty acid transportrelated proteins carbohydrate metabolismrelated proteins and energy homeostasisrelated proteinslipid metabolism and hormoneinduced lipolysis in the adipocytes were altered autophagy related gene atg4b isactivated by cebp in the process of lipid diï¬erentiationand autophagy activation is necessary for the degradationof klf2 and klf3 two negative regulators of lipid diï¬erentiation these results showed that adipose diï¬erentiation andautophagy are mutually complementary in 3t3l1cells autophagy was inhibited by aspartate ammonia or 0cstem cells internationalmethyladenine at diï¬erent lipid induction periods “ ““ and “ days and only autophagy inhibition at “days hindered the formation of lipid droplets and the expression of lipid marker genes indicating that autophagy wasvery important in the early stage of lipid diï¬erentiation recent studies showed that lc3 is overexpressed in3t3l1 cells further demonstrating the important role ofautophagy in lipid diï¬erentiation role of alternative splicing in adipogenesis selectivesplicing is ‚uenced by splicing regulators which regulateadipocyte diï¬erentiation by regulating the selective splicingof genes specific to this process lipin1 is an important regulator in the process of adipocyte diï¬erentiation and includestwo isomers lipin1α and lipin1 which have diï¬erenteï¬ects high expression of lipin1α promotes adipocyte differentiation while that of lipin1 promotes lipid droplet formation in sam68deficient mice the fifth intron ofserinethreonineprotein kinase mtor was retained resulting in unstable and rapid mtor degradation and inhibitionof adipocyte diï¬erentiation furthermore there arefour isomers of pref1 pref1a and pr Answer:

Colon_Cancer

the periparturient period is one of the most challenging periods in dairy cows and encompasses the a0wk prior to and a0wk after parturition the nutrient requirements of dairy cows change greatly during this time largely due to the exponential growth of the gravid uterus and fetus followed by the demands of lactation nrc inflammation oxidative stress and adipose tissue mobilization lead to a reduction in dry matter intake dmi during the periparturient period this reduction in dmi leads to a negative nutrient balance nnb with a shortfall in the nutrient availability for the cow and fetus ingvartsen and andersen additionally this reduction in dmi also increases the risk of metabolic ketosis fatty liver milk fever and immunerelated disorders the risk of these diseases poor reproduction and low efficiency is greatly impacted by the degree and length of time during which these systems metabolism and immune response remain out of balance loor et a0al 2013a much of the research over the last decade have examined these biological interactions to identify the mechanisms behind the metabolic physiologic and immune adaptations that occur during the periparturient period loor et a0al 2013a 2013b roche et a0al bradford et a0al it is now known that nutrients such as amino acids aa serve functional roles outside of their use as building blocks for proteins and have immunomodulatory properties and interact through common biochemical pathways eg 1carbon metabolism figure a0 this concept has been well explored in nonruminant species li et a0 al sikalidis with nutritional management during the periparturient period continuing to be an active area of research it is important to develop a system understanding the potential immunometabolic role that dietary aa may play during this period thus the objective of this review is to provide an overview of physiological adaptations during the periparturient period the immune system and methods to assess immune function and oxidative stress this will be followed by a more specific discussion of the immunometabolic roles of specific aa and their potential effects in dairy cow during the periparturient period the potential effects of enhanced aa supply during the preweaning period will also be discussed brieflybiological adaptations in the transition cow”a brief overviewduring the nnb associated with the periparturient period biological mechanisms coordinate the mobilization of body reserves in order to support fetal growth and milk production bauman and currie insulin concentrations are reduced and the response of hormonesensitive lipase in adipose tissue eg low insulin high growth hormone and catecholamines or high glucocorticoid concentrations is greater to facilitate lipid mobilization this periparturient period is also characterized by a state of inflammation encompassing an increase in hepatic production of positive acutephase proteins app such as haptoglobin and serum amyloid a a0saa and a decrease in the production ingvartsen the authors published by oxford university press on behalf of the american society of animal sciencethis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly cited for commercial reuse please contact spermissionsoupcoms175 0cs176 of animal science vol no suppl abbreviationsaa akt app asct2 bcaa bcat bcka bhmt bmec bsa cbs csad cth dmi gator1 gcl gclc gpx gsh gsr gss il inos kegg klh lps mpo mtor mtorc1 mtr nfe2l2 nfκb nnb nos pc pmn pmnl pmtor rns rom ros rpm saa sahh sam samtor sell shmt slc1a1 slc1a5 sod stat tag th tlr tnfα vldl amino acidsprotein kinase bacutephase proteinsalanineserinecysteine transporter branchedchain amino acidsbranchedchain amino transferasesbranchedchain αketoacidsbetaine homocysteine methyltransferasebovine mammary epithelial cellsbovine serum albumincystathionine synthasecysteine sulfinic acid decarboxylasecystathionine gammalyasedry matter intakegtpaseactivating protein activity towards rags glutamate cysteine ligaseglutamate cysteine ligase catalyticglutathione peroxidaseglutathioneglutathione reductaseglutathione synthaseinterleukininducible noskyoto encyclopedia of genes and genomeskeyhole limpet hemocyaninlipopolysaccharidemyeloperoxidasemechanistic target of rapamycinmtor complex 5methyltetrahydrofolatehomocysteine methyltransferasenuclear factor erythroid 2like2nuclear factor kappa bnegative nutrient balancenitric oxide synthasephosphatidylcholinepolymorphonuclear neutrophilspolymorphonuclear leukocytesphosphorylated mtorreactive nitrogen speciesreactive oxygen metabolitesreactive oxygen speciesrumenprotected metserum amyloid asadenosyl homocysteine hydrolasesadenosyl methioninesadenosylmethionine sensor upstream of mtorlselectinserine hydoxymethyltransferasesolute carrier family member solute carrier family member superoxide dismutasesignal transducer and activator of transcriptiontriacyglycerolthelpertolllike receptorstumor necrosis factorαverylowdensity lipoproteinsof negative app such as albumin bertoni et a0 al it has been well established that these responses are mediated by the proinflammatory cytokines interleukin il6 il1 and tumor necrosis factorα tnfα kindt et a0al additionally oxidative stress also occurs during this period and is driven by the imbalance between the production of reactive oxygen metabolites rom reactive nitrogen species rns and the neutralizing capacity of antioxidant mechanisms in tissues and blood some of the wellestablished cellular antioxidants include glutathione gsh taurine superoxide dismutase sod and vitamins a a0and e bernabucci et a0al when oxidative stress overwhelms cellular antioxidant capacity rom induce an inflammatory response that is controlled via changes in mrna abundance of transcription regulators eg signal transducer and activator of transcription [stat3] nuclear factorkappa b [nfκb] the increase in oxidative stress and inflammation during this period is also negatively associated with a reduction in liver functionality and measurement of app can provide a useful tool to assess liver function as well as inflammation bertoni and trevisi during the periparturient period aa metabolism is also altered with moderate carcass protein