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Prioritization schemes usually highlight species-rich areas, where many species are at imminent risk of extinction. To be ecologically relevant these schemes should also include species biological traits into area-setting methods. Furthermore, in a world of limited funds for conservation, conservation action is constrained by land acquisition costs. Hence, including economic costs into conservation priorities can substantially improve their conservation cost-effectiveness.We examined four global conservation scenarios for carnivores based on the joint mapping of economic costs and species biological traits. These scenarios identify the most cost-effective priority sets of ecoregions, indicating best investment opportunities for safeguarding every carnivore species, and also establish priority sets that can maximize species representation in areas harboring highly vulnerable species. We compared these results with a scenario that minimizes the total number of ecoregions required for conserving all species, irrespective of other factors. We found that cost-effective conservation investments should focus on 41 ecoregions highlighted in the scenario that consider simultaneously both ecoregion vulnerability and economic costs of land acquisition. Ecoregions included in priority sets under these criteria should yield best returns of investments since they harbor species with high extinction risk and have lower mean land cost.in-situ biodiversity maintenance strategy.Our study highlights ecoregions of particular importance for the conservation of the world's carnivores defining global conservation priorities in analyses that encompass socioeconomic and life-history factors. We consider the identification of a comprehensive priority-set of areas as a first step towards an Conservation assessment and planning aim to optimize the allocation of scarce conservation funds by prioritizing areas for protection et al.This happens because, albeit most of these templates prioritize irreplaceable areas, some are reactive, i.e. they usually attribute high importance to areas with the highest number of threatened or endemic species or where extensive habitat loss has already taken place et al.et al.Specifically for mammals of the order Carnivora (i.e. the carnivores), Cardillo The order Carnivora includes several major conservation icons, such as the tiger, and many other flagship, umbrella, keystone, and indicator species et al.In a world of limited conservation funds, prioritization of areas for conservation has often been limited by land acquisition In this paper, we used broad-scale biogeographical data of carnivore species distribution - occurrence in world ecoregions Carnivore species richness is especially high in southeast Asia, the Philippines, and central and southeast Africa . Other sUnder the minimum-ecoregion scenario, only 14 ecoregions occurred in all of the 100 optimal sets that represent each species at least once and thus have maximum irreplaceability . Such arIrreplaceability values of ecoregions selected in the cost-effective scenario were similar to those in the minimum-ecoregion set. Sixteen ecoregions occurred in all optimal solutions for this scenario: these are located in central Africa, and in certain Neotropical regions, such as the Valdivian temperate forests, the Yucatán moist forests and the Florida Everglades . EcoregiOnly 13 ecoregions were included in all optimal solutions for the highly vulnerable scenario for global carnivore conservation . These eFinally, the scenario seeking to simultaneously maximize vulnerability across included carnivore species while minimizing land acquisition costs had 15 eThe minimum-ecoregion scenario needed 41 ecoregions to represent all carnivore species. These areas are mainly concentrated in Africa . In the The mean predicted population density in 2015 was higher in the highly vulnerable conservation scenario . The minRecently, several studies have portrayed priority areas for conservation of various taxonomic groups at different spatial scales et al.A growing body of evidence indicates that large-bodied species, with sizeable home ranges that occur at low densities and feed at higher trophic levels, are more prone to local extinction in habitat fragments et al.et al.The disparity in economic cost among ecoregions means that there is potential for great benefit in seeking efficient financial investments The priority sets identified in this study complement and lend support to other priority-setting frameworks The necessity of developing conservation action at the landscape level – by itself or combined with broad-scale actions Protected areas are the keystone of current conservation strategies. Our results showed that mean percentage of protected area in ecoregions sets selected by different conservation scenarios vary between 14 to 17%. However, there is also great variation among individual ecoregions attaining 38% of protection whereas others have none. We should highlight the relative high proportion (>0.56) of area still available for conservation in the our combined set – which, coupled with the lowest estimate of population density in 2015, may offer concrete opportunities for designing and establishing protected areas in several key regions. Note that our analyses include average estimated land costs and human population density as socioeconomic factors, but there are clearly other cultural, economic, and political concerns which affect such policies. Furthermore, conservation implementation and outcomes are also affected by many complex interacting socioeconomic forces like those related to governance, institutional capacity, and dynamic markets.in-situ biodiversity maintenance strategy, which is part of a much more complex process of policy negotiation and implementation To conclude, we must acknowledge that prioritization analyses such as these ones should be considered more indicative than prescriptive. It should be considered by conservation planners as a rapid and coarse assessment of potential costs in achieving a particular conservation goal We used the WWF hierarchical classification of ecoregions et al.et al.For each species, we obtained five biological variables used by Purvis et al.−2 were 50.6±13.5 (mean±SE) times national mean recurrent costs km−2 y−1]. Following Underwood et al.et al.et al.'s Balmford http://devdata.worldbank.org/wdi2006/contents/Section4.htm). As the PPP term is the PPP conversion factor divided by the exchange rate, we calculated the area-weighted average after determining the costs for each country to allow the inclusion of ecoregions that span multiple countries.Where GNI is Gross National Income, and PPP is Purchasing Power Parity. We excluded an additional term for the influence of reserve size on annual management cost 2), proportion of area protected (area under IUCN category I-VI), proportion of land-use area and proportion of land available for conservation [calculated as the total area – (land-use area+protected area)]. For our measures of Human Population Density (HPD), we used the Gridded Population of the World Finally, we obtained the following data for each ecoregion from WWF species.dat file), so that the final solution for each conservation scenario included all species.We set up four different conservation-planning scenarios all of them trying to resolve the optimization problem know as “the set-covering problem”. This mathematical selection method aims to represent all natural features (e.g. species or habitats) a given number of times in the smallest possible area, fewest numbers of sites, or with the lowest overall cost. Usually, analyses of this type have concentrated on the identification of the minimum set of sites required to represent all species at least once The site-selection procedure was limited by different constraints operating at each conservation scenario. The minimum-ecoregion scenario (A) was a reference “null” scenario aimed at the representation of all species in the minimum number of ecoregions in the world; threats to species and economic cost of each ecoregion were not considered. This means that the site-selection algorithm had to find solutions that represent all species minimizing the number of ecoregions. As several solutions were tied for number of ecoregions, we chose the one with the smallest total area. Thus, this scenario minimizes the number of ecoregions and the area targeted for high-priority conservation action.−2) of land acquisition. This means that the site-selection algorithm had to find solutions that represent all species minimizing the mean acquisition cost per ecoregion in the set of high priority for conservation. Therefore, we could find the “cheapest” scenario among several options for global carnivore conservation within a macroecological framework.In the cost-effective scenario (B), all species were represented whereas the economic cost of each ecoregion was equaled to the calculated cost to allow comparison and calculations among ecoregions. The z-scores representing each variable within ecoregions were summed, so that “very vulnerable” ecoregions for conservation are those that tend to aggregate carnivore species with larger bodies, higher interbirth intervals, longer gestation periods, lower litter sizes, and smaller local populations In highly vulnerable scenario (C), we aimed to find a minimum set of ecoregions containing a greater proportion of species whose biological traits predispose them to greater extinction risk. To produce this set, we attributed a vulnerability cost for each ecoregion based on the biological variables mentioned above. We calculated mean values for these species' traits within each ecoregion and identified ecoregions in which trait values were higher or lower than expected from a null-model of equiprobable species occurrence in all ecoregions, given the observed richness found in an ecoregion see . This waz-scores described above, including z-scores for land acquisition costs. This means that mean economic costs were calculated for each ecoregion and then were shuffled assuming that costs are not geographically structured, i.e. ecoregion costs could vary randomly. We calculated z-scores for land acquisition costs indicating if an ecoregion has costs that are higher or lower than expected by chance. In the combined scenario, ecoregion vulnerability and cost had the same weight; otherwise they could not be compared nor summed. These z-scores were summed and used with those indicating species extinction risk as used as constraints to produce this combined set. This approach has been called an “iterative-stage protocol” in multi-criteria conservation planning analyses Finally, we combined in the last scenario all variables related to species biological traits as well as economic costs associated with land acquisition within ecoregions to produce an optimal combined scenario (D) capable of representing all carnivore species while favoring the inclusion of ecoregions with maximum vulnerability and lower mean economic costs, whenever possible. To use both biological traits and economic costs as constraints in such prioritization analysis, we performed the same calculation of et al.Because often there are multiple combinations of ecoregions that satisfy the representation goal in each conservation scenario, we also integrated such alternative solutions into a map in which the relative importance of each ecoregion is indicated by its rate of recurrence in optimal subsets see . This is2), total number of ecoregions, mean land acquisition costs, proportion of protected area, proportion of land-use area, and proportion of available area for conservation, as well as their predicted HDP in 2015 – a measure of indirect conservation conflict sensu Cardillo et al.a posteriori comparison of conservation planning scenario has been called a “terminal-stage protocol” in multi-criteria conservation planning analyses The summary results of each systematic planning scenario were evaluated according to their total amount of area in optimal sets under a minimum-ecoregion scenario, a cost-effective scenario, a highly vulnerable scenario, and a combined scenario - along whit their irrepleceability values. Ecoregion area obtained from (1.33 MB XLS)Click here for additional data file.
To date, functional imaging studies of treatment-induced recovery from chronic aphasia only assessed short-term treatment effects after intensive language training. In the present study, we show with functional magnetic resonance imaging (fMRI), that different brain regions may be involved in immediate versus long-term success of intensive language training in chronic post-stroke aphasia patients.Eight patients were trained daily for three hours over a period of two weeks in naming of concrete objects. Prior to, immediately after, and eight months after training, patients overtly named trained and untrained objects during event-related fMRI. On average the patients improved from zero (at baseline) to 64.4% correct naming responses immediately after training, and treatment success remained highly stable at follow-up. Regression analyses showed that the degree of short-term treatment success was predicted by increased activity (compared to the pretraining scan) bilaterally in the hippocampal formation, the right precuneus and cingulate gyrus, and bilaterally in the fusiform gyri. A different picture emerged for long-term training success, which was best predicted by activity increases in the right-sided Wernicke's homologue and to a lesser degree in perilesional temporal areas.The results show for the first time that treatment-induced language recovery in the chronic stage after stroke is a dynamic process. Initially, brain regions involved in memory encoding, attention, and multimodal integration mediated treatment success. In contrast, long-term treatment success was predicted mainly by activity increases in the so-called 'classical' language regions. The results suggest that besides perilesional and homologue language-associated regions, functional integrity of domain-unspecific memory structures may be a prerequisite for successful (intensive) language interventions. Detailed clinical and demographic information of the patients are provided in Additional file Prior to training, all patients completed a baseline assessment consisting of a neurological examination, speech and language tests, and neuropsychological testing. All patients had relatively preserved cognitive abilities except for the language domain were examined twice within a two-week time interval on an overt naming task during fMRI to (a) establish the set of brain regions activated by healthy subjects during our naming task and (b) to control for effects of repeated testing .For the intensive anomia training, 50 concrete object names were individually selected for each patient ('trained objects'). Each trained object name had been named incorrectly at least twice during three baseline runs comprising a standardized set of 344 objects served as a control set for the post assessments (immediately and eight months post training). During the post assessments, the 50 trained and 30 untrained objects were presented thrice outside the scanner.Performance for a respective object name was classified as correct when at least two of the three runs were correct . By definition, the baselines for trained and untrained object names were zero percent correct (one or less correct runs out of three for each of the object names).2* weighted single shot echo-planar (EPI) sequence . A high-resolution T1-weighted anatomical image was acquired for anatomical identification and coregistration into the Talairach space.MRI data were acquired in a 3 Tesla whole body scanner (Philips Intera T30) with nominal gradient strength 30 mT/m and maximal slew rate 150 mT/m/ms. A circularly polarized transmit/receive birdcage head coil with a HF reflecting screen at the cranial end was used for spin excitation and resonance signal acquisition. Functional images were acquired using a TThe object-naming task comprised the visual presentation of photos of concrete objects during fMRI. Patients attended three identical fMRI sessions: The first was prior to the training (pre), the second directly after the training (post1), and the third eight months after completion of training (post2). Please note that patient P06 did not attend the 'two weeks'-appointment due to health problems unrelated to the study. The 'eight months'-assessment for P08 could not be conducted due to a required scanner hardware upgrade after his initial training.During each fMRI session, the participants had to overtly name the same 90 individually selected objects Figure . Thirty ). Control subjects also overtly named ninety visually presented objects during fMRI that were selected from our standardized set of 344 object pictures, based on a name agreement of > 80 percent in a different sample of healthy adults [Naming performance was assessed during the respective fMRI session by means of a special MR dual-channel communication system, equipped with software to subtract gradient noise from the patient microphone channel .Imaging data were analysed with SPM2 process . During process . The masData were analysed in the context of the general linear model, using the canonical hemodynamic response function (hrf) to model responses during the five seconds of overt object naming for each experimental condition. To account for movement artefacts, the estimated realignment parameters were entered in the design matrix as user specified regressors. Anatomical localization of activated brain regions was determined using the Talairach Demon .Planned contrasts-of-interest were calculated for each individual subject . These included the individual activity changes from pre to post training (post1-pre) or the comparison of the baseline scan with the third scan eight months after the training (post2-pre) for each object type separately . To detect areas with the greatest short- or long-term fMRI activity changes, which linearly correlated with behavioural improvement, we performed two linear regression analyses . Dependent variable was the individual activity change from pre to post training , respectively. The respective individual training success for trained items (post1 or post2) served as behavioural regressor.Technical difficulties, which could not be resolved by the fMRI microphone manufacturer, precluded the recording of the speech samples during scanning for later off-line analysis with application of a software filter to extract the speech signal from the scanner noise. Online assessment of patient's word production was not reliably possible because of the scanner background noise. Sound quality was, however, good enough to determine that all patients complied with task instructions and tried to name the objects presented during scanning.Thus, we decided to use the patients' performance outside of the scanner on the matched set of items as behavioural regressors. Even though intra-scanner scores are considered to be the most appropriate measure of performance for language production tasks , our extTo ensure that the results are specific for overt naming of the trained objects and not due to therapy-unrelated activity changes over time , we used the results of the respective regression analyses for the matched set of untrained objects as exclusive masks . The behavioural regressor here was the performance improvement of each patient from pretraining to post1 or post2, respectively. Thus, this analysis allows assessing which areas are specifically related to training-induced improved word-retrieval in the patient group.To determine the brain activity pattern during overt picture naming in the nine healthy subjects, we performed a one sample t-test using the individual fMRI activation patterns during overt object naming (first fMRI session only). The nine healthy control subjects showed overt naming related activity in brain regions mediating the core processes of picture naming for det. UnspeciZ-score > 3.00) for the patients because of the known stronger activations seen in chronic stroke patients compared to healthy controls in fMRI studies [Maximally activated voxels within significant clusters (p < 0.05) are reported at a voxel threshold of p < 0.05 and a minimum cluster size of 10 voxels if not otherwise stated. At least one voxel within a given cluster had to additionally survive a single-voxel statistical threshold of p < 0.001 , right inferior parietal (BA 40) regions, the right fusiform gyrus (BA 19), and the right caudate nucleus.increases of brain activity ('Session2 > Session1'), even at an uncorrected threshold of p < 0.05. Only activity decreases in the right inferior parietal lobe were observed from the first to the second assessment .In healthy control subjects, the comparison between the two fMRI sessions revealed no The present fMRI study determined for the first time both short- and long-term correlates of successful treatment-induced language recovery in chronic aphasia. We here show that success of an intensive language training in chronic aphasia patients depends both on immediate activity changes in visual, attention-, and memory-related brain structures as well as on slowly evolving activity changes (over the course of eight months) in right language homologue areas of the middle temporal lobe . This longitudinal study thus demonstrates that even in the chronic stage after a stroke, language reorganization is a highly dynamic process, comparable to the time course of language or motor recovery in the acute stage after stroke ,38.Patients behaviourally improved from zero percent correct naming responses at pre-training to a mean of 64.4 percent immediately after training, which amounts to a very large treatment effect compared to previous studies with chronic aphasia patients . This deinitial language learning, i.e., the acquisition of lexical [Immediate treatment success from baseline to immediately after training was best predicted by an fMRI activity increase in brain structures orchestrating memory encoding . It may lexical , semanti lexical , and syn lexical knowledg lexical . The con lexical . Thus, iAdditionally, a right parahippocampal activity increase from pre to post training was a good correlate of language recovery in our patients. This presumably reflects the fact that language re-learning is more laborious in aphasia as compared to language learning in healthy subjects and may thus require the joint effort of both hemispheres. The right parahippocampus may also mediate the functional recruitment of right-sided homologue language regions following left hemisphere damage ,24.We also observed increased activity immediately after training in the right-sided precuneus and cingulate gyrus and bilaterally in the posterior fusiform gyri in patients with good naming recovery. This in line with previous functional imaging studies on treatment induced recovery of language functions in chronic aphasia and thesA lack of treatment-induced hippocampal activity modulation in prior aphasia intervention neuroimaging studies may be due to the smaller extent of treatment induced language recovery in chronic aphasia e.g. ,18,19). ,19. 18,1spontaneous language recovery over the course of one year depended on regional cerebral blood flow increases bilaterally in the temporal lobes [The major novel aspect of the present study was the identification of brain structures mediating the long-term retention (after eight months) of the training outcome. Behaviourally, patients' naming performance remained highly stable throughout the eight months follow-up period . The long-term treatment success, however, was mediated by different brain structures than those involved in short-term language recovery. Greater treatment success in the long run was predominantly related to an activity increase in language regions of right temporal lobe - when lowering the statistical threshold to account for the reduced statistical power in perilesional brain areas - in peral lobes ,47. The The current study aimed to investigate the neural correlates of immediate versus long-term successes of intensive naming training in chronic aphasia patients. The results can therefore not be directly generalized to other types of language training (non-intensive) or other types of aphasic symptoms . Future studies may also consider obtaining complimentary information from different imaging techniques that have a better temporal resolution. For example, Laganaro et al. used eleFurthermore, the core problem in our patients was the linking of semantic information with a particular word form . RM, MM, HK, MD, AB, HS, MT, KK, HL, AF, SK and CB contributed to the design of the study. RM, MM, HK, HS, KK and CB collected the imaging data. HL conducted the neuropsychological testing. RM, KK and MT administered the treatment. RM, MM, HK, MD and CB analyzed the fMRI data. RM, MM and CB drafted the manuscript. All authors were involved in revising the initial draft and approved the final version.Patient Information. Demographic and clinical information and language test results for the eight aphasia patientsClick here for fileSupporting information. Provides additional supportive information regarding patient characteristics, neuropsychological testing and functional imaging analyses and resultsClick here for fileBrain activity changes immediately after training and at the follow-up assessment. Brain activity changes in the patients and healthy controls: Brain areas showing a) positive and b) negative correlations between short-term training success and 'post1-pre' training activity changes for trained object names (masked with the respective results for untrained object names). Brain areas showing c) positive and d) negative correlations between long-term training success and 'post2-pre' training activity changes for trained object names (masked with the respective results for untrained object names). The corresponding results for the control group (Session1 versus Sesssion2) are displayed in italic fontClick here for file
However, the conformation of the N—H bond is syn to the meta-methyl substituent in the aromatic ring of one of the molecules and anti in the other molecule. The two independent molecules are linked through intermolecular N—H⋯O hydrogen bonding into chains parallel to the b axis.The conformation of the C=O bond in the structure of the title compound, C Å b = 9.8076 (9) Å c = 12.409 (1) Å α = 95.415 (8)°β = 96.492 (9)°γ = 97.767 (9)°V = 996.26 (16) Å3 Z = 4 Kα radiationMo −1 μ = 0.34 mmT = 299 K 0.44 × 0.36 × 0.20 mm Oxford Diffraction Xcalibur diffractometer with a Sapphire CCD detectorCrysAlis RED; Oxford Diffraction, 2007T min = 0.868, T max = 0.936Absorption correction: multi-scan (10754 measured reflections3634 independent reflectionsI > 2σ(I)2874 reflections with R int = 0.017 R[F 2 > 2σ(F 2)] = 0.035 wR(F 2) = 0.127 S = 0.97 3634 reflections240 parametersH-atom parameters constrainedmax = 0.19 e Å−3 Δρmin = −0.23 e Å−3 Δρ CrysAlis CCD used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008PLATON (Spek, 2009SHELXL97.Data collection: 10.1107/S1600536809013051/dn2441sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809013051/dn2441Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:
Apodemus peninsulae (4/70), A. agrarius (1/39), and Clethrionomys rufocanus (1/8). The hantavirus sequences identified in two seropositive A. peninsulae and two patients with hemorrhagic fever with renal syndrome (HFRS) from the Primorye region of Far East Russia were designated as Solovey and Primorye, respectively. The nucleotide sequences of the Solovey, Primorye, and Amur (obtained through GenBank) sequences were closely related (>92% identity). Solovey and Primorye sequences shared 84% nucleotide identity with the prototype Hantaan 76-118. Phylogenetic analysis also indicated a close relationship between Solovey, Primorye, Amur, and other viruses identified in Russia, China, and Korea. Our findings suggest that the Korean field mouse (A. peninsulae) is the reservoir for a hantavirus that causes HFRS over a vast area of east Asia, including Far East Russia.In an epizootiologic survey of 122 rodents captured in Vladivostok, Russia, antibodies positive for hantavirus were found in Hantavirus genus (family: Bunyaviridae) have been identified worldwide. Rodents are the natural reservoir for hantaviruses, although one virus strain has been isolated from the house shrew (Suncus murinus), an insectivore Currently, at least 20 serotypes and genotypes of the Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), and Dobrava-Belgrade virus (DOBV) Apodemus agrarius), Norway rat (Rattus norvegicus) and black rat (R. rattus), bank vole (Clethrionomys glareolus), and yellow-necked field mouse (A. flavicollis), respectively. DOBV was also found in A. agrarius in Europe (Sin Nombre virus (SNV), New York virus (NYV), Black Creek Canal virus (BCCV), Bayou virus (BAYV), Andes virus (ANDV), and other related viruses cause HPS in the New World and are carried by the deer mouse (Peromyscus maniculatus), white-footed mouse (P. leucopus), cotton rat (Sigmodon hispidus), marsh rice rat , and Oligoryzomys longicaudatus, respectively (Microtus fortis) in Far East Russia were found to harbor two novel hantaviruses, Khabarovsk virus (KHAB) and Vladivostok virus (Topografov virus (TOPV), was isolated from brown lemmings (Lemmus sibiricus). The correlation between these three viruses and their pathogenicity for humans are not yet known Hantaviruses cause two forms of human disease, hemorrhagic fever with renal syndrome (HFRS), and hantavirus pulmonary syndrome (HPS); human infection occurs after the inhalation of aerosolized rodent excreta. HFRS is manifested as high fever, renal dysfunction, and hemorrhage; HPS is characterized by an acute progressive pulmonary edema and a fatality rate of about 40%. Among the hantaviruses that cause HFRS in Eurasia are n Europe . Sin Nomectively ,6. Althook virus ,8. AnothA. peninsulae, according to a recent study on nucleotide sequence comparisons by Yashina et al. A recent study reported two novel hantaviruses, designated as Amur (AMR) and Far East (FE), that were identified from HFRS patients in Far East Russia A. peninsulae is a carrier of pathogenic hantaviruses. We detected antibodies to hantaviruses in A. peninsulae, and the viral genome characteristics were extremely similar to the newly identified genotype, AMR A. peninsulae, we were able to corroborate the assumption of Yashina et al. A. peninsulae-related viruses are pathogenic for humans and are distributed over a large area of east Asia that includes Far East Russia.In 1999, we carried out an epizootiologic survey in a suburb of Vladivostok, Russia, to determine the characteristics of hantaviruses circulating in Far East Russia and to examine the possibility that We collected sera and organs from wild rodents captured during 1999. We also collected sera and autopsy materials from HFRS patients in two rural villages in the Primorye region of Russia, located 400 km and 600 km from Vladivostok.Rodent sera were screened for antibodies to HTNV and PUUV or both by indirect immunofluorescent antibody assay (IFA). Vero E6 cells infected with the Hantaan 76-118 strain of HTNV or the Sotkamo strain of PUUV were used as antigen slides. Diluted sera (1:16 and 1:64) were spotted onto the antigen slides and incubated at 37°C for 1 h. After three washes with phosphate-buffered saline (PBS), protein G-conjugated fluorescein isothiocyanate (FITC) was spotted onto the slides. After incubation at 37°C for 1 h, the slides were washed and observed by fluorescence microscopy. Scattered, granular fluorescence in the cytoplasm of infected Vero E6 cells was considered a positive reaction. Antibodies in HFRS patient sera were detected by the same protocol, except for the substitution of FITC-conjugated antihuman immunoglobulin (Ig) G .A. peninsulae with Isogen , which is based on the acid guanidium-phenol-chloroform technique, according to manufacturer’s instructions. Similarly, total RNA was extracted from lung, liver, kidney, spleen, and brain tissues of HFRS patients. Reverse transcription (RT) was carried out at 42°C for 30 min by using Superscript II and random primer . Full-length S segments were amplified with Platinum Taq (Gibco-BRL) and HTNV–full S primer for 30 polymerase chain reaction (PCR) cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 68°C for 2 min. Amplification of M segments was identical to that of S segments, except for the use of M genome-specific primers to generate the phylogenetic trees by using the neighbor-joining method with 1000 bootstrap replicates. Hantavirus sequences used in the comparisons were obtained from GenBank. The S and M genome sequences used in this study are listed in We used the ClustalX program package A. agrarius, (8) C. rufocanus, (3) M. fortis, and (2) Tamias sibiricus. Screening by IFA showed that one A. agrarius (2.5%), four A. peninsulae (5.7%), and one C. rufocanus (12.5%) had antibodies to HTNV or PUUV or both. HTNV-antibody titers ranged from 1:32 to 1:512. All the seropositive rodents, except for C. rufocanus, lacked antibody against PUUV (A. peninsulae were subjected to RT-PCR to amplify the virus genomes. Two of the four rodents with high IFA titers to HTNV (1:256 and 1:512) were positive by PCR for both the S and M segments of hantavirus.We carried out the epizootiologic survey on 122 rodents captured in a suburb of Vladivostok; results of serologic screening of rodent sera by IFA are shown in nst PUUV . Lung tiWe obtained the clinical histories of two fatal cases of HFRS in the Primorye region. The patients, who lived in villages 400 km and 600 km from Vladivostok, died 8–13 days after the onset of illness; gastrointestinal bleeding and acute renal failure were the causes of death. Serologic screening showed that both patients were positive for hantaviral antibodies. Antibody titers to HTNV and SEOV were apparently higher than to PUUV. We used lung, liver, kidney, spleen, and brain tissues of these HFRS patients for RT-PCR analysis; the lung and kidney tissues of patient no. 1 and the spleen tissue of patient no. 2 were positive for hantaviral M segment.To examine the histopathologic changes in HFRS patients, we used light microscopy to examine sections of formalin-fixed lung, liver, kidney, spleen, and brain tissues from patient no. 2, who had died of acute renal failure . The kidA. peninsulae were amplified and sequenced. We designated these segments as Solovey/AP61/1999 and Solovey/AP63/1999 based on the name of the village closest to the survey point, the rodent species from which the sample was taken, and the year in which the epizootiologic survey was done. We compared the coding regions of these sequences with those of other hantaviruses . We also sequenced the partial M segments of genetic lineages identified in the two HFRS patients from the Primorye region, designated as Primorye/H1/2000 and Primorye/H2/2000. The M segment of Solovey and Primorye sequences were compared with those of other hantaviruses (A. peninsulae in Far East Russia (A. peninsulae) in Far East Russia and China. Our results also suggest that this genetic lineage is widely distributed throughout east Asia.To explore the genetic diversity of hantaviruses identified in aviruses . Nucleott Russia ,11. The The M segments of Solovey, Primorye, and AMR sequences formed a common phylogenetic lineage with high bootstrap support values, regardless of viral origin . FurtherTo identify signature amino acids for each virus type, we compared the deduced partial amino acid sequences of their G2 regions using ClustalX multiple-sequence alignment . The preP. maniculatus could transmit highly virulent hantavirus to humans until SNV was identified . Recently, AMR sequences were found in both HFRS patients and A. peninsulaeA. peninsulae. Comparison of the deduced hantaviral amino acid sequences showed that aspartic acid and methionine represented signature amino acids for AMR genetic lineage, regardless of the region in which the virus was identified or its origin from Primorye region, we detected pathologic changes typical of severe HFRS caused by hantavirus infection –35. We aA. peninsulae as one of the causative agents of HFRS. We think that this information may be helpful in preventing human infections in East Asia. Controversy persists over whether A. peninsulae carries a distinct virus type or a subtype of HTNV. A similar question arises with Dobrava/Slovenia and Dobrava/Saaremaa, which are carried by A. flavicollis and A. agrarius, respectively. The S segment identities between Dobrava/Slovenia and Dobrava/Saaremaa (both obtained from GenBank for comparison purposes) were 87.8% (nucleotide) and 92.7% (amino acid). Similarly, the nucleotide and amino acid sequence identities of the S segments of Solovey sequences and HTN 76-118 were 82.7% and 92.2%, respectively. We suggest that Solovey sequences belong to a sublineage within the HTNV clade.Through epizootiologic, clinical, pathologic, and sequencing studies, we identified a hantavirus carried by
A. fumigatus) allergen and diesel exhaust particles will be associated with altered IgE production, airway inflammation, airway hyperreactivity (AHR), and airway remodeling of adult offspring.Multiple studies have suggested that prenatal exposure to either allergens or air pollution may increase the risk for the development of allergic immune responses in young offspring. However, the effects of prenatal environmental exposures on adult offspring have not been well-studied. We hypothesized that combined prenatal exposure to Aspergillus fumigatus (A. fumigatus and mating, pregnant BALB/c mice were exposed to additional A. fumigatus and/or diesel exhaust particles. At age 9-10 weeks, their offspring were sensitized and challenged with A. fumigatus.Following sensitization via the airway route to A. fumigatus or diesel exhaust particles during pregnancy experienced decreases in IgE production. Adult offspring of mice that were exposed to both A. fumigatus and diesel exhaust particles during pregnancy experienced decreases in airway eosinophilia.We found that adult offspring from mice that were exposed to These results suggest that, in this model, allergen and/or diesel administration during pregnancy may be associated with protection from developing systemic and airway allergic immune responses in the adult offspring. In hu-37 days . Most re1 levels, eosinophilia in BAL fluid, and reduced phorbol 12-myristate 13-acetate (PMA), inomycin, and OVA-induced T helper (Th) 2 cytokine production in the offspring [Dermatophagoides pteronyssinus (D. pteronyssinus) allergen prior to mating developed significant decreases in total and anti-D. pteronyssinus IgE, IgG1, IgG2a and IgG2b levels upon resensitization in comparison to offspring of unexposed mice [Alternately, some prenatal exposures have induced protection from the asthma phenotype. Lipopolysaccharide (LPS or endotoxin) administered prenatally to mice led to the development of lower anti-OVA IgE and IgGffspring ,8. In epffspring . Furthersed mice . Hence, A. fumigatus mouse model that induces strong allergic responses via the airway route in the absence of adjuvants and, hence, arguably better mimics clinical asthma [A. fumigatus and diesel exhaust particles would be associated with altered IgE, airway inflammation, airway hyperreactivity (AHR), and airway remodeling in adult mice offspring.Despite these advances, many key questions still need to be elucidated. These include questions about the effects of airborne prenatal exposures to toxins of concern in the urban environment, as well as their possible long-term adverse effects on adult offspring. Our objective was to determine the effects of concomitant and chronic aerosolized prenatal exposure to allergen and diesel exhaust particles, two environmental exposures implicated in inner city asthma ,12, on pl asthma . Diesel l asthma ,15. We hA. fumigatus (62.5 ug) in 50 ul of saline or saline vehicle alone was administered five times, four days apart, beginning 20 days prior to mating. Pregnant mice were treated again with A. fumigatus or saline on day 7 and 14 after mating. Offspring were separated from their mothers at 21 days of age. At 9-10 weeks of age, all offspring were treated with either five or six dosages of A. fumigatus each dose four days apart . Males and females were housed separately prior to mating. All animals were housed at New York University (NYU) animal facility and fed a commercial pellet mouse feed. Mice were lightly anesthetized with isoflurane . Intranasal application of 3 flow-through exposure chamber where mice were exposed. Pregnant mice were exposed for 5 hours (average 5.18 hours) a day, Mondays through Fridays, to DEP or HEPA (high efficient particle) filtered ambient air (as negative control) in parallel during the second and third weeks of pregnancy that contained a 418-cc displacement engine (Model LE100EE-DEGY6), as described ,16. The um pore; Gelman Sciences, Ann Arbor, MI) for subsequent gravimetric analyses. Particle size distributions were measured with a Wide-Range Particle Spectrometer . The average particle concentration was 1.09 mg/m3. The DEP atmosphere had a count median aerodynamic diameter of 80 nm, and a mass median aerodynamic diameter of 152 nm.The mass concentrations of the DEP in the exposure chamber were recorded every 20 minutes using a real-time Personal DataRam (PDR) aerosol monitor . DEP also were collected daily onto Teflon filters dose of A. fumigatus. Sera were aliquoted and frozen. Total Ig levels were measured by ELISA using isotype specific capture antibodies for IgE, IgG1 and IgG2a , following a previously described protocol [1 or IgG2a. Sera were diluted 1:20 for IgE, 1:10,000 for IgG1, and 1:100 for IgG2a. Biotin labeled rat-mouse IgE, IgG1 and IgG2a along with AKP Streptavidan were used for detection. Specimens were run in duplicate and averaged.Sera were obtained from adult female mice immediately prior to the first dose of protocol . BrieflyMice were euthanized at median age 12.5 weeks and bronchoalveolar lavage was performed three times on each mouse with 1 ml of phosphate buffered saline (PBS) 24 hours after the last allergen challenge. Lavage fluid was centrifuged at 4°C 1500 rpm for 5 minutes. Cell pellets were resuspended in 1 ml of phosphate buffered saline. Slides were prepared using a cytocentrifuge at 500 rpm for 5 minutes then stained with Wright-Giemsa stain . 100 cells total were counted for each sample from 10 randomly chosen viewing fields and total eosinophil, lymphocyte, macrophage and neutrophil counts were quantified by a blinded reader.At median age 15.5 weeks, additional mice were anesthetized and intubated with a 20 g catheter inserted directly into the trachea via a neck dissection, then placed on a flexivent ventilator. A nebulizer attached to the flexivent apparatus exposed mice to increasing concentrations of methacholine at 8, 16, 32, 64 mg/ml . Airway resistance was determined by the flexivent-apparatus . The shaImmediately subsequent to the AHR testing, lungs were inflated and stored in 10% formalin. Lungs were paraffin-embedded and sections were stained with hematoxylin and eosin . Under blinded conditions, each lung was scored for perivascular inflammation, peribronchial inflammation and arterial remodeling as previously described . For per6/ml) were seeded in triplicate in 96 well plates and treated with A. fumigatus at 0, 20 ug/ml or 40 ug/ml and CD3 10 μg/ml and incubated with 5% CO2 for five days at 37°C. 3H-thymidine uptake was assessed on day 5 as described [Splenocytes was used to compare mean differences across treatment groups followed by Tukey HSD test except where noted. Nonparametric rank order correlations were used to compare continuous data (eg. IgE levels and eosinophil counts) between treatment groups. Differences were considered statistically significant at p < 0.05.A. fumigatus developed higher total IgE levels than those treated with only vehicle saline solution . Adult offspring from mothers who received A. fumigatus or DEP alone, or DEP and A. fumigatus together, developed lower IgE levels compared with levels from offspring whose mothers received saline alone when assessed after the sixth dose of allergen treatment as well (p < 0.0001 ANOVA). In addition, IgE levels from adult offspring of mice that were treated with DEP and A. fumigatus were lower than those from offspring of mice that were treated with A. fumigatus alone . Significant differences in IgE levels were not apparent after the third dose of A. fumigatusAdult offspring from mothers who received A. fumigatus, DEP, or both DEP and A. fumigatus, developed greater IgG1 levels compared to offspring of mothers treated with saline. This effect was significant after the fifth and sixth, but not third, doses of A. fumigatus treatment . Also, offspring from mothers exposed to both DEP and A. fumigatus, developed greater IgG2a levels compared to offspring of mothers exposed to either DEP or A. fumigatus alone (p < 0.01 Tukey HSD test). Adult offspring from mothers who received A. fumigatus alone, or DEP and A. fumigatus together, developed greater IgG2a levels compared with levels from offspring whose mothers received saline or DEP alone after the sixth dose of allergen as well (p < 0.0001 ANOVA). In contrast, after the third dose of A. fumigatus, a reduction in IgG2a was detected among offspring from mice exposed to DEP compared with those treated with saline compared to offspring from mothers that had received A. fumigatus or saline alone. The first result (A. fumigatus and DEP lower than A. fumigatus) was replicated when examining absolute numbers of eosinophils (p < 0.001 on ANOVA and p < 0.01 by Tukey HSD). Adult offspring from mothers that received both A. fumigatus and DEP also developed higher levels of macrophage counts compared to offspring of mothers that had received A. fumigatus or saline alone or sixth dose .Adult offspring from mothers that received both A. fumigatus. On histological examination, examples of perivascular and peribronchial inflammation and arterial muscularization were detected among adult offspring of mice exposed to A. fumigatus when compared to offspring of mice exposed to saline were not detected either.However, using an established semi-quantitative scoring system , only a A)Figure . Also, wA)Figure . CorrelaA. fumigatus and/or DEP can induce airway remodeling in adult sensitized offspring, pulmonary arterial remodeling was assessed in the mice offspring. Offspring from mothers who received A. fumigatus and/or DEP during pregnancy did not exhibit significant differences in the degree of arterial airway remodeling compared to offspring of mothers who received saline .A. fumigatus and/or DEP would be associated with altered antigen-specific T cell proliferation in the offspring, splenocytes were tested following induction with several doses of A. fumigatus or anti-CD3. We found that antigen-specific proliferation in the adult offspring was not affected by A. fumigatus or DEP exposure administration to the mother (data not shown).To determine whether prenatal exposure to A. fumigatus prior to and during pregnancy were associated with diminished IgE production and airway eosinophilia. The latter occurred following prenatal exposure to both A. fumigatus and diesel. The parallel increases in IgG levels suggest that the antibody responses were specific to IgE. These findings indicate that prenatal exposure to A. fumigatus, may be associated with protection from systemic and airway allergic immune responses in adult offspring.These results suggest that exposures to D. pteroynissinus was associated with lower total and D. pteroynissinus-IgE levels in the offspring. Similar to our model, exposure to allergen prior to mating reduced allergen sensitization in the offspring at the humoral level [1 and IgE and reduction of interleukin (IL)-5 and IL-13 in splenic mononuclear cells [A. fumigatus and diesel may have timed or dosed so as to favor establishment of tolerance instead of allergic sensitization.While these results may appear contradictory to several studies that show prenatal sensitization is associated with greater allergic immune responses in the offspring , they aral level . In moreal level . Prenataar cells . Besidesar cells . Hence, While postnatal exposure to mold has been associated with greater asthma severity or emergency room visits for asthma -27, receμg/m3 for railroad workers, 4 - 748 μg/m3 for firefighters, and 7 - 98 μg/m3 for public transit workers and airport crews [A. fumigatus and DEP together, developed lower levels of total IgE, and greater levels of IgG1, when assessed after the fifth and sixth dose of allergen. Paradoxically, after the third dose of A. fumigatus, a reduction in IgG2a was detected among offspring from mice exposed to DEP compared with those treated with saline. Also, adult offspring of mothers that received both A. fumigatus and DEP developed significantly less airway eosinophilia compared to offspring of mothers that had received A. fumigatus alone. Combined, these results suggest that prenatal DEP exposure independently may have conferred some protection against allergic immune responses in the adult offspring in this model. These findings were unexpected, especially given previous research using the engine byproduct residual oil fly ash as the air pollutant that induced greater airway eosinophilia and hyperreactivity in the OVA-sensitized offspring [A. fumigatus), components and dose of the air pollutants, strain of mouse, or age of the offspring following allergen sensitization (less than 5 weeks vs. 9-10 weeks).EPA has estimated occupational DEP exposures to range from 39 - 191 rt crews . So whilrt crews ,33. HoweA. fumigatus and airway arterial remodeling in adult offspring were not statistically significant, with only mild changes detected during histological examination. Exposure to A. fumigatus has been shown to exacerbate an asthma phenotype in rats by aggravating Th2 inflammation, increasing AHR, and inducing airway remodeling [A. fumigatus for a prolonged period of time developed remodeling of small to medium sized pulmonary arteries [Associations between prenatal exposure to DEP and/or modeling . Previoumodeling -37. Our arteries . In anotarteries . From a arteries . This cuA. fumigatus exposure has been shown to induce hypermethylation of multiple CpG sites of the interferon-gamma (IFNg) promoter and hypomethylation of one CpG site of the IL-4 promoter with associated changes in IgE levels, suggestive of the contribution of epigenetic regulation following environmental exposures [A. fumigatus, or diesel, may induce protection from allergy in the offspring, especially in light of past data that suggest A. fumigatus induces greater, not repressed, Th2 cytokine production [Several plausible mechanisms may explain how prenatal exposures may help modulate the development of allergic and/or airway immune response in the offspring. It has been reported that antigen-specific T cell and B cell immune responses in the fetus can occur distinctly from those of the mother, as demonstrated by our group in response to vaccination against influenza . In addixposures . These mxposures ,42. Howeoduction . In one oduction . Rats whoduction .Some limitations of this study merit discussion. First, we used only one strain of mice to obtain the above findings even though it has been shown that when comparing acute injury responses, such as airway remodeling, patterns are unique to different strains. Also, tA. fumigatus administration during pregnancy resulted in protection from systemic and airway allergic responses. Prenatal diesel exhaust particle exposure also was associated with reduced IgE levels and suppressed airway eosinophilia in the adult offspring. These results suggest that prenatal environmental exposures can induce exert systemic and airway immune changes in the adult offspring related to respiratory disease. These results highlight the need to consider the health effects of prenatal exposures on offspring, even through adulthood.In conclusion, our results indicate that AHR: airway hyperreactivity; AKP: alkaline-phosphatase; BAL: bronchoalveolar lavage; BHR: bronchial hyperresponsiveness; DEP: diesel exhaust particles; EPS: extracellular polysaccharides; ETS: environmental tobacco smoke; HDM: house dust mite; IFN: interferon; Ig: immunoglobulin; IL: interleukin; in: intranasal; LPS: lipopolysaccharide; NS: non-significant; NYU: New York University; OVA: ovalbumin; PAH: polycyclic aromatic hydrocarbon; PMA: phorbol 12-myristate 13-acetate; RUNX3: Runt-related transcription factor 3; Th: T-helperThe authors declare that they have no competing interests.LL carried out the experimental work, performed some of the statistical work, and drafted the manuscript. HZ carried out the experimental work. CQ carried out all exposure related experiments. GG participated in the design of the study and advised on the experimental work. MB carried out a significant proportion of the experimental work. XJ worked with CQ administer the diesel exposure. FPP participated in the design of the study and advised on the experimental work. PHF participated in the design of the study and advised on the experimental work. LCC participated in the design of the study and advised on the experimental work. RLM conceived of the study, performed the statistical work, and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.