losses reported even when animals are fed to their predicted metabolizable protein requirements bell et a0 al additionally circulating aa concentrations change dramatically with favorable circulating profiles of many aa not being restored until d postpartum zhou et a0al 2016b furthermore the total concentration of aa in plasma reaches a nadir around day postpartum zhou et a0al 2016b which corresponds to a peak in total disease incidence during early lactation ingvartsen this decrease in circulating aa is likely associated with the increased use of aa for gluconeogenesis as well as for hepatic production of app thus it is important to investigate how supplemental aa during the periparturient period may modulate metabolism and immune responses to promote production and reduce susceptibility to diseasegeneral overview of the immune a0systemthe immune system consists of both the adaptive and innate immune responses which are linked together through signaling molecules such as cytokines the innate immune system is the first line of defense and includes physical barriers such as epithelial cell layers that express tight cell junctions and the mucus layer of the respiratory gastrointestinal and genitourinary tracts chaplin cells involved in the innate immune response include macrophages polymorphonuclear neutrophils pmn dendritic cells mast cells eosinophils natural killer cells and natural killer t cells turvey and broide the focus of the present review is on the effects of aa on phagocytic cells that is cells that engulf and kill such as macrophages and pmn macrophages not only phagocytose invading pathogens but also produce cytokines such as ils and tnfα which initiate innate and adaptive immune responses and recruit pmn to the site of infection chaplin the adaptive immune response consists of t lymphocytes b lymphocytes and humoral factors marshall et a0 al there are two types of t lymphocytes cytotoxic t cells cd8 cells and thelper th cells cd4 cells marshall et a0al cytotoxic t cells detect and eliminate infected cells while th cells produce ils and interferonγ ingvartsen and moyes the b lymphocytes are cells that produce antibodies that bind to antigens on the surface of pathogens to mark them for destruction marshall et a0al 0ccoleman et a0al s177aa immune function and oxidative a0stressduring the periparturient period the metabolic status is associated with the inflammatory regulation of peripheral blood mononuclear cells with a more pronounced inflammatory response in those cells during the nnb immediately postpartum agrawal et a0 al mann et a0 al this period is also associated with altered signaling of nutrientsensing kinases in immune cells such as the protein kinase b akt and mechanistic target of rapamycin mtor pathway which may modulate cytokine production mann et a0al aa can directly and indirectly alter the immune system besides being used in energy metabolism reactions and the synthesis of proteins aa are critical for the synthesis of other functional molecules such as the antioxidants gsh and taurine no histamine and hydrogen peroxide li et a0 al thus this section of the review will focus on the role that aa play in modulating immune function and oxidative stress in dairy cows during the periparturient period a a0special focus will be placed on aa involved in 1carbon metabolism as this represents an interconnected route through which aa could impact molecular events such as epigenetic regulation protein synthesis via mtor energy metabolism and antioxidant synthesis a a0summary of studies investigating the effects of aa on immune function and oxidative stress in dairy cows is provided in table a0 a a0summary model of how aa might alter immune function and oxidant status is depicted in figure a0methioninemethionine not only is essential for protein synthesis but also serves as a functional nutrient that is needed for the production of the antioxidants gsh and taurine atmaca and provision of methyl groups finkelstein these features are exemplified by the central role of met in 1carbon metabolism figure a0 in these pathways met is used to synthesize sadenosyl methionine sam which can be used to methylate dna and to support phosphatidylcholine pc and carnitine synthesis for fatty acid metabolism vance et a0al during the periparturient period triacyglycerol tag accumulate in the liver leading to mitochondrial dysfunction inflammation and reduced liver function li et a0al du et a0al pc is the main phospholipid component of verylowdensity lipoproteins vldl vance thus enhancing pc synthesis through greater met supply may help improve vldl synthesis and reduce hepatic tag accumulationas part of 1carbon metabolism homocysteine can be remethylated to met using betaine or folate as methyl donors bhmt and via betaine homocysteine methyltransferase 5methyltetrahydrofolatehomocysteine methyltransferase mtr respectively homocysteine is also used to synthesize cystathionine via cystathionine synthase cbs in the first reaction of the transsulfuration pathway banerjee et a0 al cystathionine is used to make cys which is utilized to synthesize the antioxidants taurine or gsh cbs is allosterically figure interrelationships among components of the 1carbon metabolism pathway methionine cycle folate cycle and transsulfuration pathway 5mthf 5methyltetrahydrofolate amd1 adenosylmethionine decarboxylase arg arginase b12 cobalamin b2 riboflavin b6 pyridoxal ²phosphate cbs cystathionine betasynthase dcsam decarboxylated sadenosylmethionine dhfr dihydrofolate reductase dnmt dna methyltransferase dtmp thymidine monophosphate dump deoxyuridine monophosphate ftcd formimidoyltransferase cyclodeaminase gnmt glycine nmethyltransferase mat methionine adenosyltransferase mthfr methylenetetrahydrofolate reductase mtrr 5methyltetrahydrofolatehomocysteine methyltransferase reductase odc1 ornithine decarboxylase pemt phosphatidylethanolamine nmethyltransferase sahh sadenosylhomocysteine hydrolase shmt serine hydroxymethyltransferase sms spermine synthase srm spermidine synthase tdh threonine dehydrogenase thf tetrahydrofolate tyms thymidylate synthetase 0cs178 of animal science vol no suppl activated by sam banerjee et a0 al therefore enhanced sam production with increased met supply can help enhance the flux of the transsulfuration pathway increasing taurine and gsh production to help reduce oxidative a0stressin dairy cattle low levels of serum met postpartum are associated with severe hepatic lipidosis shibano and kawamura and other than his met is the only aa for which net uptake by the liver increased pre and postpartum larsen and kristensen therefore enhancing postruminal met supply during the periparturient period is of interest to increase circulating concentrations of met for its functional roles in the body the work from dalbach et a0 al demonstrated that rumenprotected met rpm can be used to increase serum concentrations of met in the first 2wk postpartum which will enhance the availability of met for protein synthesis and metabolism via the 1carbon pathways in terms of production supplementation of met during the peripartal period concomitantly increases milk yield milk protein and milk fat soon after calving ordway et a0 al osorio et a0 al these responses are in large part driven by enhancing met availability and by the additional flux of met through the met cycle in the liver which consequently increases the production of downstream compounds such as sam pc gsh and taurine additionally work feeding rpm during the periparturient period has detected positive responses in maintaining consistent rates of dmi prepartum last d and faster and greater rates of dmi during the first to d after calving osorio et a0al zhou et a0 al 2016c batistel et a0 al the same milk production response was also observed when met was supplemented postpartum as the isopropyl ester of 2hydroxy4methylthiobutanoic acid stpierre and sylvester as described earlier the transient inflammatorylike status around parturition appears to be a œnormal aspect of the adaptations to lactation as cows approach parturition those with greater but still subclinical concentrations of circulating cytokines have greater inflammation and oxidative stress and lower liver function along with lower milk yield and lower postpartum dmi bertoni et a0 al in addition to their function in the immune system cytokines interferons and tnfα also elicit pathophysiological effects leading to œsickness table summary of studies in dairy cows investigating the effects of supplemental aa on immune function oxidative stress and inflammation tissuecellstreatmentmain outcomeplasmarpm for d prepartum and d postpartumimprovements in plasma biomarkers indicating reduced oxidative stress and inflammation and enhanced liver function increased neutrophil phagocytosis and oxidative burstreferencebatistel et a0al plasmaabomasal infusion of glu for first infusions of gln increased the abundance of cd4 tcells on day doepel et a0al d postpartumpostpartum and increased the abundance of monocytesmammary rpm for d prepartum and d methionine supply upregulated expression of genes involved in han et a0al glandplasmapostpartumantioxidant metabolism and increased activation of nfe2l2intravenous infusions of gln for glutamine infusion decreased plasma haptoglobin and increased jafari et a0al d postcalvinglpsbinding protein and saa subcutaneous rpm for d prepartum and d enhanced met supply increased mrna and protein abundance of liang et a0al 2019aadiposepmnlpostpartumenzymes related to gsh metabolismincubation with met andor supplemental met coupled with adequate choline enhanced gene lopreiato et a0al cholineexpression of pathogen recognition mechanisms methionine ameliorated the increased inflammation and oxidative stress observed when cells were incubated without choline plasma and protected gln for d postpartum increased total blood protein and albumin decreased plasma nemati et a0al milkwhole bloodrpm for d prepartum and d aspartate aminotransferase and milk somatic cell countincreased whole blood neutrophil phagocytosis on day osorio et a0al 2013apostpartumpostpartum with supplemental metliverrpm for d prepartum and d methionine supply altered flux through 1carbon metabolism via osorio et a0al 2014aliver and plasmaplasmapostpartumchanges in mrna to support antioxidant and met synthesisrpm for d prepartum and d methionine increased liver gsh and decreased concentrations of osorio et a0al 2014bpostpartum plasma biomarkers of inflammationrpm for d during midlactation increased proliferative ability of peripheral blood t lymphocytes soder and holden with supplemental metplasmarpm for d prepartum and increased antioxidant capacity of plasma and cd4cd8 t sun et a0al postpartumlymphocyte ratio with met supplywhole bloodrpm for d prepartum and d methionine damped the hyperresponse of il1 during an lps vailatiriboni et a0al plasmajugular infusion of arg and lps in arginine alleviated lpstriggered inflammation by decreasing il6 zhao et a0al 2018apostpartumchallenge through improvements in oxidative stressserumjugular infusion of arg and lps in infusion of arg promoted antioxidant mechanisms during lpszhao et a0al 2018bmidlactation cowsinducible nos and lpsbinding proteinmidlactation cowsrpm for d prepartum and d postpartumrpm for d prepartum and d triggered inflammation by increasing total antioxidant capacity and gsh peroxidase activity and decreasing malondialdehyde increased hepatic gsh and improved plasma biomarkers of liver function and inflammation with met neutrophil phagocytosis capacity and oxidative burst were also increased with metenhanced met supply increased mrna expression of genes zhou et a0al 2016azhou et a0al 2017bpostpartumassociated with pc and antioxidant synthesisrpm for d prepartum and d decreased expression of genes related to inflammation and zhou et a0al 2018bliver and plasmaliver pmnlpostpartumoxidative stress 0ccoleman et a0al s179figure the theoretical model of cellular aa utilization amp adenosine monophosphate atp adenosine triphosphate ctp cytidine triphosphate dttp deoxythymidine triphosphate gmp guanosine monophosphate imp inosine monophosphate no nitric oxide r5p ribose 5phosphate tca cycle tricarboxylic acid cycle ump uridine monophosphate utp uridine5²triphosphatebehaviors whose primary manifestation is satiety larson and dunn an example of this behavior in dairy cows is the reduction in dmi around calving in mice these cytokines have been shown to reduce meal size and duration as well as decrease meal frequency and prolong intermeal intervals platasalamán furthermore cytokines directly affect the hypothalamus il1 and ifn act directly and specifically on the glucosesensitive neurons in the brain œsatiety and œhunger sites platasalamán in addition to increases in dmi and milk production rpm supplementation during the periparturient period has been associated with positive health responses across four studies osorio et a0al 2014b sun et a0al zhou et a0al 2016a batistel et a0 al there have been consistent improvements in the concentrations of plasma biomarkers of inflammation where il1 and haptoglobin have decreased and albumin has increased summarized in table a0 improvements in biomarkers of oxidative stress have also been observed with enhanced met supply during the periparturient period in the study by batistel et a0al plasma concentrations of ferricreducing antioxidant power carotene tocopherol and total and reduced gsh were increased with rpm while rom were lower sun et a0al also observed an improvement in blood antioxidant status with rpm increasing total antioxidant capacity glutathione peroxidase gpx activity and vitamin e a0a a0similar effect was observed in liver tissue by osorio et a0 al 2014b where cows fed rpm had greater hepatic concentrations of total and reduced a0gshfrom a mechanistic standpoint changes in the mrna abundance of sadenosyl homocysteine hydrolase sahh mtr sod1 glutamate cysteine ligase catalytic gclc subunit and dna methyltransferase 3a suggested alterations in flux through 1carbon metabolism osorio et a0al 2014a importantly sahh the enzyme that makes homocysteine from sadenosylhomocysteine was upregulated with met which would support a supply of homocysteine to be used for antioxidant and met synthesis furthermore in the study by zhou et a0al 2016a greater met supply compared with rumenprotected choline increased antioxidant concentration in liver tissue despite a lower concentration of pc those responses were due to the greater abundance of phosphatidylethanolamine methyltransferase the enzyme that utilizes sam and phosphatidylethanolamine to make pc and cbs zhou et a0al 2017b additionally enhanced met supply during the periparturient period was observed to increase mrna and protein abundance of enzymes related to gsh metabolism in subcutaneous adipose tissue suggesting greater activation of those pathways liang et a0al 2019a a a0greater dietary supply of choline did not change the mrna abundance of bhmt and mtr in cows zhou et a0al 2017ba positive effect of met supplementation on mammary gland antioxidant mechanisms was observed by han et a0 al mrna abundance of gpx1 gclc glutamate cysteine ligase gcl modifier subunit malic enzyme ferrochelatase and ferritin heavy chain and genes involved in gsh and iron metabolism were upregulated protein abundance of phosphorylated nuclear factor erythroid 2like2 nfe2l2total nfe2l2 was also increased han et a0al nfe2l2is a regulator of transcription of antioxidant genes hence an increase in phosphorylated nfe2l2 suggested greater activation of antioxidant systems and is likely one of the mechanisms behind the changes in mrna abundance lastly across studies there has also been a consistent improvement in the concentrations