Bone metastases are a common and dismal consequence of lung cancer that is a leading cause of death. The role of IL-7 in promoting bone metastases has been previously investigated in NSCLC, but many aspects remain to be disclosed. To further study IL-7 function in bone metastasis, we developed a human-in-mice model of bone aggression by NSCLC and analyzed human bone metastasis biopsies.We used NOD/SCID mice implanted with human bone. After bone engraftment, two groups of mice were injected subcutaneously with A549, a human NSCLC cell line, either close or at the contralateral flank to the human bone implant, while a third control group did not receive cancer cells. Tumor and bone vitality and IL-7 expression were assessed in implanted bone, affected or not by A549. Serum IL-7 levels were evaluated by ELISA. IL-7 immunohistochemistry was performed on 10 human bone NSCLC metastasis biopsies for comparison.At 12 weeks after bone implant, we observed osteogenic activity and neovascularization, confirming bone vitality. Tumor aggressive cells implanted close to human bone invaded the bone tissue. The bone-aggressive cancer cells were positive for IL-7 staining both in the mice model and in human biopsies. Higher IL-7 serum levels were found in mice injected with A549 cells close to the bone implant compared to mice injected with A549 cells in the flank opposite to the bone implant.We demonstrated that bone-invading cells express and produce IL-7, which is known to promote osteoclast activation and osteolytic lesions. Tumor-bone interaction increases IL-7 production, with an increase in IL-7 serum levels. The presented mice model of bone invasion by contiguous tumor is suitable to study bone-tumor cell interaction. IL-7 plays a role in the first steps of metastatic process. Lung cancer is a major cause of cancer-related deaths . More thThe role of IL-7 in diseases characterized by bone loss-10 has bIn the last years, important advances provided new animal models of different human cancer metastasis to human tissues -16. SomeA NSCLC cell line with osteotropic characteristics, the A549, was purchased from the American Type Culture Center (ATCC). Cells were cultured in F-12K Medium, Modified 2 mM L-glutamine e 1500 mg/L sodium bicarbonate (ATCC), 10% fetal bovine serum (FBS) with antibiotics, according to manifacturer's conditions. Sub-confluent cells were washed by PBS and harvested by trypsinization.A colony of 18 NOD/SCID 7-week-old female mice were housed under aseptic sterile conditions. Experimental animals were treated in compliance with the actual national and international guidelines and in accordance to the authorization provided by Italian Ministry of Health (as of D.M. 44/1994-A and subsequent integrations).3 cube-shaped human bone implant, obtained from the discarded head bone of an adult patient submitted to total joint replacement (after the patient's informed consent), was immediately transplanted sub-cutaneously in the left flank in all animals. Surgery was performed in sterile conditions and in general anaesthesia. Animals received antibiotics (Enrofloxacin 2.5% 1 ml/1 L) in the drinking water up to 2 weeks following all surgical procedures.Mice were given autoclaved food and water ad libitum. A 0.5 cm6of cells were resuspended in PBS and Matrigel 1:3 and injected in volume of 40 μL sub-cutaneously, using a 25-gauge needle.The bone implants were allowed to engraft in the mice for 4 weeks; then mice were divided in three groups of six animals each, as follows: a) mice subjected only to bone implant (control); b) mice injected with A549 cells close to the bone implant (group #1); c) mice injected with A549 cells in the opposite flank to the bone implant (group #2). In particular, 1 × 10Eight weeks after cancer cell injection, mice were euthanized; primary tumors, bone implants, lungs and spleens were excised and processed for immunohistochemistry and other analyses.Immunohistochemistry was performed on tissues fixed in 10% neutral buffered formalin and bone tissues were decalcified with EDTA treatment until soft. Tissues were embedded in paraffin and 4 micron sections were deparaffinized, rehydrated through graded alcohols, and subjected to antigen retrieval for immunohistochemistry. Sections were stained for H&E to study the morphology and incubated with mouse monoclonal antibodies against human-specific CD34, clone QBEnd/10 and human IL-7 . IL-7 staining was performed also on 10 human biopsies derived from bone metastasis by NSCLC. Research on human specimen was carried out in compliance with the Helsinki Declaration. IL-7 expression in mice and human tissues was quantifed based on IL-7 staining intensity.FACS analysis was performed on whole NOD/SCID mice spleens. Spleen tissue was dissociated with forceps and cells were re-suspended in D-MEM with 10% FBS. Cells were passed through a 40 μm cell strainer and re-suspended in red blood cell lysis buffer. Two million cells were incubated with antibodies specific for human IgG-phycoerythrin (PE). Spleen cells were analyzed on a FACSCalibur system to identify the human IgG-PE positive cell population after gating out PI positive cells. Samples were analyzed in a FACsCalibur instrument and elaborated by CellQuest software.To dose serum IL-7 levels we performed an ELISA assay, according to the manifacturer's instructions . The kit sensitivity was 0.27-8.7 pg/mL.in situ. The vascularization of the grafts was examined and neo-angiogenesis surrounding the implanted bone was detected. . The presence of human derived blood vessels was confirmed by CD34 staining of human blood vessels .The A549 is a NSCLC cell line with osteotropic ability. A rapid tumor growth was obtained at the implantation site in all animals. When tumor cells were injected close to the bone chip, the tumor invaded the normal implanted human bone, disrupting it. Metastases were not observed in the group where tumor cells were injected in the contralateral flank. H&E staining of bone implant with tumors showed large areas of invading tumor cells surrounding live human bone, with presence of fat cells, blood vessels, and osteoblasts Fig .IL-7 expression by A549 cells was evaluated, showing a high IL-7 expression by tumor cells of epithelial origin. IL-7 expression in tumor masses was observed in both groups. The analysis of bone invaded by tumor cells and bone contralateral to the tumor mass, showed a different intensity of IL-7 expression. In detail, bone invaded by tumor cells showed a stronger level of IL-7 expression than contralateral bone, where IL-7 was expressed only by stromal cells Fig. . In humaSince we previously demonstrated that serum IL-7 levels are higher in NSCLC patients with bone metastases than in patients without bone lesions, we tested the IL-7 production in the two groups of mice, by dosing serum IL-7.p < 0.001), while no significant difference was found between opposite flank and control group without tumor cells. The IL-7 serum value in the control group was 0.21 ± 0.03 pg/ml , rather focuses on cancer cell bone invasion capability. It allowed us to bypass technical difficulties related to obtaining NSCLC metastases through blood circulation. The opposite flank injection group was useful for serum IL-7 level comparisons, in order to discriminate production related to bone invasion from production by tumor mass alone. To the author's knowledge, this is the first human-in-mice NSCLC bone invasion model described in literature. The general validity of this model is supported by results on bone vitality and implanted tumor growth. The specific validity for studies on tumor-bone interactions in metastasis is highlighted by the histologic and cytokine production analyses. Bone invasion appeared to have the same histological characteristics of human metastasis, such as bone resorption, neo-apposition and tumor nested within the bone tissue. The immunohistochemical staining of bone invaded by NSCLC cells, showed a strong expression of IL-7; the same result was obtained by the staining performed on human tissues from bone biopsies of patients affected by NSCLC bone metastasis. Staining for IL-7 of the bone implanted contralateral to the tumor showed stromal cells positive for IL-7 expression, as expected and according to the literature ,23. In tWe speculate that the mechanism underlying interaction between cancer cells and bone closely resembles the metastatic mechanism in humans, therefore this model might be valuable and considered for further testing.We demonstrated that bone invading cells directly express and produce IL-7, which is known to promote osteoclast activation and osteolytic lesions. Both tumor cells and bone stroma can produce IL7 but cross-talk between the two increases production, as shown both in humans and in the described mice model. Our mice model of bone invasion by contiguous tumor mass showed similar histology and IL-7 patterns compared to human pathology. We believe it to be suitable for studies on bone-tumor cell interaction, being both realistic and technically feasible compared to other models.We believe that new studies on the first steps of metastatic process and on the role of IL-7 are due, considering together the fine mechanism through which IL-7 is regulated, its role in pathologies characterized by bone loss and the sudden intense increase in IL-7 production when bone is invaded by tumor cells.The authors declare that they have no competing interests.IR: conception and design of the study, analysis and interpretation of data, carried out part of laboratory assessment and the draft of the manuscript. DC: conception and design of the study, analysis and interpretation of data, carried out part of the experimental surgery and the draft of the manuscript. LG: carried out and analysis of immunohistochemical staining. LD: carried out laboratory assessment. PG: was responsible for animal care and surgical procedures. EM: carried out part of the experimental surgery. QR: critical revisal of the manuscript. LM: interpretation of data. PB: critical revisal of the manuscript. AM: critical revisal of the manuscript. RF: interpretation of data, critical revisal of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/12/prepub
The role of sensory systems is to provide an organism with information about its environment. Because sensory information is noisy and insufficient to uniquely determine the environment, natural perceptual systems have to cope with systematic uncertainty. The extent of that uncertainty is often crucial to the organism: for instance, in judging the potential threat in a stimulus. Inducing uncertainty by using visual noise, we had human observers perform a task where they could improve their performance by choosing the less uncertain among pairs of visual stimuli. Results show that observers had access to a reliable measure of visual uncertainty in their decision-making, showing that subjective uncertainty in this case is connected to objective uncertainty. Based on a Bayesian model of the task, we discuss plausible computational schemes for that ability. confident in our visual perception: in the context of a visual task, what are the physical properties of the stimulus that will make us think we are doing the task well? The mathematical framework of Bayesian statistics provides an elegant way to frame the problem, by assuming that the visual system is trying to estimate physical properties of the world from incomplete, sometimes unreliable visual information. Objective uncertainty will therefore depend on the quality of the information available in the stimulus. In our experiments we compare objective uncertainty—as computed using the Bayesian framework—with subjective uncertainty, the confidence observers report about their visual percepts. To this end, we use a visual task with well-defined statistical properties, discrimination under noise. We report a surprising degree of agreement between objective and subjective uncertainty, and discuss possible computational models that could explain this ability of the visual system.Most work in vision science focuses on the question of why we perceive what we do, and we now have many models explaining what physical properties of a stimulus make us see depth, colour, etc. Here we ask instead what makes us feel Every single human action happens in a context of uncertainty, being based on incomplete knowledge and undertaken despite unpredictable consequences. When faced with uncertainty, humans employ heuristics Uncertainty is a familiar concept in cognitive science, in particular thanks to Signal Detection Theory . In a typical psychophysical task, an observer has to detect small contrast increments near threshold. The uncertainty in this task comes mostly from internal variability: because of fluctuations in her internal representation of contrast, the observer makes mistakes and is uncertain about the correctness of her decisions. Unfortunately for the experimenter, this source of the uncertainty is internal to the observer and therefore only indirectly controllable.Now consider another difficult perceptual task: listening to a speaker among cocktail-party chatter. Here the difficulty depends not so much on variability in the brain, but rather on interactions between the different voice signals: the one emitted by the speaker you aim to listen to, and the sound of other voices. Even with the volume of the other voices staying the same over time, difficulty will depend on the languages spoken, the gender of the speakers, and other sources of confusion. More generally, background chatter plays the role of noise, and difficulty will vary based on how much signal and noise covary.objective uncertainty: objective uncertainty is inversely related to the amount of task-relevant information available in the stimulus. Concurrently, we can measure the perceived uncertainty of the observer, the level of confidence she actually reports. We introduce three experiments where we manipulate objective uncertainty and study its relationship with perceived uncertainty.An analogous visual task can be obtained by adding visual noise to a signal –random perturbations to the stimuli shown to the observer. Using visual noise, we are in a position to manipulate the In the first two experiments, observers were presented with pairs of images of oriented objects embedded in high levels of noise, and had to report the orientation of the image of their choice. Even though the two images contained the same level of noise, the particular noise structure made one image orientation more certain than the other. We found that observers reliably chose the more certain of the two images, thereby providing evidence of a capacity to accurately evaluate objective uncertainty. We confirmed this in another experiment, in which we held the objective uncertainty of one of two stimuli fixed while varying the other, and asked observers to pick the less uncertain one. The greater the difference in uncertainty was, the greater the chance that observers picked the less uncertain stimulus, showing that uncertainty discrimination behaves similarly to normal psychophysical tasks. In a third experiment, we extend our results to a letter discrimination task. We discuss plausible computational mechanisms for achieving these results.In the first two experiments, visual uncertainty was introduced in an orientation discrimination task by manipulating the amount of pixel noise added to a visual template. There were only two templates, which were always visible to the observers. The templates were left and right oriented Gabor patches that presented alternating dark and bright lines under a blurry circular aperture . We embeA decision must be reached by evaluating which of the two templates is the more probable hypothesis given the noisy stimulus. The orientation task can be understood as a classification task under noise, where stimuli correspond to items and the two templates determine the two categories: our two categories are simply defined as “stimuli generated by the left-tilted template”, and “stimuli generated by the right-tilted template”. An ideal Bayesian observer can be derived for this task, and we therefore defined the objective uncertainty of a stimulus as the entropy of the ideal observer's posterior distribution over the two classes.s be the stimulus, u and v the templates (we use boldface notation for vectors). We assume that the characteristics of the noise are known and that the prior probabilities of the templates are equal. The posterior probability of template u is written:i indexes the pixels from 1 to the total number k, and is for a Gaussian of mean iu and standard deviation σ.Stimuli and templates can be represented as vectors in a space where dimensions correspond to the contrast of each pixel (difference to background luminance). Let An ideal observer in the discrimination task will respond by choosing the most probable template. That decision function can be written using the log-likelihood ratioWhat is uncertainty for our ideal observer? A very general measure of uncertainty is given by the information entropy of a probability distribution representing a state of knowledge To measure perceived uncertainty we used comparative judgements. On every trial, observers saw a pair of noisy stimuli, one at the top of the screen, and one at the bottom. Of the two stimuli presented, they only had to make a discrimination judgement about one. In this setup, if observers want to maximize their discrimination performance, the best strategy is to choose the more certain of the two stimuli. This is precisely what observers were instructed to do: the task consisted in choosing, first, the stimulus for which they felt the more confident, and only then to make a discrimination judgment on the chosen stimulus . Note thTo determine whether observers did effectively pick the less uncertain stimuli, we contrasted two conditions. In the so-called True Choice (TC) condition, the two stimuli presented resulted from independent draws from the same noise distribution. Note that two stimuli with the same average noise level, as is the case here, can still vary in the objective uncertainty they induce, because different realizations of the same noise distribution can make the stimulus more or less ambiguous. In that case there is a benefit to be had in choosing the less uncertain of the two: this gives observers a higher chance of responding correctly than if only one stimulus is available.equal objective uncertainty in that context. In the False Choice case, there is therefore nothing to be gained by choosing one rather than the other.In the other condition, the False Choice (FC) condition, we removed that benefit: the first stimulus was computed the normal way, but the second was obtained by flipping the top one either once or twice . We tookAt no point in the experiment were observers aware of the existence of the two conditions. The two stimuli presented always had equal contrast, preventing observers from using a heuristic of selecting the lower-contrast stimulus as the most certain. The False Choice condition therefore provides the performance baseline that will be used to determine whether or not observers are able to successfully compare objective uncertainties.We measured observers' performance, defined as proportion of correct classifications, in the two conditions across five different signal-to-noise ratios, chosen to span a range of performance between approximately 60 to 85%. Both the signal-to-noise ratio and the condition each trial belonged to were randomized. If observers are able to make accurate judgments of objective uncertainty, then we expect that measured performance will be higher in the TC than in the FC condition.As expected given the nature of the task, mean performance for all observers grew with increased signal-to-noise ratio. More interestingly, however, mean performance is higher in the TC condition than in the FC condition, which translates into lower performance thresholds in the TC condition a and b.It appears then that observers were able to take advantage of the True Choice condition, by choosing the less uncertain stimulus a majority of the time. It seems reasonable that, should the ability to pick the less uncertain stimulus be present, the probability of choosing the correct stimulus ought to be an increasing function of the magnitude of the difference: the more the two stimuli differ in their uncertainty, the more likely observers are to choose the right one. We evaluate that by regressing observers' choices of stimuli on the difference of log-entropies . We founThis last result hints at a more general property: in all psychophysical discrimination tasks, the larger the difference between two stimuli, the more reliable discrimination is. For example, when asked to compare the length of two lines, an observer's responses are likely to be better predictable when the two lines differ by 20 cm rather than 1. In a second experiment, we sought to confirm our findings by checking that discrimination of uncertainty behaves in the same way. The task was identical to that of experiment 1, but instead of introducing a False Choice condition, we manipulated the stimuli such that one – the standard – had always the same level of uncertainty and the other – the test – had lower uncertainty.u,v in that space, and stimuli obtained by adding white noise to a template are other points, forming Gaussian point clouds around the templates. To decide whether a point is more likely to belong to the left-tilted template rather than the right-tilted one, a simple geometrical rule describes the ideal strategy.We show in the supplementary material that generating random stimuli with a controlled level of uncertainty can be achieved using a simple orthogonal projection. Mathematically, the space of all possible stimuli of the kind used here can be described in terms of the contrast of individual pixels by having one dimension (one axis) for each pixel. Then the two templates are two points u and v, as in u we will call “left-tilted” and any falling on the side of v we will call “right-tilted”: the plane represents the decision boundary. Stimuli falling right on the hyperplane are completely ambiguous: both categories are equally likely. In fact, it is possible to show that the uncertainty of a stimulus is given by its (unsigned) distance to the decision boundary. Then the set of stimuli of fixed uncertainty is the set of points that are of the same distance to the decision boundary, and that set is simply the union of two parallel planes.Imagine drawing a line between d to the decision boundary. Standard stimuli were always on a plane of distance standardd and test simuli were on a plane of distance testd. The difference between standardd and testd was varied parametrically between 4 different levels: we expected the observers to more reliably choose the test stimulus as the difference increased.We therefore generated our stimuli by constraining them to lie on a plane of distance t-test for Generalised Linear Models coefficients, all p-values at 10−3 or below). This confirms that uncertainty behaves in that respect just like other psychophysical quantities: the more dissimilar two stimuli are on that scale, the more predictable observers' judgments are.The results appear in In experiments 1 and 2, the underlying visual task is orientation discrimination under noise, with templates identical in every way except for one basic attribute – their orientation. To check that our results were sufficiently general, we ran a variant of experiment 2 using a letter discrimination task. Observers had to discriminate between the letters ‘T’ and ‘X’ shown on , a pair Our results imply that observers had access to some estimate of the uncertainty in the orientation task. How is that estimate computed? Do observers have effective access to a probability distribution over perceptual hypotheses, from which they can estimate their own uncertainty? Or do they rely on more limited information? To investigate that question we evaluated two distinct families of models that compute uncertainties globally over the full distribution for the first, and locally for the second.r and s be two stimuli, represented as vectors of pixel luminances. Call u and v the left-tilted and right-tilted templates. Then r is to u and v, respectively. If r is more like u than v : uncertainty is low if one hypothesis corresponds to the data much better than the other, and high otherwise. We call this model the difference of responses model.In comparing the uncertainty between two stimuli - choosing between maximum response model. The same measures of distances are computed as in the first model, but only the maximum is retained for each stimulus. The observer then compares the two maximaAnother strategy, perhaps simpler for the observer, is to evaluate uncertainty based only on how well the best hypothesis fits the data. We call this the pothesis , a stratabsd and maxd, is a simple non-linear step readily implementable in a neural system.Both hypotheses are realistic from a neural-computation point of view. Computing absd and maxd, the more likely observers are to choose the bottom stimulus. As above, we compute the decision variables for every trial and we fit a linear binomial regression model to the responses , but also to estimate how uncertain that knowledge is, at least comparatively. Why humans should be so well calibrated to what is in essence a laboratory task rather than a natural one is a question that deserves attention. It is possible that they learn the statistical properties of the task over time, although we find no conclusive evidence for that in our data Gaussian noise was added to the templates to produce the stimuli.12 observers took part in the first experiment. All observers had normal or corrected-to-normal vision and gave informed consent.General. Observers were familiarised with the task with a 20-trial run of the experiment, during which the experimenter was present. They completed a total of 1000 trials over the course of two sessions. We varied the signal-to-noise ratio of the stimuli randomly, trial by trial. Feedback on the orientation task was provided on every trial.False Choice and True Choice conditions. On each trial, a condition was chosen pseudo-randomly. In the True Choice condition, the two stimuli were generated independently from the same noise distribution. This was done to ensure that the two images had equal contrast, and that observers could not use that clue to discriminate between less certain and more certain stimuli. In the False Choice condition, the first stimulus was computed as in the True Choice condition, but the second was obtained by flipping the first either left-to-right or left-to-right followed by up-down. This made it possible to have two stimuli that were different pixel-to-pixel, and looked different to the observer, but contained the same amount of information .The experimental method was the same as in experiment one, unless indicated otherwise.The templates used were Gabor patches, presented in a square window subtending 5 degrees of visual angle. The stimuli were generated by adding uncorrelated noise, then projecting onto an equal-uncertainty hyperplane as described in 8 observers took part in the second experiment, including the first author. All observers had normal or corrected-to-normal vision and gave informed consent.Observers were familiarised with the task with a 10-trial run of the experiment, during which the experimenter was present. Observers then completed a total of 500 trials in one session. The difference in uncertainty between the test and the standard stimuli was chosen at random on every trial, between four different levels see . FeedbacThe experimental method was the same as in experiment 2, unless indicated otherwise.The templates used were a T and a X, rendered in a sans-serif font. The templates are shown on 4 observers took part in the third experiment, including the first author. All observers had normal or corrected-to-normal vision and gave informed consent.Observers were familiarised with the task with a 10-trial run of the experiment, during which the experimenter was present. Observers then completed a total of 500 trials in one session. The difference in uncertainty between the test and the standard stimuli was chosen at random on every trial, between four different levels see . FeedbacText S1Supporting Information.(1.21 MB DOC)Click here for additional data file.