of plasma biomarkers of liver function such as increases in paraoxonase and cholesterol with rpm osorio et a0al 2014b zhou et a0al 2016a batistel et a0 al which is likely linked to the reduction in inflammation and oxidative stress thus the consistent changes across studies in metabolites and plasma biomarkers as well as mrna abundance across tissues indicate that enhanced met supply during the periparturient period reduces oxidative stress and inflammation however more work is needed to verify the exact mechanisms behind the observed changes 0cs180 of animal science vol no suppl enhanced postruminal supply in the form of rpm during the periparturient period has been associated with improvements in immune cell function when rpm was provided for d prepartum and d postpartum whole blood neutrophil phagocytosis was increased compared with control cows at d postpartum osorio et a0 al in the study by zhou et a0 al 2016c rpm supplementation from day ˆ’ prepartum to day postpartum increased neutrophil phagocytosis capacity and oxidative burst activity zhou et a0 al 2016a this same improvement in neutrophil immune function was observed when rpm was supplied from day ˆ’ prepartum to day postpartum batistel et a0 al furthermore an increase in the proliferative ability of peripheral blood t lymphocytes was observed when rpm was supplemented to midlactation cows soder and holden zhou et a0 al 2018b isolated polymorphonuclear leukocytes pmnl and observed lower abundance of genes related to inflammation il1b tlr2 nfκb and stat3 and oxidative stress cbs gpx1 glutathione synthase [gss] and sod2 as well as an increase in plasma taurine with supplemental met suggesting a better redox and inflammatory status of those cells additionally those same cows were used for an ex vivo whole blood challenge with lipopolysaccharide lps to further investigate immune cell responses during this challenge a table summary of additional beneficial effects of feeding rpm during the transition period and early lactationbiomarkerresponse12sitebiological functionmetabolism carnitine cholesterolonecarbon metabolism cystathionine betasynthase activity cystathionine cystine homocysteineinflammation il1beta haptoglobin albumin oxidative stress rom†‘†‘†‘†‘†‘†‘†‘†“†“†“†‘†‘liveroxidation of fatty acidsplasmalipoprotein metabolismliverplasmaplasmaplasmaantioxidant synthesish2s biosynthesis redox statusredox statusmethylation reactionsplasmaproinflammatory cytokineplasmainflammation signalplasmaacutephase response†”†“plasmaperoxides gsh taurine antioxidant capacity paraoxonase†‘†‘†”†‘†”†‘†‘†‘superoxide ohradicalsliver blood antioxidantantioxidantplasmaplasmatotal antioxidants in bloodplasmaantioxidant enzyme†‘ beneficial increase †“ beneficial decrease †” no change in concentration2relative to a control or rumenprotected choline supplemented diet osorio et a0al 2014b zhou et a0al 2016a sun et a0al batistel et a0al vailatiriboni et a0al hyperresponse in il1 was observed around parturition which likely arose from oxidative stress vailatiriboni et a0 al however rpm supplementation dampened this hyperresponse likely through improvements in oxidative stress vailatiriboni et a0al the recent work by lopreiato et a0 al investigated the effects of incubating bovine pmnl with met andor choline and observed that supplemental met coupled with adequate choline enhanced gene expression of tlr2 and lselectin sell which are pathogen recognition mechanisms in the same experiment cells incubated without choline had greater mrna abundance of il1b il6 il10 and myeloperoxidase mpo glutathione reductase gsr gss cystathionine gammalyase cth and cysteine sulfinic acid decarboxylase csad indicating greater inflammation and oxidative stress this effect however was ameliorated by supplementing additional met lopreiato et a0 al thus the increased dmi and milk production observed when feeding rpm can be partly explained by a reduction in inflammation as it directly at the hepatic level and by dampening the immune cell overresponse and indirectly reducing oxidative stress decreases circulating proinflammatory cytokinesenhanced met supply during the periparturient period has also been associated with changes in mtor signaling mtor is a serinethreonine kinase that plays a central role in integrating environmental cues from growth factors nutrients particularly aa and energy powell et a0al in dairy cattle mtor has traditionally been studied in the context of its role in regulating protein synthesis however work from humans and nonruminant species has indicated that mtor is an important regulator of immune responses powell et a0 al jones and pearce when activated by aa mtor directs an activation of anabolic metabolism which allows growth proliferation and development this makes the activation of mtor in immune cells particularly important for maintaining proliferation and without proper activation cells may enter periods of growth arrest jones and pearce methionine specifically may interact with mtor via the production of sam specifically sam can bind to sadenosylmethionine sensor upstream of mtor samtor a protein that inhibits mtor complex mtorc1 by interacting with gtpaseactivating protein activity towards rags gator1 gu et a0al when sam binds to samtor it inhibits the association of samtor and gator1 allowing mtorc1 to be activated gu et a0al to our knowledge there is only one study investigating the expression of mtor signaling proteins in immune cells in dairy cattle and work is also lacking in nonruminant species in periparturient cows the activation of aktmtor signaling in immune cells was reduced postpartum compared with prepartum mann et a0al importantly agrawal et a0al also identified the expression of aa transporters and the kyoto encyclopedia of genes and genomes kegg pathways related to 1carbon metabolism and mtor in pmnl from peripartal cows a a0 list of these transporters and kegg pathways related to aa and 1carbon metabolism are summarized in table a0 together these studies support the potential importance of aa for nutrient signaling in immune a0cellsrecent work indicating that enhanced met supply activates mtor signaling in the mammary gland supports its role in enhancing protein synthesis for example in vitro enhancing met supply to immortalized bovine mammary epithelial cells bmec by varying the ratio of lys to met increased the concentration and utilization of essential aa particularly branchedchain aa bcaa dong et a0 al this change 0cwas potentially mediated by alterations in aa transporters controlled by mtor dong et a0al other studies with bmec have also revealed a potential for greater mtor activation when met supply is enhanced nan et a0al sala ma et a0al in vivo the effects of supplemental met during the periparturient period on mtor signaling were explored using cows from the study by batistel et a0al in the mammary gland cows receiving rpm had lower protein abundance of total mtor and phosphorylated mtor pmtor compared with control cows on day postpartum but the ratio of pmtortotal mtor was not different suggesting that there was no difference in mtor activation between treatments ma et a0 al however changes in aa transporters and insulin signaling indicated that insulin sensitivity of the mammary gland was enhanced with supplemental met ma et a0al in subcutaneous adipose tissue the mrna and protein abundance of some aa transporters and pmtor were upregulated with enhanced met supply while changes in insulin signaling and plasma glucose also indicated that met helped improve insulin sensitivity liang et a0 al 2019b thus enhancing met supply during the periparturient period may lead to mtor activation in immune cells as well as improved nutrient uptake which could help to support proliferation and development additional work in ruminants and nonruminants is needed to understand whether met modulates mtor signaling in immune cellscysteinecysteine can be synthesized endogenously from homocysteine as described earlier and is needed to synthesize gsh and taurine gsh is synthesized via two enzymes gcl and gss lushchak to synthesize taurine cysteine sulfinic acid is first synthesized from cysteine by cysteine dioxygenase and can then be utilized by csad to produce taurine park et a0al dietary cys has been explored as a way to improve health in nonruminant species and humans under a variety of conditions including type2 diabetes cardiovascular disease and liver disease to name a few yin et a0 al across these studies increased dietary cys increased the concentrati

cancer7569

You are a medical assistant specializing in cancer diagnosis. Based on the given information, you must classify the cancer type as one of the following three options: 'Thyroid_Cancer', 'Colon_Cancer', or 'Lung_Cancer'. Please choose the most appropriate category based on the provided text. Do not include any other cancer types or responses outside of these three categories. Text: the periparturient period is one of the most challenging periods in dairy cows and encompasses the a0wk prior to and a0wk after parturition the nutrient requirements of dairy cows change greatly during this time largely due to the exponential growth of the gravid uterus and fetus followed by the demands of lactation nrc inflammation oxidative stress and adipose tissue mobilization lead to a reduction in dry matter intake dmi during the periparturient period this reduction in dmi leads to a negative nutrient balance nnb with a shortfall in the nutrient availability for the cow and fetus ingvartsen and andersen additionally this reduction in dmi also increases the risk of metabolic ketosis fatty liver milk fever and immunerelated disorders the risk of these diseases poor reproduction and low efficiency is greatly impacted by the degree and length of time during which these systems metabolism and immune response remain out of balance loor et a0al 2013a much of the research over the last decade have examined these biological interactions to identify the mechanisms behind the metabolic physiologic and immune adaptations that occur during the periparturient period loor et a0al 2013a 2013b roche et a0al bradford et a0al it is now known that nutrients such as amino acids aa serve functional roles outside of their use as building blocks for proteins and have immunomodulatory properties and interact through common biochemical pathways eg 1carbon metabolism figure a0 this concept has been well explored in nonruminant species li et a0 al sikalidis with nutritional management during the periparturient period continuing to be an active area of research it is important to develop a system understanding the potential immunometabolic role that dietary aa may play during this period thus the objective of this review is to provide an overview of physiological adaptations during the periparturient period the immune system and methods to assess immune function and oxidative stress this will be followed by a more specific discussion of the immunometabolic roles of specific aa and their potential effects in dairy cow during the periparturient period the potential effects of enhanced aa supply during the preweaning period will also be discussed brieflybiological adaptations in the transition cow”a brief overviewduring the nnb associated with the periparturient period biological mechanisms coordinate the mobilization of body reserves in order to support fetal growth and milk production bauman and currie insulin concentrations are reduced and the response of hormonesensitive lipase in adipose tissue eg low insulin high growth hormone and catecholamines or high glucocorticoid concentrations is greater to facilitate lipid mobilization this periparturient period is also characterized by a state of inflammation encompassing an increase in hepatic production of positive acutephase proteins app such as haptoglobin and serum amyloid a a0saa and a decrease in the production ingvartsen the authors published by oxford university press on behalf of the american society of animal sciencethis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly cited for commercial reuse please contact spermissionsoupcoms175 0cs176 of animal science vol no suppl abbreviationsaa akt app asct2 bcaa bcat bcka bhmt bmec bsa cbs csad cth dmi gator1 gcl gclc gpx gsh gsr gss il inos kegg klh lps mpo mtor mtorc1 mtr nfe2l2 nfκb nnb nos pc pmn pmnl pmtor rns rom ros rpm saa sahh sam samtor sell shmt slc1a1 slc1a5 sod stat tag th tlr tnfα vldl amino acidsprotein kinase bacutephase proteinsalanineserinecysteine transporter branchedchain amino acidsbranchedchain amino transferasesbranchedchain αketoacidsbetaine homocysteine methyltransferasebovine mammary epithelial cellsbovine serum albumincystathionine synthasecysteine sulfinic acid decarboxylasecystathionine gammalyasedry matter intakegtpaseactivating protein activity towards rags glutamate cysteine ligaseglutamate cysteine ligase catalyticglutathione peroxidaseglutathioneglutathione reductaseglutathione synthaseinterleukininducible noskyoto encyclopedia of genes and genomeskeyhole limpet hemocyaninlipopolysaccharidemyeloperoxidasemechanistic target of rapamycinmtor complex 5methyltetrahydrofolatehomocysteine methyltransferasenuclear factor erythroid 2like2nuclear factor kappa bnegative nutrient balancenitric oxide synthasephosphatidylcholinepolymorphonuclear neutrophilspolymorphonuclear leukocytesphosphorylated mtorreactive nitrogen speciesreactive oxygen metabolitesreactive oxygen speciesrumenprotected metserum amyloid asadenosyl homocysteine hydrolasesadenosyl methioninesadenosylmethionine sensor upstream of mtorlselectinserine hydoxymethyltransferasesolute carrier family member solute carrier family member superoxide dismutasesignal transducer and activator of transcriptiontriacyglycerolthelpertolllike receptorstumor necrosis factorαverylowdensity lipoproteinsof negative app such as albumin bertoni et a0 al it has been well established that these responses are mediated by the proinflammatory cytokines interleukin il6 il1 and tumor necrosis factorα tnfα kindt et a0al additionally oxidative stress also occurs during this period and is driven by the imbalance between the production of reactive oxygen metabolites rom reactive nitrogen species rns and the neutralizing capacity of antioxidant mechanisms in tissues and blood some of the wellestablished cellular antioxidants include glutathione gsh taurine superoxide dismutase sod and vitamins a a0and e bernabucci et a0al when oxidative stress overwhelms cellular antioxidant capacity rom induce an inflammatory response that is controlled via changes in mrna abundance of transcription regulators eg signal transducer and activator of transcription [stat3] nuclear factorkappa b [nfκb] the increase in oxidative stress and inflammation during this period is also negatively associated with a reduction in liver functionality and measurement of app can provide a useful tool to assess liver function as well as inflammation bertoni and trevisi during the periparturient period aa metabolism is also altered with moderate carcass protein losses reported even when animals are fed to their predicted metabolizable protein requirements bell et a0 al additionally circulating aa concentrations change dramatically with favorable circulating profiles of many aa not being restored until d postpartum zhou et a0al 2016b furthermore the total concentration of aa in plasma reaches a nadir around day postpartum zhou et a0al 2016b which corresponds to a peak in total disease incidence during early lactation ingvartsen this decrease in circulating aa is likely associated with the increased use of aa for gluconeogenesis as well as for hepatic production of app thus it is important to investigate how supplemental