Ixodes scapularis and addressed the vaccine potential of this antigen in the context of tick engorgement and B. burgdorferi transmission. Results from western blotting and quantitative Reverse Transcription-PCR showed that tHRF is secreted in tick saliva, and upregulated in Borrelia burgdorferi-infected ticks. Further, the expression of tHRF was coincident with the rapid feeding phase of the tick, suggesting a role for tHRF in tick engorgement and concomitantly, for efficient B. burgdorferi transmission. Silencing tHRF by RNA interference (RNAi) significantly impaired tick feeding and decreased B. burgdorferi burden in mice. Interfering with tHRF by actively immunizing mice with recombinant tHRF, or passively transferring tHRF antiserum, also markedly reduced the efficiency of tick feeding and B. burgdorferi burden in mice. Recombinant tHRF was able to bind to host basophils and stimulate histamine release. Therefore, we speculate that tHRF might function in vivo to modulate vascular permeability and increase blood flow to the tick bite-site, facilitating tick engorgement. These findings suggest that blocking tHRF might offer a viable strategy to complement ongoing efforts to develop vaccines to block tick feeding and transmission of tick-borne pathogens.Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Effective vaccines against most tick-borne pathogens are not currently available. In this study, we characterized a tick histamine release factor (tHRF) from I. scapularis ticks, the predominant vector of B. burgdorferi, the agent of Lyme disease in North America. Our results suggested that tHRF is presented in tick saliva and critical for tick feeding; blocking tHRF markedly reduced the efficiency of tick feeding, and reduced the B. burgdorferi burden in mice. This finding provides novel insights into the molecular mechanisms of tick feeding and provides a potential vaccine target to block tick feeding and pathogen transmission.Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Safe and effective vaccines against most tick-borne pathogens are not currently available. Typical vaccines target microbes directly, using extracts of the organism, or recombinant antigens as the immunogen; the transmission of tick-borne pathogens can also theoretically be prevented by interfering with the ability of ticks to feed on a mammalian host. In this study, we have characterized a putative histamine release factor (tHRF) from Borrelia burgdorferi (the Lyme disease agent), Anaplasma phagocytophilum (the cause of human granulocytic anaplasmosis), Babesia microti, and tick-borne encephalitis virus (TBEV), among other pathogens Ticks are distributed worldwide and affect human and animal health by transmitting diverse infectious agents. Ticks are considered to be second to mosquitoes as major vectors of human diseases B. burgdorferi outer surface protein A has been extensively studied and resulted in an Federal Drug Administration-approved vaccine that was commercially available from 1998 until 2002 I. ricinus in Northern Europe and Asia Effective vaccines against most tick-borne pathogens are not currently available and there is an urgent need for the control of ticks and their associated pathogens Boophilus engorgement on cattle Ixodes scapularis nymphs to feed Anaplasma marginale infection in some regions B. burgdorferi infection B. burgdorferi need to replicate within the ticks during blood feeding and are transmitted to the host after 36–48 h of tick attachment I. scapularis feeding could be another useful strategy to reduce B. burgdorferi transmission.The transmission of tick-borne pathogens can also theoretically be prevented by interfering with the ability of ticks to feed on a mammalian host Dermacentor variabilis ticks also express a protein in their saliva, which shares high homology with mammalian histamine release factor in vivo during tick feeding is not understood and warrants detailed examination.Tick saliva contains molecules that are important for formation and maintenance of the feeding cavity in the host dermis, as well as the transmission of tick-borne pathogens I. scapularis salivary gland proteome changes during these early and late phases of feeding Tick feeding can be divided into a series of 9 stages I. scapularis, the predominant vector of B. burgdorferi, the agent of Lyme disease in North America. We invoke a pivotal role for I. scapularis HRF during the rapid phase of tick feeding, and address the vaccine potential of this antigen in the context of tick engorgement and B. burgdorferi transmission.In this study, we have characterized a putative histamine release factor from B. burgdorferi to facilitate transmission, 2-dimensional fluorescence difference gel electrophoresis (DIGE) was performed using extracts from B. burgdorferi-infected, and uninfected, I. scapularis salivary glands. Seventeen differentially expressed proteins were selected for mass spectrometric analysis, and 4 I. scapularis proteins were unambiguously identified with significant MASCOT scores (p<0.05) of 79 . Immunoblots using tHRF antiserum further demonstrated a ∼2.5 fold up-regulation of tHRF in B. burgdorferi-infected ticks (To identify tick proteins that may be utilized by 5) of 79 . In thised ticks . tHRF waed ticks .B. burgdorferi transmission, tHRF-deficient I. scapularis nymphs were generated by RNA interference (RNAi). Buffer-injected (MOCK), SSRB or tHRF double-stranded RNA (dsRNA)-injected B. burgdorferi-infected nymphs were allowed to engorge on mice. The silencing of tHRF and SSRB were confirmed by quantitative RT-PCR , while 100% of the control animals were infected (N = 30) .I. scapularis nymphs also fed less efficiently on tHRF antiserum-treated mice . Immunoblots confirmed that mice generated antibodies against tHRF following active immunization . The ticrol mice . The spirol mice and in mrol mice and in jrol mice . 20–33% amplicon .Our above experiments focused on 72 h post tick attachment- a specific time point at which 30–40% of the ticks from the control groups successfully complete engorgement and drop off the mice, and the remaining ticks nearing engorgement. To address the role of tHRF on 72–96h post tick attachment, all the ticks were allowed to feed to repletion on tHRF antiserum immunized mice or control mice. While, ticks in the control group fed to repletion and detached around 72–84 h of attachment, ticks fed on tHRF-immunized animals fed to repletion around 96 h after attachment. Further, 10–20% of ticks from the tHRF group remained attached to the mouse even after 96 h, . The engin vitro binding assay was performed using a rat basophil cell line and recombinant tHRF . Flow cytometry and confocal imaging showed that recombinant tHRF bound to rat basophils . The last decade has seen an increased functional understanding of tick salivary proteins and their critical interactions with the host and pathogen The incidence of tick-borne diseases has steadily increased over the past few years, and effective vaccines against most tick-borne pathogens are not currently available B. burgdorferi within ticks may alter the expression level of selected genes that encode antigens in saliva salp15B. burgdorferi from infected ticks to mice, although Salp15 antibodies did not influence the ability of ticks to feed. The action is mainly due to the interaction between Salp15 antibody, Salp15 and BorreliaThe identification of tick proteins potentially involved in pathogen transmission is an important step in the development of effective tick vaccines Borrelia-infected tick salivary glands. HRF is an evolutionally conserved multiple-function protein Plasmodium falciparum parasite Dermacentor variabilisDermanyssus gallinaeWe performed a 2DIGE analysis to identify additional tick salivary proteins modulated by spirochetes. We found that tHRF was up-regulated in HBP) in I. scapularis nymphal salivary glands during this early phase of tick feeding and might be critical to counter the effect of histamine mice, 4 to 6 weeks of age, were obtained from the Jackson Laboratory.An infectious and low passage isolate of Animals were housed and handled under the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal experimental protocol was approved by the Yale University's Institutional Animal Care & Use Committee (Protocol Permit Number: 2008-07941). All animal infection experiments were performed in a Bio-safety Level 2 animal facility, according to the regulations of Yale University.I. scapularis salivary gland proteome was carried out by 2D fluorescence differential gel electrophoresis (DIGE) at the W.M. Keck Facility at Yale University. Salivary gland extracts from 200 clean and Borrelia-infected I. scapularis nymphs fed for 66–72 h were suspended in a cell lysis buffer and equal amounts of protein (50 µg) from Borrelia-infected and clean salivary gland extracts were then differentially labeled in vitro with Cy3 and Cy5 N-hydroxysuccinimidyl ester dyes as described in the Ettan DIGE manual and electrophoresis and analysis performed essentially as described earlier Borrelia-infected salivary glands were excised for identification. The gel spots of interest were robotically digested using trypsin prior to analysis on an Applied Biosystems 4800 MALDI-Tof/Tof mass spectrometer. The data was analyzed using the Applied Biosystems GPS Explorer software with Mascot analysis against the NCBI nr database, and a combined peptide mass fingerprint/MS/MS search was done. Spots identified with significant MASCOT scores (P<0.05) of 79 were tabulated.A quantitative analysis of the tHRF (GenBank accession no. DQ066335), and SSRB (GenBank accession no. DQ066202). The primer sequences are indicated in SacII-XhoI sites of the L4440 double T7 Script II vector in vitro transcription using the Megascript RNAi kit . The dsRNA was purified and quantified spectroscopically. The microinjection of dsRNA was performed as described previously 9 molecules per nl) or buffer alone (MOCK) into the ventral torso of the idiosoma of nymphal I. scapularis. The ticks were allowed to rest for 4∼6 hrs before feeding on mice.Fed-nymph salivary gland cDNA was prepared as described DNA was extracted from mouse tissues and ticks using a DNeasy tissue kit according to the manufacturer's protocol. The nymphal ticks were dissected under the microscope to get the tick salivary gland and midgut. Total RNA was extracted using RNeasy mini spin columns (QIAGEN). RNA was converted into first-strand cDNA using random hexamers and Superscript III reverse transcriptase according to the manufacturer's protocol.All quantitative PCR (Q-PCR) assays were performed with an iCycler using gene-specific primers, and IQ SYBR green quantitative PCR system (Bio-Rad) or a Taqman quantitative PCR system with a program consisting of an initial denaturing step of 3 min at 95°C and 45 amplification cycles consisting of 30 s at 95°C followed by 1 min at 60°C. The gene-specific primers used for Q-PCR were indicated in tHRF was amplified from the tick salivary cDNA library using gene specific primers P15, 16 using primers P17, 18 . The animals were boosted twice at 3-week intervals with the same dose of antigen in incomplete Freund's adjuvant, and the sera were collected 2 week after the second boost.To generate polyclonal antisera, tHRF (without the GST tag) produced in A western blot was performed to analyze the protein expression of tHRF in adult tick saliva, nymphal salivary gland extract, midgut extract and whole nymphs. The tick saliva and tissue extract were prepared as described B. burgdorferi-infected nymphal ticks or 10 non-infected nymphs were placed on each mouse. After 72h, the ticks were collected and weighed to analyze the feeding efficiency. For the B. burgdorferi-infected tick experiment, the Borrelia burden in ticks as well as in the localized skin specimen at 7 post tick repletion and in the murine heart and joints at 3 weeks post-infection were determined by measuring flaB copies using quantitative PCR.Groups of 5 mice were passively immunized with 200 µl of normal rabbit serum, anti-Salp25D antiserum (as controls) or anti-tHRF antiserum, respectively . After 60 h post tick attachment, the mice were examined every 12 h and the number of tick detached from the mice were recorded. The weights of ticks after repletion were measured as described above.In the active immunization study, groups of 5 mice were immunized by subcutaneously injecting 10 µg of purified recombinant tHRF suspended in complete Freund's adjuvant, or adjuvant alone (mock control). Mice were boosted with 5 µg of antigen suspended in incomplete Freund's adjuvant every two weeks. Before tick challenge, mice were bled and the anti-tHRF antibody titer was analyzed by western blot. The tick challenge and pathogen burden analysis were performed using the same methods described above.in vitro binding assay was performed as described previously E. coli) or GST in 1% FCS or buffer alone at 4°C for 2hrs. After 3 washes with PBST (PBS+ 0.1% Tween 20), the cells were incubated with purified Alexa 488 labeled anti-tHRF IgG or Alexa 488-anti-GST IgG in 1% FCS plus 1% rat isotype IgG buffer. The IgG labeling was performed using the Alex488 easy labeling kit according to manufacture's direction. After 3 washes with PBST, the cells were fixed with 4% PFA and permeablized with 1% Triton X-100, and the nuclei were stained with TOPRO3. The cells were then analyzed by microscopy and Flow Cytometry.To test whether tHRF binds to mammalian basophils, an −1 of endotoxin-free HRF (DES) or GST (as negative control) were added to confluent RBL-2H3 cells (in 2 ml media) and incubated at 37°C for 30 min. To determine whether, native tHRF in tick tissue extracts could also induce histamine release, 1.0 µg ml−1 nymphal tick salivary gland extracts were assayed for histamine release. Substance C48/80 , a calcium ionophore was used at 0.5 µg ml−1 for positive control. A histamine ELISA kit purchased from Research diagnostic Inc. was used to determine histamine concentrations in culture supernatants.To investigate whether tHRF can induce histamine secretion from basophils, a histamine release assay was performed using the method described B. burgdorferi infected nymphal ticks were fed on naïve mice for 60h. Then 10mM of histamine or 10 µg of recombinant tHRF were injected into the mouse skin at the tick bite site . Control mice were given the same amount of PBS or recombinant GST. At 72h post-tick attachment, the tick weights were measured and B. burgdorferi burden in ticks were analyzed by Q-PCR.To investigate the role of histamine and tHRF on late stage of tick feeding, t test with StatView software (SAS Institute).Results are expressed as the mean ± the SEM. The significance of the difference between the mean values of the groups was evaluated by Student's The GenBank accession numbers for the genes related with this study: tHRF/DQ066335; SSRB/DQ066202; Salp25D/AF209911; HBP1/DQ066014; HBP2/DQ066128; HBP3/DQ066002.Figure S1p<0.05. Representative results from at least 3 independent experiments were shown.tHRF antiserum significantly delays tick feeding. A) Assessment of percentage of replete ticks at different time points post tick attachment on immunized mice. . Results are expressed as the mean + the SEM. B) The weights of replete ticks. Horizontal lines and bars represent the mean values ± the SEM. * (0.57 MB TIF)Click here for additional data file.Table S1Borrelia burgdorferi-infected nymphal salivary glands by Matrix-Assisted Laser Desorption/Ionization.2D Fluorescence Differential Gel Electrophoresis (DIGE) analysis and identification of proteins with increased expression in (0.03 MB DOC)Click here for additional data file.Table S2Oligonucleotide primers and probes.(0.05 MB DOC)Click here for additional data file.
We attempted to clone candidate genes on 10p14–15 which may regulate hTERT expression, through exon trapping using 3 BAC clones covering the region. After obtaining 20 exons, we examined the function of RGM249 (RGM: RNA gene for miRNAs) we cloned from primary cultured human hepatocytes and hepatoma cell lines. We confirmed approximately 20 bp products digested by Dicer, and investigated the function of this cloned gene and its involvement in hTERT expression by transfecting the hepatoma cell lines with full-length dsRNA, gene-specific designed siRNA, and shRNA-generating plasmid.RGM249 showed cancer-dominant intense expression similar to hTERT in cancer cell lines, whereas very weak expression was evident in human primary hepatocytes without telomerase activity. This gene was predicted to be a noncoding precursor RNA gene. Interestingly, RGM249 dsRNA, siRNA, and shRNA inhibited more than 80% of hTERT mRNA expression. In contrast, primary cultured cells overexpressing the gene showed no significant change in hTERT mRNA expression; the overexpression of the gene strongly suppressed hTERT mRNA in poorly differentiated cells.These findings indicate that RGM249 might be a microRNA precursor gene involved in the differentiation and function upstream of hTERT. Telomerase, which adds repeated telomere sequences to chromosome ends, is a ribonucleoprotein enzyme that includes an endogeneous RNA (hTR) which acts as a template and a reverse transcriptase (hTERT), and are crucial molecules -4. TelomWe used the RNA interference (RNAi) method, which is a sequence-specific post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA), to cause degradation of mRNAs homologous in sequence to dsRNA and to effectively inhibit gene-specific expression . BecauseIn this report, we present a summary of our cloning research and introduce functional analyses regarding the identified gene regulating hTERT mRNA.We investigated the 3 BAC clones using a sequence-tagged-site marker (D10S1728) by PCR, based on previous reports ,19. FromApproximately 90%~95% of the BAC clone H-11 sequence and supercontig were determined (studies were performed by the University of Colorado Cancer Center Sequencing Core). D10S1728 was regarded as the sequence on 10p15.3. We reconfirmed that the sequence and supercontig were derived from the original BAC clones by Southern blotting and PCR, and 20 exons were thereafter identified fig . We perft-test and P = 0.040 by paired t-test) (fig Using a probe against each exon sequence (47–376 bp), Northern blotting was performed, which demonstrated that several exons showed a very faint signal (data not shown). In particular, a very faint single hybridization signal in human Multiple Tissue Northern (MTN) blot (Clontech) migrating at about 250 bp was determined for RGM249. One-step real-time RT-PCR showed a very low expression rate, that is, less than 20,000 copies per 50 ng of total RNA extracted from human organs fig . In normest) fig .. Although their functions remain unknown because we have not fully clarified whether RGM249 is actually processed by Dicer in vivo, given that it can be processed by Dicer in vitro. On the other hand, a motif search predicted no motifs in the RGM249 gene. Because this gene generated no protein, the secondary structure of RGM249 was drawn based on the structure provided at (fig 5' and 3' RACE was performed using liver c DNA libraries (Clontech). Screening of the cDNA libraries yielded a clone with a full sequence length of 249 bp. A comparison of the cDNA sequence with the genomic sequence from H-11 revealed that the gene of interest was extended across 9 kb. The cDNA named RGM249 consisted of 2 exons and the RNA was not poly-adenylated fig . The seq at fig . Moreove at fig .EF433558.Sequence data have been deposited with the EMBL/GenBank Data Libraries under accession no. In many hTERT- or hTR-regulating molecules reported previously, only two factors, hTERT and ribosomal protein L22 (RPL22), were transcriptionally suppressed in the hepatoma-derived cell lines (HLF) transfected with full-length RGM249 dsRNA fig 31]. To . To 31].RGM249 siRNA suppressed more than 90% of hTERT mRNA, approximately 50% of RPL22 mRNA, and RGM249 transcripts fig unlike hSubsequently, we examined the inhibitory effect of shRNA transfection. Compared with LacZ shRNA, we found that RGM249 shRNA and mt-1RGM249 shRNA significantly downregulated hTERT mRNA . FACS analysis showed that RGM249 mRNA expression introduced more than half of RGM249 pRNAT/U6.1/neo/GFP-transfected populations into the preG1 phase is rare, except at 10q26.11-qter, which is observed in 25% of HCC cases (32). However, relatively frequent alterations and deletions of chromosome 10p have been identified in human tumors, including glioblastoma multiforme (GBM) and malignant melanoma ,34. In Gin vitro trial)It is well known that a miRNA, generated from a miRNA precursor gene, can act as a new regulator which induces cancer cell lines to adopt a suppressive phenotype, except for the tumor suppressor gene. Because the phenotype we observed in a series of transfections with a chromosome (fragment) or BAC was suppressed, this miRNA might be a Suppressor-miR or it might have demonstrated a miRNA-directed inhibition. Actually, RGM249 repressed hTERT mRNA in A172 cells derived from GBM, moderately differentiated HCC (HMc-Li7), and poorly differentiated HLF. Beyond 60S RPL22, which we referred to in this study, several ribosomal protein mRNAs changed their expression level after the introduction of siRNA or shRNA into cell lines or tumors inoculated in nude mice (data not shown). In a miRNA-mediated mRNA repression study, RPL22, of which Greider et al. previously referred to as one of the binding protein to hTR that is essentially required for telomere maintenance and telomerase activity in normal tissues and disease, showed no significant alteration in expression level, whereas it showed an obvious change in siRNA-directed RNAi with reproducibility . We do nmiRNA is physiologically generated by both Drosha and Dicer. We can not digest RGM249 mRNA by Drosha because Drosha protein is now unavailable commercially. RGM249 RNA processed by DICER, results in at least 3 small fragments of a product (17–23 bp) related to RNA species fig . The seqAlthough the way in which RGM249 regulates hTERT remains unidentified, the physiological expression of RGM249 synchronizes the upregulation of hTERT mRNA, and its excess expression using the RNA-expression vector downregulated hTERT in specific cancer cells. Moreover, its siRNA or shRNA suppressed hTERT. Taken together, RGM249 may function negatively or positively, reversibly or indirectly on unknown mediators upstream of hTERT. Using microarray analysis between transfectants with RGM249 siRNA and those with LacZ siRNA fig , the comin vitro and is essential in vivo. Beyer et al. reported that the downregulation of another mediator complex (MED28) expression in NIH3T3 cells results in a significant induction of several genes associated with smooth muscle cell (SMC) differentiation and functions as a repressor of SMC differentiation [This novel noncoding gene is strongly expressed in poorly differentiated tumor cells. The genes (mRNAs) targeted commonly by both miRNAs and siRNAs in our study were MAPK6 and PRKCA, which can induce hTERT upregulation fig . Representiation . A mediantiation . MED18 mTaken together, the enhanced cell death in RGM249 transfectants likely resulted from an hTERT-independent mechanism or the presence of a mediator with crucial responsibility for oncogenesis, arguing that some other targets instead of hTERT might be the real players mediating these contradictory oncogenic and growth suppressive functions. It should be noted that mRNA studies do not always reflect processes at the protein level of gene regulation.Further studies will be required to investigate these expression levels. Several ncRNAs in the exons we trapped might provide new insights into the coordinated and dynamic mechanisms of development, stress, transformation, and (de)differentiation, resulting in the possible prevention of oncogenesis and suppression of tumor progression.BAC clones were selected on the basis of results from a previous experiment . BAC DNAPurified BAC DNAs were shared using the AERO-MIST nebulizer and after ligation of adapters, these DNAs were subcloned into the BstXI sites of pSHOT II, which was constructed by subcloning the BstXI sites from pcDNA II, prior to the disclosure of the endosequences of the 3 BAC clones fig . A seque. In 1999 to 2005, the sequenced reference human genome draft was not definitely determined on 10p14–15 and altered several times during this interval.For all exon trapping experiments, we used the exon-trapping system kit and the supplements recommended therein. We shotgunned M-6, H-11, and J-21 into pSPL3, employing BamHI and BglII or BamHI, BglII, and PstI for 2 ways of cutting BAC DNA using restriction enzymes. This method was performed as described previously [The probe, consisting of the respective exons, including two exons forming RGM249, radiolabeled by random priming using the Radprime kit (Gibco-BRL), was incubated with human multiple tissue northern blot (Human 12-Lane MTN Blot) . One-step real-time RT-PCR experiments were performed using Human Total RNA Master Panels (Clontech) according to methods we previously reported using LightCycler . The priUsing the exons identified by Northern blotting or RT-PCR, we amplified cDNA fragments from human skeletal muscle and liver "Marathon-ready" cDNA, according to the manufacturer's protocol (Clontech). Amplicons were subcloned into the pcDNA3 vector . A comparison of cDNA and genomic sequences revealed a total of 20 exons and detected 3 alternative splice donor sites for 6 exons.To screen for point mutations in trapped exons, we performed PCR-SSCP assays using flanking intron primers, as previously described .To confirm telomerase activity in transfectants after siRNA transfection, a telomerase detection kit, TRAP-eze, used for RGM249 cloning and the TeloChaser kit used for the expression analysis of shRNA transfectants, were employed for the telomeric repeat amplification protocol assay according to the manufacturer's protocol. Quantification of TRAP was estimated using an image analyzer provided by ATTO Corp. .in vitro translation assay according to the manufacturer's instructions (Clontech). After examining the motif and homology ; ; ), we predicted the secondary structure of RGM249 using the Vienna RNA Secondary prediction and comparison program ; ). Subsequently, we screened the micro RNA) (miRNA) database among species. To confirm whether the RGM249 gene functions as a miRNA precursor gene, we digested the full-length RNA with a Dicer enzyme using a Dicer siRNA generation kit according to the manufacturer's protocol .To examine whether RGM249 can generate a protein, we performed an To confirm the transcriptional level (copy number) in human organs, liver-related normal cells, and immortalized cells, we performed one-step real-time RT-PCR using human hepatocytes , hepatoma cell lines , and 74 surgically resected liver tissue samples, including 33 pairs of hepatomas and corresponding adjacent tissues. We measured each copy number 3 times and calculated the average.After ligasing a synthesized RGM249 into the pRNAT/U6.1/Neo/GFP vector digested at the KpnI/SacI site, it was transfected into several cell lines , with NH-12 as senescence-programmed cell lines, and keratinocytes and TIG 1–20 as primary cells. Immortalized cells were transfected with FuGene to obtain stable cells overexpressing RGM249. After enhanced green fluorescent protein expression in the presence of a neomycin-resistant gene in all cell types was confirmed, β-gal staining and RT-PCR were performed. Primary cultured cells were electroporated using Nucleofector technology under optimal conditions. At least 5 transfectants were examined in mock or RGM249 transfectants. The respective gene expression (GE) ratio to β-actin mRNA in mocks or RGM249 tranfectants was analyzed using the Mann-Whitney test. Between both transfectants, FACS analysis of HLF cells was performed using Guava EasyCyte Mini according to the manufacturer's instructions .in vitro transcription method and was transfected into 4 hepatoma cell lines at a concentration range of 10–40 μg/ml medium. Subsequently, to avoid unspecific inhibition of dsRNA through interferon (INF) activation [dsRNA derived from the RGM249 sequence was generated from the expression vector with RGM249 in the MCS of Litmas28i using an tivation ,24, steativation . The expTwo kinds of siRNAs (siRNA ① and siRNA ②) were designed using the Stealth RNAi designer and synthesized by Invitrogen . The sequences are shown in Table Expressing vector (Invitrogen) which was designed to generate shRNA in transfectants. To examine the effect on hTERT expression 7 to 10 days later after selection with 200 μg/ml Zeocin, an shRNA-generating lentiviral vector with the functional sequence of RGM249 fig was tran and typical GE changes are shown in fig .Microarray analysis was performed for total RNA purified from transfectants with LacZ shRNA and from those with RGM249 shRNA following confirmation of the downregulation of both RGM249 and hTERT mRNA expression by RT-PCR after strict quality control in accordance with the guidelines of the Hokkaido System Science . Data for genes that had changed more than twofold were additionally analyzed using Gene Ontology Tree Machine hTR: telomerase RNA component; BAC: Bacterial artificial chromosome; hTERT: human telomerase reverse transcriptase; siRNA: short interfering RNA; shRNA: short hairpin RNA; RPL22: ribosomal protein L22; ncRNA: noncoding RNA; GE: gene expression.The authors declare that they have no competing interests.MN and SR carried out the molecular genetic studies. WJ and DH participated in the sequence alignment and drafted the manuscript. MJ kindly provided the exon trapping vector. SG kindly provided the clinical specimens. MN and VA participated in the study design and performed the statistical analysis. Other authors conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. All contributors who do not meet the criteria for authorship are listed in Acknowledgements.A comparison of hTERT mRNA expression between mock and RGM249 transfectants. In response to upregulated RGM249 mRNA, hTERT mRNA did not induce any significant change in senescence-programmed cells except for KMST-6 cells. The dotted line indicates hTERT mRNA expression in parental cells. Six transfectants were examined in both mock and RGM249 transfectants. (b) Actin mRNA and RPL22 mRNA showed no significant difference among untransfected cells and mock or RGM249 transfectants in the 4 cell lines described in (a). (c) Comparison of hTERT mRNA expression between mock and RGM249 transfectants. In response to upregulated RGM249, hTERT mRNA induced a significant change in suppression in (left) HLF cells (poorly differentiated hepatoma) and (right) A172 cells (glioblastoma). The dotted line indicates mean mRNA expression in untransfected parental cells. hTR mRNA and b-actin mRNA showed no significant differences in b-actin, hTERT, RGM249, and hTR among untransfected cells and mock or RGM249 transfectants. (d) By transfection of MED18 siRNA or MED18/hTERT siRNA into A172 cells, an insight into the regulative network among 3 genes could be obtained. The number of respective inductions was more than 5. Following transfection of MED18 siRNA, both TERT mRNA and RGM249 mRNA were significantly upregulated (P < 0.01). Following co-transfection of MED18 siRNA and TERT siRNA, RGM249 mRNA was significantly upregulated (P < 0.05).Click here for fileA representative case of microarray analysis and microRNAarray analysis. Microarray analysis (a) and miRNA array analysis (b) were performed for total RNA purified from transfectants with LacZ shRNA and from those with RGM249 shRNA. The representative analyses are demonstrated, respectively.Click here for file
Organised crime and political violence (OPV) and human rights violations have marred Bangladesh history since 1971. Little is known about the consequences for the oppressed population. This study describes the patterns of OPV and human rights violations in a disturbed area of Bangladesh and assesses the physical, emotional and social functioning of victims.A total of 236 of selected participants in a household survey in Meherpur district were recruited for a detailed study. Interviews and physical examinations were used to obtain information about history of torture and other cruel, inhuman or degrading treatment or punishment (TCIDTP), and about injuries, pain frequency and intensity. Handgrip strength and standing balance performance were measured. The "WHO-5 Well-being" scale was used to assess the subjective emotional well-being of study participants.The majority of the reported cases of TCIDTP occurred in 2000-2008, 51% of incidents occurred during winter; 32.0% between 20:00 and midnight. Police involvement was reported in 75% of cases. Incidents took place at victims' homes (46.7%), or at the police station, military camp, in custody or in prison (21.9%). Participants experienced 1-10 TCIDTP methods and reported 0-6 injury locations on their bodies; 77.5% reported having at least two injuries. Less than half of the participants were able to stand on one leg for 30 seconds. Only 7.5% of males aged 25-44 had handgrip strength in both hands exceeding average values for healthy people at the same age. Over 85% of participants scored low (<13) on the 25-point "WHO-5 Well-being" scale. The number of years since the TCIDTP event, pain frequency, the need to quit a job to take care of an injured family member, political involvement, personal conflicts and the fear of neighbourhood violence strongly affected emotional well-being. Good emotional well-being correlated with increased political and social participation.A detailed picture of characteristics of the victimisation is presented. The participants showed poor emotional well-being and reduced physical capacity. The results indicated that the simple and rapid method of assessment used here is a promising tool that could be used to monitor the quality and outcome of rehabilitation. A clinical and functional assessment of the victims is essential when evaluating the effects of organised crime and political violence (OPV). Many studies have suggested that OPV has long-term psychological effects on an individual that persist throughout his or her lifespan. However, research documenting whether OPV results in the deterioration of physical and functional fitness, as well as social relationships, is limited -10.et al. [Bangladesh has been a victim of a power struggle between its two major parties since it gained independence. The dominant parties have used armed groups and militias to fight for power ,12. The et al. measuredThe aims of the present study were to describe the history and pattern of OPV and human rights violations in a border region of Bangladesh, and to measure their consequences for the physical and emotional fitness and social functioning of the victims. It was a pilot study using a simple and rapid method of assessment, which could be further developed for use in countries with limited resources. Such a tool would be valuable for generating a baseline for monitoring the quality and outcome of rehabilitation services.Our hypothesis in developing the method was that various personal factors, inter-personal relationships, and the extent of political involvement and social participation play a crucial role and interact with the current physical and emotional fitness and functional capacity in victims of OPV and human rights violations. Another hypothesis of the study was that reduced handgrip strength, and poor performance in standing balance measurements - both of which can be simply and inexpensively measured - would be related to having suffered from OPV and human rights violations.Meherpur district, located within the Khulna division and bordered by India's West Bengal to the north, has an area of 716 square kilometres. It is the smallest district in Bangladesh, with an estimated population of 680,000 in 2008. Khulna division has been heavily affected by different forms of violence since the government launched two security operations: Operation Clean Heart in 2002 and Operation Spider Web in 2004.A protocol was generated based on the World Health Organization (WHO) document "Guideline for conducting community surveys on injury and violence" , with a The second part of the study, reported in this paper, was a retrospective study in which a detailed OPV screening was carried out. The families that participated in the household survey and reported one or more of the following experiences were selected for OPV screening: 1) torture and other cruel, inhuman or degrading treatment or punishment (TCIDTP) (n = 343); 2) sexual harassment, molestation, rape or inserting blunt object into a genital organ and/or rectum (n = 9); 3) arrest and detention without warrant or order (n = 317); or 4) extrajudicial execution of family members (n = 78), perpetrated by members of law enforcement agency. This study used the definition of TCIDTP adopted by the United Nations Convention against Torture and Other Cruel, Inhuman or Degrading Treatment or Punishment. Altogether, 495 families were identified.The selected families were given vouchers that outlined the aims and the process, including the offer of free medical examination and treatment at mobile clinics. We deployed four mobile clinics: one in Meherpur upazila, and one in Mujibnagar upazila on 12 March, 2008, and two in Gangni upazila on 13 March. The mobile clinics teams consisted of two coordinators, five medical doctors, four physiotherapists, two pharmacists from Dhaka and 12 social workers recruited from a Meherpur-based non-governmental organisation (NGO) Manab Unnayan Kendra, and from Dhaka University.The 356 people who volunteered to participate were taken in buses to one of the mobile clinics. At the entrance to the clinic the participants were screened again by one of the coordinators. Only people who were victims or secondary victims were included in the study. After the screening process, we excluded 120 participants who were either mentally ill or reported no experience of TCIDTP or others forms of OPV and human rights violations mentioned above. The excluded participants were not interviewed, but they were given a routine medical examination and treatment. Altogether, 236 victims were identified and recruited for the study. Their personal information was collected, including age, sex, education, religion, occupation and area of residence. The questionnaire was developed in English and translated into Bengali. After structured interviews, a physiotherapist or a specially trained interviewer measured height and weight, handgrip strength, and standing balance. A medical doctor performed a physical examination including a routine medical check-up, pulse measurement, and injury examination and provided a consultation. Injuries were recorded on a body map adapted from the Istanbul Protocol: international guidelines for the investigation and documentation of torture. Information about pain frequency and intensity was also obtained.For the collection of data on body function, activity, participation and environmental factors a 14-point scale was constructed, based on the WHO International Classification of Functioning, Disability and Health , were also recorded. Under malnutrition conditions, muscular strength decreases before changes in anthropometric and laboratory parameters are noticeable -20. Handherapists. We usedi.e., the central vestibular system, vision, and proprioception (giving information to the brain about leg muscle position). Participants were excluded from testing if they were blind or had amputations or deformities of lower limbs. Participants were asked to stand barefoot on a flat floor on one foot with their eyes open. The physiotherapist/interviewer instructed the participants to adjust the standing position. A silent stopwatch was used to measure the time that each participant could balance on one foot without assistance; participants were asked to stop after 90 seconds.The equilibrium function of the participants was measured by the standing balance test. The test measures the ability to stand on one leg, making use of all the sensory inputs that contribute to balance, The "WHO-5 Well-being" questionnaire was used to assess the subjective emotional well-being of study participants. The raw score is calculated by summating the figures of the five answers. Raw scores ranged from 0 to 25. A score of 0 represents the worst possible, and 25 the best possible quality of life. A raw score under 13 is considered indicative of poor emotional well-being.Data were entered and validated in Microsoft Access 2000 and Epi Info™ 6.04 . For quality assurance, the data set was checked three times for typing discrepancies and transposition errors. All analyses were carried out using Stata software, version 9.2 . The 5% significance level (P < 0.05) was used for all analyses. Explanatory variables for personal factors were age group, sex, residence, education, occupation, religion, political party affiliation and level of political and social participation. A generalised linear model was used to study the association between binary outcomes and explanatory variables, adjusted for the effects of other variables.The study is compliant with the Declaration of Helsinki and with Danish law. The Bangladesh NGO Bureau gave approval for the study. All of the participants gave informed consent. Treatment was free and severely traumatised cases were referred to the Bangladesh Rehabilitation Centre for Trauma Victims. The confidentiality and safety of participants is our primary concern. All the procedures used were designed to protect the privacy of the participants and the confidentiality of the information. It can be a risk for the oppressed population to participate in such a study; therefore, both the Bangladesh Rehabilitation Centre for Trauma Victims and the Task Force against Torture will be following up on the legal and health needs of victims once our study is completed. The sponsor had no role in the study design, data collection, analysis, and interpretation, and writing of the report.The sample population characteristics are presented in Tables The level of political involvement was considered because during the household survey, the affiliation of a family member to a political party was found to be a risk factor for victimisation . OverallOver two-thirds of participants reported having personal, financial or political conflicts with other people. Over half of participants had been out to visit their friends within 7 days. When they were asked about their fear of violence in the community, 22.1% of participants said they had no fear of violence in the community, but 7.5% said they were often afraid, and 5.0% that they were always afraid of violence.The earliest cases of TCIDTP reported by the participants occurred as early as 1971 when the liberation war against Pakistan took place Figure . Only a i.e., in a police station, in a military camp, in custody or in prison (21.9%). Only 10.7% incidents occurred in open areas on streets or motorways were subjected to TCIDTP during the 4.5-month cool and dry winter season (mid October to February), while 76 participants (32.9%) recalled the incident being during the 4-month hot and humid summer season (March to June) and 37 participants (16.0%) during the 3.5-month rainy monsoon season (July to mid October). Nearly one third of participants alleged that they were subjected to TCIDTP between 20:00 and midnight, which is the peak hour Figure . Events s Figure . The parThe mean number of TCIDTP methods reported by each participant was 3.0 with a SD of 2.0. The maximum was 10 (reported by two members of Awami League). Around 20% of the participants (n = 46) were subjected to at least five methods. Among them, 19 were from the village called Shibpur and eight were from the village of Monakali; both of which are located along the Bangladesh-Indian border in Mujibnagar upazila. The five most frequently applied methods of TCIDTP were: 1) kicking; 2) beating with a blunt object; 3) falanga (beating the soles of the feet); and 4) threats to the family and mock execution; 5) other psychological torture , while 20 acts of TCIDTP (8.2%) were committed by the Bangladesh Rifles (Bangladesh border force), 15 (6.2%) by the Rapid Action Battalion (anti-crime and anti-terrorism elite force) and five (2.1%) by terrorists Figure . Among fThe number of reported injury locations ranged from 0 to 6: 33.5% of participants reported having one injury location, and 66.5% reported having at least two Table . The thr2) recommended for a Southeast Asian population by Singaporean and Indian researchers [Height and weight were measured for 228 participants , right hand strength dominated left hand strength. We found that five participants had no strength in either hand, and two had no strength in their left hands. Lacking the national average for the general population in Bangladesh, we compared the mean handgrip strength of male participants aged 25-44 years old with that of a reference group of 100 healthy males aged 27-42 years in West Bengal: that of 50 office workers was 43 kg and that of 50 metal workers performing intensive manual labour was 41 kg . The meaIn total, 229 participants were able to take the standing balance test. The mean duration of standing balance on the right foot was 44.5 seconds (95% CI: 40.1-48.8) and the mean duration for the left foot was 44.4 seconds (95% CI: 40.1-48.8). The difference between the mean duration of standing balance between male and female participants is shown in Table Standing balance is negatively correlated with age over 55 years , but there is no association with gender. Obese people (BMI≥27) have more difficulty to maintain a standing balance , as compared to people of normal size (18.5≤ BMI<23.0) or underweight and malnourished people (BMI<18.5). After adjusting for age and BMI and the cluster effect of villages [One of the questions we asked participants was whether they had difficulty in walking to the other side of their village, or whether they needed help. The distances involved depended on which village a participant lived in. Odds ratios for good standing balance adjusted for age, BMI and the cluster effect of village were 2.94 for people who reported no difficulty in walking to another side of their village, and 2.67 for people who needed assistance to walk this distance. The self-reported difficulty of walking thus correlates with the objective measurement on standing balance. No statistical association was found between standing balance and the number of years that had passed since the incidence of TCIDTP or between standing balance and the self-reported pain intensity and frequency during the two weeks preceding the survey, or to being subjected to falanga and being kicked.Of the 225 participants who completed the "WHO-5 Well-being" questionnaire (one participant was missing), 194 (85.84%) scored less than 13 (Table We categorised the victims into four different groups based on the methods of TCIDTP they were subjected to. The "basic level" consists of 1) beating with a stick or a blunt object, 2) blindfolding and 3) kicking. The "basic + psychological torture level" indicates the "basic level" plus psychological torture. Victims who were subjected to torture at a "basic level" and in addition to any severe torture method were grouped as "basic + severe torture cases". The victims who were subjected to torture at a "basic level" and in addition to psychological torture plus any of the severe torture methods were grouped as "all". Out of 224 victims (two participants missed reporting any TCIDTP method), 46 (20.5%) had been subjected to the "basic level" of TCIDTP while 41 (18.3%) and underwent "basic + psychological torture" and 35 (18.3%) underwent "basic + severe torture". A further 102 of the 224 victims (45.4%) were subjected to "all" TCIDTP methods. No statistical association was found between the types/levels of TCIDTP methods victims were subjected to and either their physical performance , or their emotional well-being.The present study provides a detailed clinical and functional assessment of a sample of 236 participants in a border area in Bangladesh who reported during a household survey that theThe participants' reports did not only provide information about individuals, but about the wider history of OPV and human rights violations. The earliest reported incident in our study was in 1971. The first arrest reported in the patient records of the Bangladesh Rehabilitation Centre for Trauma Victims was also in 1971. There may have been incidents before the liberation war, but there is some doubt as to whether the victims are still alive. In our study, only a few incidents were reported from 1971 to 1990. From 1991 to 1995 there were still relatively few, but the frequency doubled in 1996-1999, and since 2000, the reported numbers have increased sharply. The years 1971, 1990-91, 1996-97 and 2000-01 are crucial in the history of OPV and human rights violations in the whole of Bangladesh. In these years, there was a high level of political tension and violence; this pattern is shown in our study and also reported by other institutions ,12,25,26Human rights violations including the use of TCIDTP have intensified in the last five years in Meherpur district since Operation Spider Web was launched, and violence and crime have been aggravated. Since 2006, the enforcement of strict emergency regulations by a care-taker government has increased the brutality of violent confrontations.The results also showed a seasonal pattern. The number of reported cases was three times higher during the winter season than during the rainy season. It is clear that the police and the military are hampered by the flooding and uncomfortable conditions during the rainy season.From the results of our study it appeared that the police were the major perpetrators. Individuals could be attacked by armed militias associated with party politics, but such incidents were not reported here. Such individuals were not likely to present in the mobile clinic, because the recruitment criteria were primary or secondary victims subjected to any of four categories of OPV and human rights violations perpetrated by the members of law enforcement agency. Mitchell (2004) pointed out that the degree of political control that the political authorities exercise over a law enforcement agency varies across time and political system. It is plausible that if individuals in a law enforcement agency have goals independent of those of the authority, or have private interests, this may influences the choice, level and method of violence .The survey provided detailed information about the methods used by the perpetrators. A previous study in Sri Lanka suggested that perpetrators generally use readily available materials as TCIDTP instruments . Methodsi.e. detention and prison, has been neglected by the authorities. A low level of control of corruption in the administration is likely to provide the members of law enforcement agency with a wealth of opportunities for hidden actions including the perpetration of sexual violence [Forced sexual contact and sexual abuse are also frequently-used forms of torture. It was reported that all of the female refugees and one-third of the male refugees from Bangladesh examined at the Centre for Torture and Trauma Survivors in Stockholm alleged that they had been raped . In our violence . It is aVery little is known about the physical and emotional consequences and social functioning of an oppressed population experiencing collective exposure to OPV and human rights violations. The rehabilitation of TCIDTP victims has been mainly based on clinical experiences seen from an illness perspective. For people who continue to live in their communities, it is most important that they should be able to maintain daily life after being traumatised. A basic level of muscle strength and physical mobility is required, simply to be able to cope with the activities of daily life.In our study we assessed muscular function by measuring handgrip strength, which is required in many daily activities in a rural area. Hand dynamometer testing is recommended to determine the loss of handgrip strength -38. ThisOur study was also the first to assess standing balance performance in an oppressed population. Impaired lower extremity performance is associated with reduced physical activity levels, which may contribute to subsequent disability in elderly persons ,42. StanThe measures used to test muscle strength and physical mobility in our survey are rapid and inexpensive, so they are appropriate for use in countries with limited resources. Such tests can be used to produce essential information for purposes of diagnosis and prognosis, as well as for prevention and rehabilitation. The results could also contribute to quantifying the economic burden in terms of disability and manpower lost due to OPV and human rights violations, and their association with poverty in a country such as Bangladesh.A person's functioning does not only depend on muscular strength but also on emotional factors. We used the brief questionnaire "WHO-5 Well-being" to estimate the percentage of victims with poor subjective emotional well-being, and to examine the association of subjective emotional well-being with social participation. Emotional, physical, and social vulnerability as a consequence of being abused is related to the development of post-trauma stress disorder and other mental disorders. The inter-personal and inter-family conflicts are high in the study area. In order to develop programmes which can help to reduce the damage to emotional health and to prevent its harsher effect on mental health, we need to understand what factors may empower victims to cope with their vulnerability.One factor that we considered was active participation in political or social movements. The effects of political and social participation on well-being are complex. On the one hand, in the household survey we found that if a family member was affiliated to a political party or participated in a demonstration, a strike or a human rights rally, this was a risk factor for victimisation . On the Our findings concerning perpetrators and years of perpetrations were consistent with reports from other international institutions ,12,25,26One limitation was that owing to logistical and political constraints, we did not recruit people without prior TCIDTP experience as a healthy control group while taking into account neighbourhood effect on OPV or human rights violations. In addition, there was inevitably some risk of bias in the recruitment of the study group. The health-seeking behaviour of the individuals concerned is one possible source of bias. We do not know why some who had vouchers decided not to come to the mobile clinic. Some of them may have been so severely depressed that they lacked any motivation to come - and others may have seen no need to interrupt their work to come to the clinic. It is also possible that there was some bias because a few participants exaggerated their injuries and pain experiences in order to receive more treatment. Memory bias does exist, but the main increase in OPV and human rights violations in this area took place within the last 10 years, and ten-year recall is considered reliable .We had hoped to use the results as a baseline for a large-scale intervention in this community, and to repeat the measurements afterwards to monitor the quality and outcome of rehabilitation. We had already developed plans for various interventions including setting up a platform, Victim Association, where the survivors can talk about their fears and stigmas and re-construct their self-images, and which will also serve as a place for social participation and political empowerment. The members should assist the Victim Association to raise the community awareness and spread knowledge about human rights by organising various community activities. Unfortunately, these cannot immediately be realised for unexpected reasonsThis study presents trends and patterns of OPV and human rights violations in the Meherpur district. Generally, the oppressed population showed poor emotional well-being and a reduced level of physical fitness. Among study participants, good subjective emotional well-being correlated with increased political and social participation. To monitor the quality and outcome of rehabilitation, the simple and rapid methods used here could be developed into a valuable tool for initial assessment. Further studies are also needed to establish reference values in the general population and oppressed populations within this complex setting.BNP: Bangladesh Nationalist Party; BRCT: Bangladesh Rehabilitation Centre for Trauma Victims; BMI: Body Mass Index; OPV: organised crime and political violence; OR: odds ratio; NGO: non-governmental organisation; RCT: Rehabilitation and Research Centre for Torture Victims; SD: standard deviation; TCIDTP: torture and other cruel, inhuman or degrading treatment or punishment; WHO: World Health Organization.The authors declare that they have no competing interests.SJW participated in the design of the study, conducted the field work, analysed and interpreted data and drafted the manuscript. MAH, SDM and SB participated in the coordination in the field, managed and supervised the data collection. JM participated in the conception of the work, helped to draft the manuscript and revised it critically. All the authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-698X/9/31/prepub
Schistosoma japonicum in order to shed light on their role in schistosome biology.Schistosomes depend for growth and development on host hormonal signals, which may include the insulin signalling pathway. We cloned and assessed the function of two insulin receptors from S. japonicum, insulin receptors 1 (SjIR-1) and 2 (SjIR-2) sharing close sequence identity to their S. mansoni homologues (SmIR-1 and SmIR-2). SjIR-1 is located on the tegument basal membrane and the internal epithelium of adult worms, whereas SjIR-2 is located in the parenchyma of males and the vitelline tissue of females. Phylogenetic analysis showed that SjIR-2 and SmIR-2 are close to Echinococcus multilocularis insulin receptor (EmIR), suggesting that SjIR-2, SmIR-2 and EmIR share similar roles in growth and development in the three taxa. Structure homology modelling recovered the conserved structure between the SjIRs and Homo sapiens IR (HIR) implying a common predicted binding mechanism in the ligand domain and the same downstream signal transduction processing in the tyrosine kinase domain as in HIR. Two-hybrid analysis was used to confirm that the ligand domains of SjIR-1 and SjIR-2 contain the insulin binding site. Incubation of adult worms in vitro, both with a specific insulin receptor inhibitor and anti-SjIRs antibodies, resulted in a significant decrease in worm glucose levels, suggesting again the same function for SjIRs in regulating glucose uptake as described for mammalian cells.We isolated, from S. japonicum possess insulin receptors that can specifically bind to insulin, indicating that the parasite can utilize host insulin for development and growth by sharing the same pathway as mammalian cells in regulating glucose uptake. A complete understanding of the role of SjIRs in the biology of S. japonicum may result in their use as new targets for drug and vaccine development against schistosomiasis.Adult worms of Schistosomes are parasitic blood flukes infecting approximately 200 million people globally As sensing and responding to environmental factors are essential in the complex life cycle of schistosomes, studying on signal transduction molecules and their functional mechanisms will be necessary for elucidating schistosome host-parasite interactions and parasite biology Schistosoma mansoni epidermal growth factor receptor (SER), which contains a conserved intracellular tyrosine kinase domain and an extracellular domain for binding epidermal growth factor (EGF) ligands, was the first RTK described in schistosomes S. mansoni in vitro, indicating a possible role for EGF signaling in the host-parasite relationship and in parasite development S. mansoniDiverse molecular pathways dependent on kinase signalling have been described in schistosomes and shown to be involved in the host-parasite relationship Transcriptomic and proteomic profiling has revealed that, similar to their mammalian counterparts, schistosomes encode a panel of growth factors and cytokines including EGF-like peptides and fibroblast growth factor (FGF)-like peptides, as well as receptors for thyroid and steroid hormones S. japonicum were up- or down-regulated in response to insulin; these genes were involved predominantly in males worms in the promotion of protein synthesis and the control of protein degradation, as observed in classical mammalian systems, and in female fecundity through up-regulation of genes associated with the mitogenic-activated protein kinase (MAPK) sub-pathway S. japonicum shares sequences with its mammalian host for insulin receptor or insulin-like growth factor receptor and can accept host hormone signals for development Insulin is a key hormone which has profound effects on both carbohydrate and lipid metabolism, and has a significant influence on protein (increases amino acid transport into cells) and mineral metabolism. We showed recently, using microarray analysis, that 1,101 genes of adult Echinococcus miltilocularisS. mansoniE. multilocularis IR (EmIR) and HIR Taenia crassiceps and Taenia soliumThe first parasitic helminth insulin receptor (IR) was isolated from S. japonicum (SjIRs), an analysis of their relationship with other IRs, assays showing the binding ability of the SjIRs with human insulin, and the incubation of adult worms in vitro both with a specific insulin receptor inhibitor and anti-SjIRs antibodies. Understanding the roles of SjIRs in S. japonicum will reveal new information on the molecular interchange between host and parasite that may provide a keystone for the future design of a drug or vaccine that would exclusively bind to the parasite IR, blocking the insulin effect and the signal transduction pathways mediated by the hormone.In the present work, we describe the presence of two discrete IRs in All work was conducted with the approval of the Queensland Institute of Medical Research Animal Ethics Committee.S. japonicum adult worms were collected by perfusion of ARC Swiss mice or rabbits infected percutaneously with 40 or 1000 cercariae of S. japonicum , respectively, shed from Oncomelania hupensis snails as described S. japonicum. A one step RT-PCR (Qiagen) kit was employed to amplify specific cDNA. Based on the conserved regions of the catalytic domains of IRs in S. japonicum and S. mansoni and partial S. japonicum sequences available at http://function.chgc.sh.cn/sj-proteome/index.htm, four pairs of primers for SjIR-1 and five primer pairs for SjIR-2 (S. japonicum transcriptome and proteome database (http://function.chgc.sh.cn/sj-proteome/index.htm) to search for 5′ and 3′ terminal sequences.A Qiagen RNeasy kit was used to purify total RNA from adult r SjIR-2 were deshttp://www.biomanager.angis.org.au) and Clustal W program , with default parameters. The alignment of intracellular regions was manually modified in order to take into account the presence of inserts in the tyrosine kinase (TK) domains of SjIRs, SmIRs and EmIR. Structural analysis was performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?), SMART (http://smart.embl-heidelberg.de.), ScanProsite (http://www.expasy.org/tools/scanprosite/) and MotifScan (http://myhits.isb-sib.ch/cgi-bin/motif_scan), and the Clustal W program for sequence alignments.Sequence alignment was performed using Biomanager . Phylogenetic trees were produced in MrBayes The three dimensional-structures of the insulin receptor domains of SjIR-1 and SjIR-2 were predicted based on the crystal structure of the insulin receptor domain of the murine insulin receptor (PDB code 2DTG) using the program Modeller in vivo and used the Gal4-based MATCHMAKER™ system according to the manufacturer's instructions. For constructing translational fusions to the Gal4-activation domain (AD), we inserted the ligand domain sequence of SjIR-1 or SjIR-2 into the vector pGADT7 after the fragments were amplified by PCR using adult S. japonicum cDNA as template with primers SjIR-1-TF1(5′-GGCATATGGACTGTTCCGGACGTTTACTGAATTTAC-3′) and SjIR-1-TR1 (5′-CTGCAGCTTCACAATCACGAATACTAATAAGGATTGATTG-3′); or SjIR-2-TF2 (5′-GGCATATGCAACATGATTCAACAGATCTCGGTTCC-3′) and SjIR-2-TR2 (5′-CTGCAGTTTTCAAATTTTTAAACGCTCTGTCCAATAAGTTAG-3′) via NdeI and Pst I restriction sites incorporated into the primer sequences. A positive control was constructed with the ligand binding domain of the HIR (from Cys-8 to Ser-340) and a negative control was constructed with the C-terminal region of the E. multilocularis ERM (ezrin/radixin/moesin)-like protein homologue (ElpC) NdeI and EcoRI restriction sites as described Yeast two hybrid analyses was employed to determine whether the SjIRs were able to bind human insulin All vector constructs were confirmed as having correct reading frames by DNA sequencing. The AD vector and BD vector containing human pre-insulin fragment were transformed into yeast strain AH109. Interaction trap analysis of diploids resulting from the mating of AH109 was performed according to the MATCHMAKER™ instructions using low stringency (SD/-Leu/-Trp), medium stringency (SD/-His/-Leu/-Trp) and high stringency agar plates. Formation of colonies was assessed after 3 days of incubation at 30°C. Positive colonies were picked for genomic DNA extraction and purification using a Y-DER Yeast DNA extraction Reagent Kit . Hot-PCR (Qiaqen) was used to amplify the LD1 or LD2 and human pre-insulin fragments from the DNA of the same colony with the specific primers for the pGADT7(T7 and 3′AD primers) and GBKT7 (T7 and 3′ BD primers) vectors. Bands of the correct size were analysed by DNA sequencing.5′-GCATATGGACTGTTCCGGACGTTTACTGAATTTACGT-3′) and reverse (5′-CGGAGCTCGTTCACAATCACGAATACTAATAAGGATTG-3′) primers for LD1 and forward (5′-GGGATCCGCGTTGCACTGTCATAGAAGG-3′) and reverse (5′-GCTCGAGTCACCAATTACAATAAGCTAAATCTCCATTTGT-3′) primers for LD2 were used for amplification and cloning into the pET28c and pET28b vectors , respectively, and transformed into Escherichia coli (BL21 strain) for expression induced with 1mM IPTG at 37°C for 3h. Recombinant proteins were purified by chromatography using a Ni-NTA His-tag affinity kit (Novagen) under denaturing conditions using 6M guanidine according to the manufacturer's instructions. To produce anti-sera, mice were immunized subcutaneously with 20 µg recombinant protein in complete Freund's adjuvant as primary immunisation, and given 3 boost immunisations at intervals of two weeks with the same quantity of protein emulsified with incomplete Freund's adjuvant. Bloods for sera were collected two weeks after the last boost.Antibodies were raised against the ligand domains of the SjIRs and were produced in mice against the N-terminal fragments of SjIR-1 (from D59 to E411) (termed LD1) and SjIR-2 (from R37 to C525) (termed LD2). Forward (Escherichia coli (BL21 strain) for expression as GST fusion proteins induced with IPTG. Recombinant proteins were purified by chromatography using a Ni-NTA His-tag affinity kit (Novagen) according to the manufacturer's instructions. The process of generating anti-T1 and T2 antibodies in mice was identical to that described above for LD1 and LD2.To generate antibodies against the TK domains of the SjIRs, we used a truncated strategy to express soluble peptides having a relatively high antigenicity based on analysis by MacVector 8.0. We combined the C-terminal peptide fragments of SjIR-1 termed as T1; and the C-terminal fragments of SjIR-2 termed as T2. The synthesised T1 and T2 cDNA fragments were inserted into the pET41b vector (Novagen) and transformed into All the anti-T1, T2 and LD1, LD2 antisera were adsorbed against lysates of bacterial cells transformed with appropriate pET expression vector expressing the GST or His tag to remove anti-GST or - His antibodies S. japonicum extracts as follows: 1) 30 ng of recombinant T1, T2 and LD1 or LD2 protein was mixed in the same volume of loading buffer, separated on SDS/PAGE gels, and then transferred onto nitrocellulose membrane. 2) Crude adult worm antigen was prepared from adult worms of S. japonicum freshly perfused from rabbits percutaneously infected with 1000 cercariae six weeks previously. After five washes in PBS to minimise contamination of the schistosome protein extracts with host components, the worms were homogenised on ice in 20 mM Tris-buffer (PH 7.4) containing 1 mM EDTA and a protease inhibitor cocktail , and the homogenate was then centrifuged at 16,000 g for 1 h at 4°C. The supernatant was collected as soluble adult worm antigen preparation (SWAP) and stored at −70°C. The pellet was suspended and, after 2 washes with PBS, was dissolved in 1% (w/v) SDS in the same volume as that of the SWAP. The preparation was then centrifuged at 16,000 g for 15 min at 25°C, the supernatant collected as insoluble protein, and stored at −70°C. For Western blot analysis, the soluble and insoluble protein preparations were mixed separately in the same volume of loading buffer, separated on SDS/PAGE gels and then transferred onto nitrocellulose membrane.Anti-T1, T2 and LD1, LD2 antisera were used to detect the four purified recombinant proteins and to probe native T1, T2 and LD1, LD2 proteins in crude After blocking in 5% (v/v) milk in PBS, the membrane was incubated overnight at 4°C with mouse anti-T1 or -T2 (dilution 1∶50) or anti-LD1 or -LD2 (dilution 1∶100) anti-sera, respectively. After 3 washes with PBS containing 0.05% (v/v) Tween-20, the membrane was soaked in goat anti-mouse HRP-conjugate IgG (Bio-Rad) (1 in 2000 dilution) and incubated at 37°C for 1 h. Following washing (5 x), detection of immunoreactive bands was obtained using 4-chloro-1-naphanol substrate solution.S. japonicum were freshly perfused with warm RPMI (37°C) from mice, 7 weeks post-infection with 40 cercariae. Following washing (3x) in the same medium, worms were incubated in RPMI1640 supplemented with 100 U/ml penicillin, 100 ug/ml streptomycin and with 20% (v/v) foetal bovine serum (as a source of insulin) (Sigma) at 37°C in an atmosphere of 5% CO2 in air overnight. The worms were then cultured in two ways:Adult worms of g for 30 min at 4°C, and the supernatants used to measure the concentration of protein by the Bio-Rad protein assay . The glucose levels of worms (ng glucose/µg protein) were measured using a glucose assay kit (Sigma) according to the manufacturers' protocols. Samples of worms were also used for extraction of total RNA in order to determine the transcription levels of the GTP4 gene by real time PCR analysis (details below).(1). In the presence or absence (control) of 100 µM insulin receptor inhibitor- HNMPA-(AM)3 for 30 min, after which glucose levels in worms of different groups were determined. Worm aliquots were homogenised in 20 mM Tris/Hcl buffer for 2–4 min on ice, then boiled for 5 min, centrifuged at 15,800 2 in air. The worms in each group were collected after 24 h culture with the different sera. Glucose levels and transcription levels of the GTP4 gene of worms were determined for each group as described above.(2) In the presence of: A) With 10% (v/v) anti-LD1 +10% anti-LD2 anti-sera; or B) With 20% (v/v) normal mouse serum (control). To inactivate complement, each serum was incubated at 56°C for 30 min before use. Cultures were established at 37°C in 24-well culture plates with each well having 3 ml RPMI 1640 containing anti-serum and 10 worms. The culture medium was gassed with a mixture of 5% COGTP4 transcription level were calculated using t-test of repeated measures. The statistical significance of differences between anti-sera treated and control groups (0 h and 24 h) in glucose level and GTP4 transcription level were calculated using one-way ANOVA of repeated measures. P values≤0.05 were considered statistically significant. GraphPad Prism software was used for all statistical analyses.The statistical significance of differences between HNMPA-treated and control groups in glucose level and SjIR-1 and SjIR-2 transcript levels were determined in different developmental stages of S. japonicum using real-time (RT)-PCR. Total RNA was isolated separately from miracidia, sporocysts, cercariae, schistosomulum, and male and female adult worms. First strand cDNA was synthesized from total RNA using a Sensiscript Reverse Transcription for First-stand cDNA synthesis Kit (QIAGEN) with oligo(dT)15 primers. Forward and reverse primers (Sigma-Aldrich) were designed for SjIR-1: SjIR-1F 5′-GCCTCATTACCATATCCTGGTT-3′ and SjIR-1R 5′- GCTGGTTCAAATTCCCAACA-3′; and SjIR-2: SjIR-2F 5′-TGGTGTTGTTTTGTGGGAGA-3′ and SjIR-2R 5′-GCCATTGGAGAGTAGCGTTT -3′. NADH-ubiquinone reductase was employed as a housekeeping gene SjIR-1 and SjIR-2 transcripts were also quantified in different female worm tissues using real time PCR with the same primers as above. A PALM microbeam laser catapult microscope was used to microdissect the gastrodermis, ovary and vitelline tissue from frozen female worm sections GTP4 transcripts were quantified in cultured worms using the forward and reverse primers (Sigma-Aldrich): GTP4F 5′-TAAGCTCTTTACTCAGAAAGATTTACGTATGC-3′ and GTP4R 5′- AACAACACAAAACTGTATGTAGTCAAGTGGGAT-3′.2O2 0.1% sodium azide in PBS for 10 min followed by 3 washes with PBS for 5 min each. Non-specific antibody binding was inhibited by incubating the section in 10% (v/v) normal goat serum in PBS for 30 min; then the sections were incubated with mouse anti-T1 (1∶25 diluted in PBS) or anti-T2 anti-serum (1∶50 diluted in PBS) for 1 h at room temperature. After 3 washes with PBS, the slides were incubated with anti-mouse HRP at room temperature for 30 min. After a further 3 washes with PBS, colour was developed for 5 min with a NovaRED subtract Kit , which produces a positive red reaction product.. Sections were slightly counterstained in Mayer's haematoxylin, then dehydrated, cleared, mounted and scanned using an Aperio scanner .Horseradish peroxidise (HRP) labelling was used for the immunolocalisation of SjIR-1 and SjIR-2. Freshly perfused adult male and female worms were fixed in 100% methanol, washed again in PBS, then embedded in Tissue-Tek Optimal Cutting Temperature (OCT) compound and sectioned with a cryostat into 7.0-µm sections. Frozen sections were fixed in ice cold acetone for 5 min followed by drying and then rehydrating in PBS for 5 minutes. Endogenous peroxidise activity was blocked by incubating the sections in 3% HIRs for S. mansoniE. multilocularisS. japonicum adult worm cDNA as template. Two types of IR cDNA were isolated from S. japonicum, SjIR-1 and SjIR-2. SjIR-1 has a complete cDNA sequence and comprises an open reading frame (ORF) of 4,599 bp (GenBank accession number GQ214553) encoding 1,533 amino acids (aa). We used a number of different strategies to amplify the distant 3′ terminal sequence of SjIR-2 and were able to obtain the complete sequence except the last exon of 21bp shown to be presented in the S. japonicum transcriptome and proteome database (http://function.chgc.sh.cn/sj-proteome/index.htm). SjIR-2 contains an ORF of 5,096 bp (GenBank accession number GQ214554) encoding 1,698 aa. Protein sequence comparisons confirmed SjIR-1 and SjIR-2 encode insulin receptors.Based on the highly conserved sequences of S. mansoni, E. multilocularis, Homo sapiens and Drosophila, in both the extracellular ligand domain (LD) and intrain (LD) . DomainsS. mansoni, E. multilocularis, Homo sapiens and Drosophila (2β2 structure). The α-subunits are extracellular and contain the insulin ligand domain, which is responsible for binding. The N-terminal of the extracellular domain contains two loops (L1 and L2) separated by a cysteine-rich region (CR) shown in Similar to IRs from osophila , SjIR-1 Alignment analysis showed a short insert of 27 amino acids (SQQILIDTNGNGRESTANSKHVISLGS1276) is present in the TK domain of SjIR-1 between sub-domain I and II when compared with HIR. The insert contains no tyrosine residues or known motifs. MacVector™ 8.0 analysis (data not shown) predicted this insert region to be highly antigenic and hydrophilic. Further comparison of SjIR-2 and HIR indicated the presence of an insert of 102 amino acids in the SjIR-2 TK domain between sub-domains IV and V; this is equivalent to the 172 amino acid stretch described by Konrad et al S. japonicum and S. mansoni are likely to be similar.Overall, the sequence analysis showed clearly that the predicted functions of IR-1 and IR-2 in SjIR-1 and SjIR-2 with genomic sequences (http://function.chgc.sh.cn/sj-proteome/index.htm) and recognised their exon-intron genomic structures whereas exon sizes in SjIR-2 range between 20bp and 617 bp (with an average of 204±120 bp). Similar complexity is evident for SmIR-1 (24 exons), SmIR-2 (25 exons), HIR (22 exons), and IGF-1 receptor (21 exons) genes SjIR-1 and one exon/intron in SjIR-2 are missing compared with genome sequence data presented for S. japonicum at http://function.chgc.sh.cn/sj-proteome/index.htm. The sizes of introns were variable with the average sizes being 2414±1616bp and 1464±1067bp for SjIR-1 and SjIR-2, respectively. The G+C% intronic content is 33.5% ±2% and 33.2%±3% for SjIR-1 and SjIR-2, respectively, similar to that in most other sequenced genomes of the Metazoa SjIR-1 and 2 are single copy genes in S. japonicum.We compared the cDNA sequences of ructures . SjIR-1 Amino acid sequences of SjIR-1 and SjIR-2 have 12.0–40.8% and 17.3–44.6% identity to HIR in the different sub-domains , respectThe overlay of the TK domain of HIR and SjIR-1 is shown in in vivo. The ligand binding domains (LD) of SjIR-1 and SjIR-2 were fused to the Gal4 activation domain , respectively and tested for interaction with human pro-insulin fused to the Gal4 DNA binding domain . The LD of HIR and the C-terminal region of the E. multilocularis ERM (ezrin/radixin/moesin)-like protein homologue (ElpC) in vivo (in yeast) with human pro-insulin at medium stringency; none of the receptor LDs, including HIR were able to bind insulin at high stringency conditions. SjIRs showed similar insulin binding ability to HIR and SmIRs SjIR sequences in one colony. To test the binding ability of SjIR-1 and SjIR-2 to human insulin, we employed yeast two hybrid analysis to identify their binding interaction E. coli and the recombinant proteins were purified from the TK domains and the N-terminal fragments of SjIR-1 (termed LD1) and SjIR-2 (termed LD2) to insert into a pET vector, respectively for expression of adult female S. japonicum. SjIR-1 had a higher transcription level in the vitelline tissue and ovary than in the gut in the vitelline grands compared with the ovary; no transcription of SjIR-2 was detected in the gut. Overall, the transcription level of SjIR-2 was higher than that of SjIR-1 in females.Real time PCR was also used to quantity the transcription levels of the gut . SjIR-2 S. japonicum. Real time PCR was employed to determine whether the inhibition/blocking could impact on the transcription of glucose transporter 4 (GTP4), the indirect target protein of IR. Incubation with HNMPA (100 µM) for 30 min resulted in a reduction in glucose levels of 40% in worms , compared to the control groups (We used an insulin receptor TK inhibitor (HNMPA) and antibodies against the LDs of the SjIRs to inhibit/block their function and to determine their role in glucose consumption by l groups . After 3l groups .S. japonicum for 24 h with 20% anti-LD1+ anti-LD2 anti-sera resulted in a 65% reduction in glucose (P≤0.0001) compared with the level in worms cultured in medium containing normal serum S. mansoni (SmIR-1 and SmIR-2) S. japonicum. We used sequence analysis and binding affinity studies to confirm that the sequences encoded insulin receptors. SjIR-2 and SmIR-2 are phylogenetically closer to EmIR than to SjIR-1 and SmIR-1, indicating that SjIR-1 and SmIR-1 might perform specific functions in schistosomes, while SjIR-2 (with higher similarity to HIR), SmIR-2 and EmIR might share similar roles in parasite growth and development in the three parasitic flatworms. A BLAST search in GenBank detected only a partial sequence with similarity to SjIR-2 and SmIR-2 in Schmidtea mediterranea, a free-living flatworm (data not shown), suggesting IR-2 orthologues occur more widely among flatworms. However since this was not a full length match we could not apply this to our phylogenetic analyses. Our data combined with the previous S. mansoni study Metazoans ranging from cnidarians to chordates employ insulin-like hormones and corresponding receptors for intercellular communication Sequence and structural analysis of SjIR-1 and SjIR-2 revealed that both receptors exhibit most of the features of IR family members. The extracellular domains of insulin receptors contain insulin binding sites within two characteristic loops (L1 and L2) flanked by a central cysteine-rich (CR) region , 1B. IdeHomo sapiens. The conserved sub-domains or motifs are important for catalytic function, either directly as components of the active site or indirectly by contributing to the formation of the active site through constraints imposed on secondary structure 2+ binding site, which plays a critical and conserved role in kinase function 2+ binding site (DFG). Despite the absence of the first Tyr of the autophosphorylation sites and the NPXY motif, the TK domain of SjIR-1 adopted a similar fold to that of HIR indicated there were 24 genes with high similarity to mammalian components involved in the insulin pathway suggesting it is functional in S. japonicum. The identification of two IRs (SjIR-1 and SjIR-2) from S. japonicum supports the hypothesis that these molecules are primary targets of host insulin.Transcription levels of the SjIRs were up-regulated in schistosomula and adult worms, further supporting their involvement in the mammalian host/parasite interaction. This might reflect differences in the demand for glucose uptake and energy requirements during the sexual and asexual (in the snail intermediate host) reproductive phases of the life cycle of S. japonicum by laser microdissection microscopy and microarray analysis demonstrated that transcripts encoding proteins localised to the outer tegument of the parasite are also enriched for the gastrodermis relative to other tissues The localisation of SjIR-1 in the tegument basal membrane and intestinal epithelium of adult worms, was similar to that observed with SmIR-1 S. japonicum adult worm than SjIR-1. This data implied that SjIR-1 and SjIR-2 could have distinct and specialized functions. Given that two IRs have been isolated only from schistosomes to date and combined with the sequence and phylogenetic analysis and the results of the immunolocalisation studies, IR-1 may be involved in utilizing host insulin, which is a specialized function used by the parasite to exploit a host hormone, with the IR-1 homologue having been lost by other taxa during the course of evolution. The diffuse expression of SjIR-2 and SmIR-2 observed in the parenchyma of males or for SjIR-2 in the vitelline cells of females suggests their possible function in the control of growth, similar to that described for HIR Immunolocalisation revealed that SjIR-2 is located in the parenchyma in males, which occupies most of the male tissue volume, and in the vitelline glands of female worms, which occupy 81.6% of female tissue Real-time PCR, using the different female tissues as templates, showed a high level of transcription of both SjIR-1 and SjIR-2 in vitelline gland tissue suggesting that both may be involved in development of vitelline for providing nutrients and shell precursors for the egg production. The high level transcription of SjIRs in vitelline tissue provides strong support for our suggestion from our earlier microarray analysis that insulin may play a role in increasing the fecundity of female parasites by activation of the MAPK sub-pathway The characterisation of IRs from schistosomes may have implications for understanding the host-parasite crosstalk, mediated by host hormones, activating receptors and second messenger cascades in these parasites that could lead to the design of new drugs against schistosomiasis. Recent success in cancer treatment has shown that receptor tyrosine kinases are attractive drug targets. The development of tyrosine phosphorylation inhibitors has greatly modified the approach to cancer therapy. Two possible approaches to intercepting these signaling pathways to develop biological reagents that block the binding of insulin to its receptors;to inhibit the interaction of an activated intracellular tyrosine kinase with its downstream targets.S. japonicum adult male and female worms 1.7- and 2.9-fold, respectively, in vitroGTP4 transcription level of S. japonicum adult worms incubated with HNMPA (100 µM) for 30 min, and showed that this compound could induce a decrease of glucose levels in worms, indicating conservation between the parasite and mammalian receptors. The reason why GTP4 transcription in adult worms increased on exposure to HNMPA can be explained by a previous investigation showing that insulin rapidly represses expression of the GTP4 gene in 3T3-L1 mouse adipocytes, as a result of both rapid repression of transcription of the GLUT4 gene, and an increased rate of turnover of the GLUT4 message S. japonicum is dependent on phosphorylation processes and could be regulated by insulin-dependent pathways, which implies that the mechanism of glucose uptake is similar to that occurring in mammals S. mansoni in vitroWe have previously shown that the presence of insulin increases the glucose content of S. japonicum with a mixture of the polyclonal antibodies prepared against LD1 and LD2, as these anti-sera cross-reacted with both LD recombinant proteins (data not shown). The mixture of antibodies significantly decreased glucose levels by up to 65% in adult worms in vitro, a result supported by a previous study whereby an anti-HIR antibody was shown capable of blocking the access of insulin to the liver receptor GTP4 of treatment groups became increased significantly compared to the controls. This result is supported by a previous study showing that the level of glucose transport 4 mRNA was diminished by prolonged (24h) administration of insulin in L6 muscle cells in culture suggesting the regulation of glucose transport activity by insulin involves post-translational mechanisms and subcellular redistribution of glucose transporter proteins S. japonicum IRs and the HIR have similar signaling transduction functions thereby interfering with the insulin pathway, which may affect glucose uptake in schistosomes and their growth. Blocking the LDs of the SjIRs, resulted in a significant decrease in glucose levels, and this can help explain previous work on S. mansoni showing that host environmental changes caused by malnutrition or by streptozotocin-induced diabetes affected the worm's ability to produce viable eggs S. japonicum and suggest that the SjIRs, especially the LDs, are likely targets for anti-schistosome vaccines.To determine whether blocking the LDs of the SjIRs would result in changes in worm glucose uptake, we incubated adult S. japonicum possesses insulin receptors that can specifically bind to insulin, indicating that the parasite can utilize host insulin for development and growth by sharing the same pathway as mammalian cells for the control of cell differentiation and proliferation. Further studies are necessary for precise characterisation of the signalling cascades of these receptors in schistosomes, particularly in the case of insulin-receptor signalling. Our findings have implications for the understanding of the host-parasite cross-talk and may lead to the design of vaccines or drugs that recognize parasitic molecules exclusively, with no collateral effects on the host.Overall, our findings demonstrate that Figure S1S. mansoni (SmIR-1 and SmIR-2), E. multilocularis (EmIR), D. melanogaster (DmIR), and Homo sapiens (HIR) IRs. Black boxes indicate identical amino acids and grey boxes denote similarity. (A) L1, CR, L2 and FnIII1-3 sub-domains are indicated by black lines spanning the regions and the insert inside the FnIII-2 domain is shown by a dotted line. Tribasic cleavage sites, RKR857 and KK839 in SjIR-1 and SjIR-2, respectively, are underlined. Cysteine residues for the α2β2 structure in SjIR-1 and in SjIR-2 , are shown in bold and italics. (B) A transmembrane domain indicated by a black bar. The 11 sub-domains numbered I-XI of TK domain are indicated by black lines over the regions. Crucial residues for tyrosine kinase activity are boxed in red including an ATP binding site (GxGxxG in I region), a sequence required for ATP stabilisation (VAV/IK-(16X)-E in the II and III regions), a motif for phosphotransfer (HRD LAARN in VIb region), a Mg2+ binding site (DFG in VII region), and a consensus PV/IRWMAPE sequence (in the VIII region). The insert of 102 aa between the kinase sub-domains IV and V in SjIR-2, SmIR-2, EmIR and the insert of 27aa between the sub-domains I and II in SjIR-1, SmIR-1, are shown in italics. (C) Exon-intron structures of SjIR-1 and SjIR-2 loci. Exons are numbered above the black boxes. Positions for translational start (ATG) and stop (TAA or TGA) codons are marked by arrows. The stop codon and its immediate region upstream were not amplified in SjIR-2 and are marked as “?”. Encoding regions for the different domains are indicated by black bars.Alignment of difference IR extracellular, intracellular regions and exon-intron structures of the SjIRs loci. The SjIR-1 and SjIR-2 extracellular (A) and intracellular regions (B) were aligned with those from (4.88 MB EPS)Click here for additional data file.Figure S2Echinoccocus multilocularis, EmIR CAD30260; Caenorhabditis elegans, CeIR AAC47715), Insects , molluscs , echinoderm , sponge , Hydra , Vertebrates , mammals and of IGF-1 receptor kinase domains of mammals . The catalytic domain of Homo Ros (NP_002935) and the Mus musculus EGFR (MmEGFR AAA17899) were used as outgroup sequences in Phylogenetic analysis showing relationships between SjIRs and homologues from other taxa. Phylogenetic trees of the tyrosine kinase domains (A) and ligand domains (B) for each receptor were generated as described in (10.20 MB TIF)Click here for additional data file.Figure S3Yeast two-hybrid analysis. Upper panel: PCR confirming the positive colonies from medium stringency contained both the ligand domain sequences of SjIR-1 (or SjIR-2) and human insulin. Three and two positive colonies were picked up from SjIR-1 and SjIR-2 separately, the genomic DNAs were extracted and used as template . T7 and 3′ BD primers were used to amplify human pro-insulin . T7 and 3′ AD primers were used to amplify LBD of SjIR-1 and SjIR-2 . The ligand binding domains of SjIR-1, SmIR-2 and HIR were fused to the Gal4 activation domain . Human pro-insulin was fused to the Gal4 DNA binding domain . The C-terminal region of the E. multilocularis factor Elp (ElpC) was fused to Gal4 AD or Gal4 BD and used as control LBD or ligand construct, respectively. Double transformants obtained from mating of AH109 and Y187 yeast strains were assessed for colony growth after 5 days of incubation in low, medium and high stringency conditions. (++), growth in medium conditions; (-), no growth in any condition.(5.72 MB TIF)Click here for additional data file.Table S1Schistosoma japonicum insulin receptors 1 and 2 (SjIR-1 and SjIR-2).Primers used for amplification of (0.04 MB DOC)Click here for additional data file.Table S2Schistosoma japonicum insulin receptors 1 and 2 and sequence identities with other insulin receptors.Domains of (0.03 MB DOC)Click here for additional data file.
Cerebral palsy (CP) is an upper motor neuron disease that results in a progressive movement disorder. Secondary to the neurological insult, muscles from CP patients often become spastic. Spastic muscle is characterized by an increased resistance to stretch, but often develops the further complication of contracture which represents a prominent disability in children with CP. This study's purpose is to characterize alterations of spastic muscle on the transcriptional level. Increased knowledge of spastic muscle may lead to novel therapies to improve the quality of life for children with CP.The transcriptional profile of spastic muscles were defined in children with cerebral palsy and compared to control patients using Affymetrix U133A chips. Expression data were verified using quantitative-PCR (QPCR) and validated with SDS-PAGE for select genes. Significant genes were determined using a 2 × 2 ANOVA and results required congruence between 3 preprocessing algorithms.CP patients clustered independently and 205 genes were significantly altered, covering a range of cellular processes. Placing gene expression in the context of physiological pathways, the results demonstrated that spastic muscle in CP adapts transcriptionally by altering extracellular matrix, fiber type, and myogenic potential. Extracellular matrix adaptations occur primarily in the basal lamina although there is increase in fibrillar collagen components. Fiber type is predominately fast compared to normal muscle as evidenced by contractile gene isoforms and decrease in oxidative metabolic gene transcription, despite a paradoxical increased transcription of slow fiber pathway genes. We also found competing pathways of fiber hypertrophy with an increase in the anabolic IGF1 gene in parallel with a paradoxical increase in myostatin, a gene responsible for stopping muscle growth. We found evidence that excitation-contraction coupling genes are altered in muscles from patients with CP and may be a significant component of disease.This is the first transcriptional profile performed on spastic muscle of CP patients and these adaptations were not characteristic of those observed in other disease states such as Duchenne muscular dystrophy and immobilization-induced muscle atrophy. Further research is required to understand the mechanism of muscle adaptation to this upper motor neuron lesion that could lead to the development of innovative therapies. Cerebral palsy (CP) is a disorder in which children experience a non-progressive brain lesion that results in permanent and progressive secondary postural and movement disorders . CP has It is clear that skeletal muscles from CP patients are altered secondary to the neurological lesion. There are many neurological symptoms secondary to the brain lesion including dystonia, ataxia, athetosis and particularly spasticity ,8. Loss Skeletal muscle from children with CP has been characterized at a variety of levels, with most studies reporting muscle tissue and muscle fiber atrophy, decreased muscle cross-sectional area, muscle shortening, and decreased specific tension ,11. All To develop an understanding of the physiological processes altered in spastic muscle secondary to CP, we exploited the fact that muscle tissue from a previous study, in which the clinical severity of the spasticity was clearly established, was available for transcriptional profiling . We usedin vivo. While the wrist was held in neutral, a small fiber bundle was transilluminated with a HeNe light. The sarcomere length could be calculated from the diffraction pattern obtained . RNA processing for the GeneChip, including stringent quality control measures, was performed by the Gene Chip Core at the Department of Veterans Affairs San Diego Health Care System, . GeneSpring software was used to identify those genes that were significantly altered in CP. Initially, a 12.5% (2/16 chips) present call on MAS5 (Affymetrix) was used to filter out poorly performing probe sets in the analyses. Three independent probe set algorithms were used for signal generation and normalization: MAS5, RMA, and GCRMA. Recent reports support requiring concordance among different probe set algorithms as an approach to reduce false positives in data sets -24. EachThe condition tree was created using a Pearson Correlation similarity score and average linkage clustering algorithm for all samples on present features. For severity analysis, a Welch ANOVA for each severity parameter was run on the MAS5 data of the features deemed significantly altered in CP without the control patients and a required statistical significance of (P < 0.05) also with an FDR multiple testing correction.Promoter sequence analysis was conducted using GeneSpring on the list of genes altered in CP. The upstream sequence from -10 to -1000 base pairs was analyzed for a nucleotide sequence of from 6 to 10 nucleotides long and containing at most 2 N values in the middle. Significance was determined based on the number of times the given sequence appeared in the upstream sequence of all other genes and was corrected for multiple testing. The analysis was performed on the whole list of genes altered in CP and the sub lists of up- or down-regulated.2 (Invitrogen), 0.2 mM sense and antisense primers, 0.2 mM dNTP, 0.2× SYBRgreen, and 1 U of platinum Taq polymerase (Invitrogen). Amplification conditions were as follows: An initial hold at 95°C for 2 min was followed by 40 cycles of denaturing at 95°C for 15 s, followed by annealing/extension at 68°C for 40 s. The success of each reaction was deduced based on the observation of a single reaction product on an agarose gel and a single peak on the DNA melting temperature curve determined at the end of the reaction. To express QPCR results, we used the standard curve method with the "cycles to threshold" value representing the number of PCR cycles at which the SYBRgreen signal was increased above the threshold. Each sample's value was measured in triplicate, normalized to the housekeeping gene GAPDH, and then averaged. QPCR data were normalized to the median value of the gene to permit comparison to the GeneChip data.QPCR was performed to validate expression levels of selected genes to the GeneChip data and to provide mRNA expression levels for genes not contained on the HG-U133A chip. After RNA was extracted from the muscle as described previously and diluted 1:5 with DNase/RNase free water (Invitrogen), 1 μl of each sample was reverse transcribed using standard protocols . cDNA was amplified with the Cepheid SmartCycler with primers specific to the genes of interest Table . All priMyosin heavy chain protein content was measured Table for comp; i can be inferred by the up-regulation of TRDN, which reclaims Ca2+ to the SR by localizing calsequestrin within the SR [i a huge 63-fold increase in PVALB, a Ca2+ binding protein was induced in order to force muscle relaxation [I and may even lower it below control levels and alter muscle contractile properties.We also uncovered significant evidence of altered calcium handling secondary to CP. Our data appear to reflect chronically increased intracellular calcium since the L-type voltage gated Can the SR . Chronicn the SR ,54. Perhlaxation . This drCALM1) and calcineurin (PPP3CA) [Of the proteins involved in calcium induced force generation, MHC isoforms are the most responsive to CP. They are primarily responsible for determining muscle fibers type and unde(PPP3CA) . A poten(PPP3CA) ,59.IGF1) and myostatin (GDF8). Interestingly they produce opposing effects on myogenesis with IGF1 leading to hypertrophy and myostatin opposing growth [The slow fiber program represents only one segment of myogenesis that is controlled by many other genes. While the majority of the pathway elements involved in myogenesis were not changed, two of the most important initial factors were both up-regulated – insulin-like growth factor and supported by an up-regulation of CACYBP, a gene involved in calcium dependent ubiquitination. The opposing actions of IGF1 to increase muscle mass are also controlled by a number of IGF binding proteins and we revealed IGFBP5 was significantly up-regulated in CP, however the effects of IGFBP5 in muscle have been questioned [Muscle development was indicated in the ontology analysis could also lead to extensive ECM through the reduced activation of MMPs [One of IGF1's broad anabolic effects could be a contribution to the increased ECM in muscle from CP patients ,65. Whil of MMPs . Basigin of MMPs .IGF1 and GDF8 acted alternatively – IGF1 increased while GDF8 decreased in DMD and conversely for IMB and HSP. This highlights the unique adaptation of CP, where myogenesis is turned on and off simultaneously.It is important to note the distinct pathology of CP, as spastic muscle does not fit neatly into any of the other "altered use" muscle models . The traWhile we are able to demonstrate the transcriptional effects of CP we also investigated this effect on two separate muscles and at different levels of clinical severity. Tendon transfer surgery is relatively common procedure for CP patients and is implicated when there is a muscular imbalance around a joint. It involves transferring the distal tendon of a muscle on the side of a joint considered to have a contracture or relative over activity to a tendon on the opposing side of the joint. Transfer of FCU to ECRB to correct wrist position is one of the common tendon transfer surgeries. Thus FCU is considered the more pathologic muscle and we might have expected a different transcriptional profile. However, we were unable to show any transcriptional differences between the muscles, indicating that both wrist flexors and extensors have a similar adaptation to CP. While the FCU is known to exhibit contractures in CP, we conclude that the contracture is developed due to its architecture, not due to a fundamental difference is secondary adaptation to the altered neuronal input of CP. The FCU is a larger muscle than the ECRB and the larger wrist flexor muscles may simply dominate the disease state based on their size. We were also unable to show significant transcriptional differences among various clinical severity scores in CP patients. This may be because CP transcriptional profiles are either on or off. More likely our study was unable to resolve a severity effect as the study is biased towards the most severe cases (patients recruited based on corrective surgery) or the study is simply underpowered. We would likely need more patients across the range of clinical severity scores to define the genes most closely correlated with severity. However the low power of the severity analysis is increased in our comparisons of CP vs. control muscle. Further, a discussion of statistical power does not apply to significant differences detected in CP vs. control muscle. We do acknowledge, however, that we are clearly not detecting all transcripts that are altered in CP.Our study has some inherent limitations, one of which is the small sample size noted above, especially in the case of control patients. As with any human study there is a high degree of heterogeneity among the samples. These patients have been treated in a variety of ways, and it is important to note that our transcriptional profile is not solely based on CP, but includes conservative treatment. We must also point out that this muscle is in a chronic disease state, making it difficult to discern the primary effects of CP from compensatory mechanisms that have taken place. As with any GeneChip study, we discuss only transcriptional control and any observation is subject to post-transcriptional modification.Despite these inherent limitations we have been able to highlight areas where future work on spastic CP muscle may lead to innovative therapies. Our altered calcium handling data points to chronically elevated calcium levels which are highly dangerous since they may activate endogenous proteases. Fortunately a variety of calcium channel blockers have been developed and tested which could be of use in treating CP. Another potential application of current techniques could come from antifibrotic therapy to combat the increase in ECM components which is suggested by the transcriptomes. Of the most promising may be myostatin inhibiters, currently under investigation, since growth is inhibited in muscle from CP patients and myostatin, a major inhibiter of muscle growth is significantly up-regulated. This transcriptional study helps point the way to these and other areas of protein modifications, cell signaling, and biomechanics where future investigations should be focused.2+ handling occur in CP that was not suggested previously. Together we are able to postulate the mechanisms known to affect muscle function in CP and predict new ones. This will aid future research into CP muscle and therapies to treat CP patients.Dramatic transcriptional alterations occur in muscle secondary to CP. These transcriptional changes ultimately lead to derangement of the ECM components of spastic muscle along with alteration of transcripts involved in myogenesis. A number of genes alter their expression in order to create a slow-to-fast transition of MHC isoforms and metabolic profile. GeneChip analysis has also allowed us to demonstrate the many changes in Ca(CP): Cerebral palsy; (ECC): excitation contraction coupling pathway; (ECM): extra-cellular matrix pathway; (ECRB): extensor carpi radialis brevis; (DMD): Duchenne muscular dystrophy; (FCU): flexor carpi ulnaris; (FT): fiber type pathway; (GCRMA): GC robust multichip analysis; (GEO): Gene Expression Omnibus; (HSP): hereditary spastic paraplegia; (IGF1): IGF1 pathway; (IMB): immobilization; (LMN): lower motor neuron; (MAS5): microarray suite version 5.0; (MC): muscle contraction pathway; (MYG): myogenesis pathway; (NMJ): neuromuscular junction pathway; (QPCR): quantitative polymerase chain reaction; (RMA): robust multichip analysis; (SCA): satellite cell activation markers; (SCQ): satellite cell quiescence markers; (SR): sarcoplasmic reticulum; (UMN): upper motor neuron.The authors declare that they have no competing interests.LRS carried out the RNA isolation, qPCR experiments, genechip analysis, and drafted the manuscript. EP provided the biopsies and assisted in review of the manuscript. YH carried out the myosin heavy chain content experiments. SRW participated in critical review of the manuscript. HC provided expertise on CP and critical review of the manuscript. SS provided expertise on genechip analysis and critical review of the manuscript. RLL conceived of the study, and participated in its design and coordination and supervised the writing of the manuscript. All authors have read and approve of this manuscript.The pre-publication history for this paper can be accessed here:Significantly altered genes in CP. List of all features with a significant p-value (< 0.05) using each preprocessing algorithm . Ratio of CP/CTRL is determined using MAS5.Click here for fileSignificantly up-regulated Gene Ontologies in CP. List of all Gene Ontologies significantly up-regulated genes in CP. O: observed genes, E: expected genes, R: ratio of observed/expected, P: p-value.Click here for fileSignificantly up-regulated Gene Ontologies tree in CP. Hierarchical list of Gene Ontologies in CP with red lettering representing significantly up-regulated Gene Ontologies.Click here for fileSignificantly down-regulated Gene Ontologies in CP. List of all Gene Ontologies significantly down-regulated genes in CP. O: observed genes, E: expected genes, R: ratio of observed/expected, P: p-value.Click here for fileSignificantly down-regulated Gene Ontologies in CP. Hierarchical list of Gene Ontologies in CP with red lettering representing significantly down-regulated Gene Ontologies.Click here for file
Inferring Gene Regulatory Networks (GRNs) from time course microarray data suffers from the dimensionality problem created by the short length of available time series compared to the large number of genes in the network. To overcome this, data integration from diverse sources is mandatory. Microarray data from different sources and platforms are publicly available, but integration is not straightforward, due to platform and experimental differences.We analyse here different normalisation approaches for microarray data integration, in the context of reverse engineering of GRN quantitative models. We introduce two preprocessing approaches based on existing normalisation techniques and provide a comprehensive comparison of normalised datasets.Loess normalisation and iterative K-means as best for time series normalisation for this problem.Results identify a method based on a combination of Quantitative models provide more information on the interactions and dynamics of the system, compared to qualitative models, but despite considerable attention in the literature Identifying biological networks is an important aspect of Systems Biology, as these give insight on the complex behaviour of an organism and help find disease markers and treatments, important precepts to establish for Synthetic Biology One way of overcoming the dimensionality problem, widely recognized in the literature Saccharomyces Cerevisiae cell cycle. These include three dual-channel, . The PramilaL dataset contains 25 time points, sampled every 5 minutes on the same Amplicon platform, and features a dye-swap replicate, which is used in our experiments as a second time series of the same length. The Hasse dataset contains 15 time points, sampled with Affymetrix arrays every 16 minutes, and a replicate that is again used as a separate time series during inference. This results in six time series measurements of the cell cycle, sampled at different intervals. The common genes in these datasets were extracted, resulting in 5337 genes for analysis.Normalisation analysis has been performed on four distinct, publicly available, raw datasets, representing microarray time series measurements during the Yeast Spellman , and oneSpellman . Each ofThe normalisation performed in this study consists of two stages. Initially, noise pre-processing was performed, using three different approaches. On the resulting datasets, three cross-platform normalisation techniques were applied, resulting in a total of nine normalised datasets for comparison. Additionally, the time spans were scaled so that the cell cycle length is the same across datasets.The first pre-processing stage aims at noise reduction within each dataset, to prepare it for cross-platform normalisation. Several normalisation techniques exist in the literature PMLoess, applies different normalisation techniques depending on platform type: PMOnly, requires positive expression values for all genes, and facilitates computation by restricting its output to the same interval. (ii) ComBat across platforms, after a preliminary normalisation within each dataset. The implementations, made available by the authors, were used for the latter two methods. The final datasets are identified in this paper by the name of the normalisation techniques used for each stage: PMLoess methods , PMOnly methods and LoessOnly methods . The rest of this section briefly describes the cross-platform normalisation procedures ComBat and XPN.For each dataset resulting from the first pre-processing stage, we applied cross-platform normalisation techniques, as follows. (i) A simple standardisation on each dataset, ComBatThe method consists of three steps. (i) The data is standardised to obtain similar overall mean and variance for genes. This involves fitting of parameters XPNblock mean, Evaluation of the normalisation methods applied has been carried out using four different criteria. Firstly, (i) variability between replicates has been computed, as the average over all genes of the rMSE (squared root of Mean Squared Error) between replicate expression values, normalised by the average gene expression level for each gene, (Equation 6).levels). This type of decomposition uses a set of functions, called wavelets, which are generated by contracting and dilating a base function, i.e. the mother wavelet, in discrete steps levels for coefficients, with low levels corresponding to small scale i.e. high frequencies, and high levels to high scale i.e. low frequencies. Having Secondly, (ii) wavelet analysis was used to compare the normalisation techniques used. Wavelets Here, we have used Daubechies mother wavelets for analysis Thirdly, (iii) a correlation analysis was performed to test whether correlations vary between normalisation techniques, as well as to determine whether genes known to interact are correlated after normalisation. This is important for GRN model inference, as interacting genes will have correlated expression levels across time within each normalised dataset was computed, for each gene pair. Ideally, the same pair of genes should have similar correlation across microarray datasets, but, due to platform differences and normalisation, these can vary. The correlations common to the different datasets are most reliable, while others are more likely to be spurious, i.e. introduced by the platform or normalisation technique, . In this work, the correlation differences between microarray datasets was computed as indicated in Equation 10:correlation variability matrix in the rest of this paper, which shows how correlations between pairs of genes differ from one microarray dataset to another. This can be viewed as an indicator of the amount of spurious correlation in each normalised dataset. Here, we use the average of the values in the correlation variability matrix to quantify this, because of similar high dimensionality of these matrices.The first aggregated criterion used was the number of gene pairs with absolute correlation larger than 0.9, which was compared across normalised datasets, to determine how each normalisation technique affects high correlations. Secondly, the average of absolute correlations for each gene A different method of identifying spurious correlations would be to analyse partial correlation coefficients in the data, i.e. the correlation seen after removing effects from other genes, as opposed to zero-order coefficients such as Pearson, which consider the correlation in isolation. However, this is difficult to assess here, as the pattern of covariance is very complex, with many gene pairs having high zero-order correlation and known existence of circuits in the causality networks, hence our use of the correlation variability matrix, described above, as a (weaker) criterion.The fourth evaluation criterion used was (iv) the capability of single gene models to translate between datasets. For this, models were built from each dataset individually, and then were applied to simulate the same genes in the other three datasets. An inferential algorithm, based on evolutionary computation, Additionally, models have been inferred from combining two datasets, and testing on a third, to analyse how the data fit changes compared to using each training dataset individually. This demonstrates that data integration can improve inference, and enables analysis of the effect of each normalisation technique. The same error measure, i.e. rMSE/Mean, has been used to evaluate the difference between simulated and experimental data.Variability analysis), and shows, again, that cross platform normalisation may come with a cost from the variability viewpoint.A first analysis, based on wavelet decomposition, measures the amplitude of high frequencies in the different normalised datasets. High amplitudes indicate stronger noise compared to low amplitudes. Model translation), are compared across the four datasets, and results are summarised in A second application of wavelet decomposition, for assessment of pre-processing methods, compares coefficients, at different scales, for signals describing expression levels for the same gene occurring in different datasets. Here, nine genes known to be involved in the cell cycle, and PramilaL . These indicate that Loess and XPN methods decrease pair-wise correlations compared to PM and Standardisation approaches, with much larger differences seen in the single-channel (Hasse) dataset. Due to the large dimensionality of these heatmaps, two aggregated criteria have been used for further analysis. In order to provide an overall view of the correlation distribution in the normalised datasets, Firstly, the number of highly correlated gene pairs has been studied in each dataset. The correlation threshold used was 0.9, and Secondly, the average of absolute correlations for a subset of nine genes was computed, with results for gene SWI4, (which is a known transcription factor involved in cell cycle regulation), shown in correlation variability matrix (Equation 10) was computed for each normalisation procedure, and averages over all gene pairs , are shown in In order to assess the amount of spurious correlation for each normalisation technique, the In the context of GRN modelling, it is very important that interactions between genes correspond to correlations seen in the data. To analyse this, we have chosen a set of 5 gene pairs, which are known to interact in reality . These iVariability analysis and Wavelet analysis). For the other gene pairs, positive correlations are decreased using Loess, (Hasse dataset), but agreement with values obtained for dual-channel datasets remains good.For the first three datasets, , Loess normalisation displays better behaviour, with PM-only methods giving significant decrease in correlations between genes known to interact. It is important to note that the correlation values do correctly indicate the nature of these interactions, with positive values for (a), (b), (c), and negative for (d) and (e). However, correlations between CLN1/2 are higher than those corresponding to activation/repression pairs, which can be explained by the regulatory time delay, which represents the time elapsed between the expression of the regulator and that of the regulated gene, causing a shift in the expression signal of the target, compared to the regulator, and, consequently, decreasing correlation values between the corresponding time series. For the Hasse dataset, on the other hand, the negative correlation between FAR1/CLN1/2 is not present, except after Loess normalisation, and, even then, absolute values are very small. This supports the hypothesis that PMOnly methods introduce spurious correlations into the data, probably due to the high variability, from datasets PramilaL and Hasse together and testing these on the Spellman dataset. The resulting error, (averaged over 20 runs), has been compared to that obtained by models inferred from PramilaL and Hasse individually, with results displayed in Based on lowest error obtained for model inference, (using two datasets as opposed to one), Loess_XPN performs best, providing strong indication of its suitability as a normalisation method for data integration in GRN modelling.Three pre-processing approaches have been applied to integrated raw microarray data from 3 different platforms. This has included application of techniques developed for dual-channel, Loess , on singFrom the variability viewpoint, LoessOnly methods performed better than PMOnly, although combined PMLoess methods exhibited best performance overall. Wavelet analysis and model translation indicated that a second normalisation stage, (cross-platform), as opposed to simple standardisation, is required in order to align the datasets for the same inferential process. However, variance is increased for experimental replicates by cross-platform processing. Additionally, combining datasets was shown to increase performance on a test dataset, especially when using integrated-within dataset normalisation, with best data fit obtained by Loess_XPN. Analysis of correlation between genes showed that Loess methods decrease high values, although patterns between genes that are known to interact are preserved. XPN also reduces some highly correlated gene values, but, in many cases, correlations between genes known to interact are amplified, even for those gene pairs for which other methods failed to obtain the correct correlation sign. This suggests that it is a fairly sensitive probe for determining true interaction patterns in the data.In conclusion, results indicate that Loess_XPN was found to be best for normalisation of time-series data for quantitative model inference, as variability is acceptably low, datasets are well aligned, correlations between interacting genes are enhanced and models, obtained from combined datasets, perform better on test data than models inferred from one dataset only. The method permits integrated pre-processing across platforms, facilitating model inference from heterogeneous datasets.
In this paper, we make amodest exploration to determine the ontological requirements for this extended versionof GO. Using the terms within the three GO hierarchies , we investigate the facility with whichGO concepts can be ontologized, using available tools from the philosophical andontological engineering literature.We present an analysis of some considerations involved in expressing the Gene Ontology (GO) as a machine-processible ontology, reflecting principles of formal ontology.GO is a controlled vocabulary that is intended to facilitate communication betweenbiologists by standardizing usage of terms in database annotations. Making suchcontrolled vocabularies maximally useful in support of bioinformatics applicationsrequires explicating in machine-processible form the implicit background informationthat enables human users to interpret the meaning of the vocabulary terms.In the case of GO, this process would involve rendering the meanings of GO intoa formal language with the help of domain experts, and adding additionalinformation required to support the chosen formalization. A controlled vocabularyaugmented in these ways is commonly called an
KRT12) gene in two generations of a German family diagnosed with Meesmann`s corneal dystrophy.To report a novel missense mutation of the cornea specific keratin 12 (KRT3) and KRT12 of the proband and three other family members were performed. Restriction enzyme analysis was used to confirm the detected mutation in affected individuals of the family.Ophthalmologic examination of the proband and sequencing of keratin 3 in exon 1 of KRT12 causing Meesmann`s corneal dystrophy in a German family.We have identified a novel missense mutation within the highly conserved helix-initiation motif of The cornea represents the anterior surface of the eye and must maintain its structural integrity as well as its transparency to retain good vision. The outermost layer of the cornea is presented by the corneal epithelium.122100) in 1964. They were followed by Fine et al. [In 1939 the German ophthalmologists Meesmann and Wilke described a dominantly inherited dystrophy affecting the corneal epithelium in three families living in northern Germany publishee et al. in 1982.Keratins are a group of water-insoluble proteins that form intermediate filaments in epithelial cells. They can be divided into acidic and basic-neutral subfamilies according to their relative charges, immunoreactivity, and sequence homologies to type I and II wool keratins, respectively. In vivo, a type I keratin (acidic) pairs with a specific type II keratin to form a heterodimer . The strKRT3) and keratin 12 (KRT12) [KRT12 locus in Meesmann`s original German kindred. Furthermore, in the two pedigrees from Northern Ireland, they found that the disorder cosegregated with KRT12 in one pedigree and KRT3 in the other.A breakthrough approached in 1997 when McLean and coworkers ,9 examin (KRT12) ,9. Irvin (KRT12) postulatKRT3 and 19 mutations in KRT12 in patients showing MECD have been identified so far , an 82-year-old woman, was examined by slit-lamp biomicroscopy. Peripheral blood samples of patient I/2, her unaffected husband (I/1) and the two affected sons were obtained. The ophthalmological history of the proband`s deceased parents was unknown. Fifty unrelated volunteers were recruited to serve as controls. All subjects were treated in accordance with the tenets of the Declaration of Helsinki and signed a consent form.KRT3 and exons 1–8 of KRT12 were amplified using the primers shown in