aa during the periparturient period may modulate metabolism and immune responses to promote production and reduce susceptibility to diseasegeneral overview of the immune a0systemthe immune system consists of both the adaptive and innate immune responses which are linked together through signaling molecules such as cytokines the innate immune system is the first line of defense and includes physical barriers such as epithelial cell layers that express tight cell junctions and the mucus layer of the respiratory gastrointestinal and genitourinary tracts chaplin cells involved in the innate immune response include macrophages polymorphonuclear neutrophils pmn dendritic cells mast cells eosinophils natural killer cells and natural killer t cells turvey and broide the focus of the present review is on the effects of aa on phagocytic cells that is cells that engulf and kill such as macrophages and pmn macrophages not only phagocytose invading pathogens but also produce cytokines such as ils and tnfα which initiate innate and adaptive immune responses and recruit pmn to the site of infection chaplin the adaptive immune response consists of t lymphocytes b lymphocytes and humoral factors marshall et a0 al there are two types of t lymphocytes cytotoxic t cells cd8 cells and thelper th cells cd4 cells marshall et a0al cytotoxic t cells detect and eliminate infected cells while th cells produce ils and interferonγ ingvartsen and moyes the b lymphocytes are cells that produce antibodies that bind to antigens on the surface of pathogens to mark them for destruction marshall et a0al 0ccoleman et a0al s177aa immune function and oxidative a0stressduring the periparturient period the metabolic status is associated with the inflammatory regulation of peripheral blood mononuclear cells with a more pronounced inflammatory response in those cells during the nnb immediately postpartum agrawal et a0 al mann et a0 al this period is also associated with altered signaling of nutrientsensing kinases in immune cells such as the protein kinase b akt and mechanistic target of rapamycin mtor pathway which may modulate cytokine production mann et a0al aa can directly and indirectly alter the immune system besides being used in energy metabolism reactions and the synthesis of proteins aa are critical for the synthesis of other functional molecules such as the antioxidants gsh and taurine no histamine and hydrogen peroxide li et a0 al thus this section of the review will focus on the role that aa play in modulating immune function and oxidative stress in dairy cows during the periparturient period a a0special focus will be placed on aa involved in 1carbon metabolism as this represents an interconnected route through which aa could impact molecular events such as epigenetic regulation protein synthesis via mtor energy metabolism and antioxidant synthesis a a0summary of studies investigating the effects of aa on immune function and oxidative stress in dairy cows is provided in table a0 a a0summary model of how aa might alter immune function and oxidant status is depicted in figure a0methioninemethionine not only is essential for protein synthesis but also serves as a functional nutrient that is needed for the production of the antioxidants gsh and taurine atmaca and provision of methyl groups finkelstein these features are exemplified by the central role of met in 1carbon metabolism figure a0 in these pathways met is used to synthesize sadenosyl methionine sam which can be used to methylate dna and to support phosphatidylcholine pc and carnitine synthesis for fatty acid metabolism vance et a0al during the periparturient period triacyglycerol tag accumulate in the liver leading to mitochondrial dysfunction inflammation and reduced liver function li et a0al du et a0al pc is the main phospholipid component of verylowdensity lipoproteins vldl vance thus enhancing pc synthesis through greater met supply may help improve vldl synthesis and reduce hepatic tag accumulationas part of 1carbon metabolism homocysteine can be remethylated to met using betaine or folate as methyl donors bhmt and via betaine homocysteine methyltransferase 5methyltetrahydrofolatehomocysteine methyltransferase mtr respectively homocysteine is also used to synthesize cystathionine via cystathionine synthase cbs in the first reaction of the transsulfuration pathway banerjee et a0 al cystathionine is used to make cys which is utilized to synthesize the antioxidants taurine or gsh cbs is allosterically figure interrelationships among components of the 1carbon metabolism pathway methionine cycle folate cycle and transsulfuration pathway 5mthf 5methyltetrahydrofolate amd1 adenosylmethionine decarboxylase arg arginase b12 cobalamin b2 riboflavin b6 pyridoxal ²phosphate cbs cystathionine betasynthase dcsam decarboxylated sadenosylmethionine dhfr dihydrofolate reductase dnmt dna methyltransferase dtmp thymidine monophosphate dump deoxyuridine monophosphate ftcd formimidoyltransferase cyclodeaminase gnmt glycine nmethyltransferase mat methionine adenosyltransferase mthfr methylenetetrahydrofolate reductase mtrr 5methyltetrahydrofolatehomocysteine methyltransferase reductase odc1 ornithine decarboxylase pemt phosphatidylethanolamine nmethyltransferase sahh sadenosylhomocysteine hydrolase shmt serine hydroxymethyltransferase sms spermine synthase srm spermidine synthase tdh threonine dehydrogenase thf tetrahydrofolate tyms thymidylate synthetase 0cs178 of animal science vol no suppl activated by sam banerjee et a0 al therefore enhanced sam production with increased met supply can help enhance the flux of the transsulfuration pathway increasing taurine and gsh production to help reduce oxidative a0stressin dairy cattle low levels of serum met postpartum are associated with severe hepatic lipidosis shibano and kawamura and other than his met is the only aa for which net uptake by the liver increased pre and postpartum larsen and kristensen therefore enhancing postruminal met supply during the periparturient period is of interest to increase circulating concentrations of met for its functional roles in the body the work from dalbach et a0 al demonstrated that rumenprotected met rpm can be used to increase serum concentrations of met in the first 2wk postpartum which will enhance the availability of met for protein synthesis and metabolism via the 1carbon pathways in terms of production supplementation of met during the peripartal period concomitantly increases milk yield milk protein and milk fat soon after calving ordway et a0 al osorio et a0 al these responses are in large part driven by enhancing met availability and by the additional flux of met through the met cycle in the liver which consequently increases the production of downstream compounds such as sam pc gsh and taurine additionally work feeding rpm during the periparturient period has detected positive responses in maintaining consistent rates of dmi prepartum last d and faster and greater rates of dmi during the first to d after calving osorio et a0al zhou et a0 al 2016c batistel et a0 al the same milk production response was also observed when met was supplemented postpartum as the isopropyl ester of 2hydroxy4methylthiobutanoic acid stpierre and sylvester as described earlier the transient inflammatorylike status around parturition appears to be a œnormal aspect of the adaptations to lactation as cows approach parturition those with greater but still subclinical concentrations of circulating cytokines have greater inflammation and oxidative stress and lower liver function along with lower milk yield and lower postpartum dmi bertoni et a0 al in addition to their function in the immune system cytokines interferons and tnfα also elicit pathophysiological effects leading to œsickness table summary of studies in dairy cows investigating the effects of supplemental aa on immune function oxidative stress and inflammation tissuecellstreatmentmain outcomeplasmarpm for d prepartum and d postpartumimprovements in plasma biomarkers indicating reduced oxidative stress and inflammation and enhanced liver function increased neutrophil phagocytosis and oxidative burstreferencebatistel et a0al plasmaabomasal infusion of glu for first infusions of gln increased the abundance of cd4 tcells on day doepel et a0al d postpartumpostpartum and increased the abundance of monocytesmammary rpm for d prepartum and d methionine supply upregulated expression of genes involved in han et a0al glandplasmapostpartumantioxidant metabolism and increased activation of nfe2l2intravenous infusions of gln for glutamine infusion decreased plasma haptoglobin and increased jafari et a0al d postcalvinglpsbinding protein and saa subcutaneous rpm for d prepartum and d enhanced met supply increased mrna and protein abundance of liang et a0al 2019aadiposepmnlpostpartumenzymes related to gsh metabolismincubation with met andor supplemental met coupled with adequate choline enhanced gene lopreiato et a0al cholineexpression of pathogen recognition mechanisms methionine ameliorated the increased inflammation and oxidative stress observed when cells were incubated without choline plasma and protected gln for d postpartum increased total blood protein and albumin decreased plasma nemati et a0al milkwhole bloodrpm for d prepartum and d aspartate aminotransferase and milk somatic cell countincreased whole blood neutrophil phagocytosis on day osorio et a0al 2013apostpartumpostpartum with supplemental metliverrpm for d prepartum and d methionine supply altered flux through 1carbon metabolism via osorio et a0al 2014aliver and plasmaplasmapostpartumchanges in mrna to support antioxidant and met synthesisrpm for d prepartum and d methionine increased liver gsh and decreased concentrations of osorio et a0al 2014bpostpartum plasma biomarkers of inflammationrpm for d during midlactation increased proliferative ability of peripheral blood t lymphocytes soder and holden with supplemental metplasmarpm for d prepartum and increased antioxidant capacity of plasma and cd4cd8 t sun et a0al postpartumlymphocyte ratio with met supplywhole bloodrpm for d prepartum and d methionine damped the hyperresponse of il1 during an lps vailatiriboni et a0al plasmajugular infusion of arg and lps in arginine alleviated lpstriggered inflammation by decreasing il6 zhao et a0al 2018apostpartumchallenge through improvements in oxidative stressserumjugular infusion of arg and lps in infusion of arg promoted antioxidant mechanisms during lpszhao et a0al 2018bmidlactation cowsinducible nos and lpsbinding proteinmidlactation cowsrpm for d prepartum and d postpartumrpm for d prepartum and d triggered inflammation by increasing total antioxidant capacity and gsh peroxidase activity and decreasing malondialdehyde increased hepatic gsh and improved plasma biomarkers of liver function and inflammation with met neutrophil phagocytosis capacity and oxidative burst were also increased with metenhanced met supply increased mrna expression of genes zhou et a0al 2016azhou et a0al 2017bpostpartumassociated with pc and antioxidant synthesisrpm for d prepartum and d decreased expression of genes related to inflammation and zhou et a0al 2018bliver and plasmaliver pmnlpostpartumoxidative stress 0ccoleman et a0al s179figure the theoretical model of cellular aa utilization amp adenosine monophosphate atp adenosine triphosphate ctp cytidine triphosphate dttp deoxythymidine triphosphate gmp guanosine monophosphate imp inosine monophosphate no nitric oxide r5p ribose 5phosphate tca cycle tricarboxylic acid cycle ump uridine monophosphate utp uridine5²triphosphatebehaviors whose primary manifestation is satiety larson and dunn an example of this behavior in dairy cows is the reduction in dmi around calving in mice these cytokines have been shown to reduce meal size and duration as well as decrease meal frequency and prolong intermeal intervals platasalamán furthermore cytokines directly affect the hypothalamus il1 and ifn act directly and specifically on the glucosesensitive neurons in the brain œsatiety and œhunger sites platasalamán in addition to increases in dmi and milk production rpm supplementation during the periparturient period has been associated with positive health responses across four studies osorio et a0al 2014b sun et a0al zhou et a0al 2016a batistel et a0 al there have been consistent improvements in the concentrations of plasma biomarkers of inflammation where il1 and haptoglobin have decreased and albumin has increased summarized in table a0 improvements in biomarkers of oxidative stress have also been observed with enhanced met supply during the periparturient period in the study by batistel et a0al plasma concentrations of ferricreducing antioxidant power carotene tocopherol and total and reduced gsh were increased with rpm while rom were lower sun et a0al also observed an improvement in blood antioxidant status with rpm increasing total antioxidant capacity glutathione peroxidase gpx activity and vitamin e a0a a0similar effect was observed in liver tissue by osorio et a0 al 2014b where cows fed rpm had greater hepatic concentrations of total and reduced a0gshfrom a mechanistic standpoint changes in the mrna abundance of sadenosyl homocysteine hydrolase sahh mtr sod1 glutamate cysteine ligase catalytic gclc subunit and dna methyltransferase 3a suggested alterations in flux through 1carbon metabolism osorio et a0al 2014a importantly sahh the enzyme that makes homocysteine from sadenosylhomocysteine was upregulated with met which would support a supply of homocysteine to be used for antioxidant and met synthesis furthermore in the study by zhou et a0al 2016a greater met supply compared with rumenprotected choline increased antioxidant concentration in liver tissue despite a lower concentration of pc those responses were due to the greater abundance of phosphatidylethanolamine methyltransferase the enzyme that utilizes sam and phosphatidylethanolamine to make pc and cbs zhou et a0al 2017b additionally enhanced met supply during the periparturient period was observed to increase mrna and protein abundance of enzymes related to gsh metabolism in subcutaneous adipose tissue suggesting greater activation of those pathways liang et a0al 2019a a a0greater dietary supply of choline did not change the mrna abundance of bhmt and mtr in cows zhou et a0al 2017ba positive effect of met supplementation on mammary gland antioxidant mechanisms was observed by han et a0 al mrna abundance of gpx1 gclc glutamate cysteine ligase gcl modifier subunit malic enzyme ferrochelatase and ferritin heavy chain and genes involved in gsh and iron metabolism were upregulated protein abundance of phosphorylated nuclear factor erythroid 2like2 nfe2l2total nfe2l2 was also increased han et a0al nfe2l2is a regulator of transcription of antioxidant genes hence an increase in phosphorylated nfe2l2 suggested greater activation of antioxidant systems and is likely one of the mechanisms behind the changes in mrna abundance lastly across studies there has also been a consistent improvement in the concentrations of plasma biomarkers of liver function such as increases in paraoxonase and cholesterol with rpm osorio et a0al 2014b zhou et a0al 2016a batistel et a0 al which is likely linked to the reduction in inflammation and oxidative stress thus the consistent changes across studies in metabolites and plasma biomarkers as well as mrna abundance across tissues indicate that enhanced met supply during the periparturient period reduces oxidative stress and inflammation however more work is needed to verify the exact mechanisms behind the observed changes 0cs180 of animal science vol no suppl enhanced postruminal supply in the form of rpm during the periparturient period has been associated with improvements in immune cell function when rpm was provided for d prepartum and d postpartum whole blood neutrophil phagocytosis was increased compared with control cows at d postpartum osorio et a0 al in the study by zhou et a0 al 2016c rpm supplementation from day ˆ’ prepartum to day postpartum increased neutrophil phagocytosis capacity and oxidative burst activity zhou et a0 al 2016a this same improvement in neutrophil immune function was observed when rpm was supplied from day ˆ’ prepartum to day postpartum batistel et a0 al furthermore an increase in the proliferative ability of peripheral blood t lymphocytes was observed when rpm was supplemented to midlactation cows soder and holden zhou et a0 al 2018b isolated polymorphonuclear leukocytes pmnl and observed lower abundance of genes related to inflammation il1b tlr2 nfκb and stat3 and oxidative stress cbs gpx1 glutathione synthase [gss] and sod2 as well as an increase in plasma taurine with supplemental met suggesting a better redox and inflammatory status of those cells additionally those same cows were used for an ex vivo whole blood challenge with lipopolysaccharide lps to further investigate immune cell responses during this challenge a table summary of additional beneficial effects of feeding rpm during the transition period and early lactationbiomarkerresponse12sitebiological functionmetabolism carnitine cholesterolonecarbon metabolism cystathionine betasynthase activity cystathionine cystine homocysteineinflammation il1beta haptoglobin albumin oxidative stress rom†‘†‘†‘†‘†‘†‘†‘†“†“†“†‘†‘liveroxidation of fatty acidsplasmalipoprotein metabolismliverplasmaplasmaplasmaantioxidant synthesish2s biosynthesis redox statusredox statusmethylation reactionsplasmaproinflammatory cytokineplasmainflammation signalplasmaacutephase response†”†“plasmaperoxides gsh taurine antioxidant capacity paraoxonase†‘†‘†”†‘†”†‘†‘†‘superoxide ohradicalsliver blood antioxidantantioxidantplasmaplasmatotal antioxidants in bloodplasmaantioxidant enzyme†‘ beneficial increase †“ beneficial decrease †” no change in concentration2relative to a control or rumenprotected choline supplemented diet osorio et a0al 2014b zhou et a0al 2016a sun et a0al batistel et a0al vailatiriboni et a0al hyperresponse in il1 was observed around parturition which likely arose from oxidative stress vailatiriboni et a0 al however rpm supplementation dampened this hyperresponse likely through improvements in oxidative stress vailatiriboni et a0al the recent work by lopreiato et a0 al investigated the effects of incubating bovine pmnl with met andor choline and observed that supplemental met coupled with adequate choline enhanced gene expression of tlr2 and lselectin sell which are pathogen recognition mechanisms in the same experiment cells incubated without choline had greater mrna abundance of il1b il6 il10 and myeloperoxidase mpo glutathione reductase gsr gss cystathionine gammalyase cth and cysteine sulfinic acid decarboxylase csad indicating greater inflammation and oxidative stress this effect however was ameliorated by supplementing additional met lopreiato et a0 al thus the increased dmi and milk production observed when feeding rpm can be partly explained by a reduction in inflammation as it directly at the hepatic level and by dampening the immune cell overresponse and indirectly reducing oxidative stress decreases circulating proinflammatory cytokinesenhanced met supply during the periparturient period has also been associated with changes in mtor signaling mtor is a serinethreonine kinase that plays a central role in integrating environmental cues from growth factors nutrients particularly aa and energy powell et a0al in dairy cattle mtor has traditionally been studied in the context of its role in regulating protein synthesis however work from humans and nonruminant species has indicated that mtor is an important regulator of immune responses powell et a0 al jones and pearce when activated by aa mtor directs an activation of anabolic metabolism which allows growth proliferation and development this makes the activation of mtor in immune cells particularly important for maintaining proliferation and without proper activation cells may enter periods of growth arrest jones and pearce methionine specifically may interact with mtor via the production of sam specifically sam can bind to sadenosylmethionine sensor upstream of mtor samtor a protein that inhibits mtor complex mtorc1 by interacting with gtpaseactivating protein activity towards rags gator1 gu et a0al when sam binds to samtor it inhibits the association of samtor and gator1 allowing mtorc1 to be activated gu et a0al to our knowledge there is only one study investigating the expression of mtor signaling proteins in immune cells in dairy cattle and work is also lacking in nonruminant species in periparturient cows the activation of aktmtor signaling in immune cells was reduced postpartum compared with prepartum mann et a0al importantly agrawal et a0al also identified the expression of aa transporters and the kyoto encyclopedia of genes and genomes kegg pathways related to 1carbon metabolism and mtor in pmnl from peripartal cows a a0 list of these transporters and kegg pathways related to aa and 1carbon metabolism are summarized in table a0 together these studies support the potential importance of aa for nutrient signaling in immune a0cellsrecent work indicating that enhanced met supply activates mtor signaling in the mammary gland supports its role in enhancing protein synthesis for example in vitro enhancing met supply to immortalized bovine mammary epithelial cells bmec by varying the ratio of lys to met increased the concentration and utilization of essential aa particularly branchedchain aa bcaa dong et a0 al this change 0cwas potentially mediated by alterations in aa transporters controlled by mtor dong et a0al other studies with bmec have also revealed a potential for greater mtor activation when met supply is enhanced nan et a0al sala ma et a0al in vivo the effects of supplemental met during the periparturient period on mtor signaling were explored using cows from the study by batistel et a0al in the mammary gland cows receiving rpm had lower protein abundance of total mtor and phosphorylated mtor pmtor compared with control cows on day postpartum but the ratio of pmtortotal mtor was not different suggesting that there was no difference in mtor activation between treatments ma et a0 al however changes in aa transporters and insulin signaling indicated that insulin sensitivity of the mammary gland was enhanced with supplemental met ma et a0al in subcutaneous adipose tissue the mrna and protein abundance of some aa transporters and pmtor were upregulated with enhanced met supply while changes in insulin signaling and plasma glucose also indicated that met helped improve insulin sensitivity liang et a0 al 2019b thus enhancing met supply during the periparturient period may lead to mtor activation in immune cells as well as improved nutrient uptake which could help to support proliferation and development additional work in ruminants and nonruminants is needed to understand whether met modulates mtor signaling in immune cellscysteinecysteine can be synthesized endogenously from homocysteine as described earlier and is needed to synthesize gsh and taurine gsh is synthesized via two enzymes gcl and gss lushchak to synthesize taurine cysteine sulfinic acid is first synthesized from cysteine by cysteine dioxygenase and can then be utilized by csad to produce taurine park et a0al dietary cys has been explored as a way to improve health in nonruminant species and humans under a variety of conditions including type2 diabetes cardiovascular disease and liver disease to name a few yin et a0 al across these studies increased dietary cys increased the concentrati Answer: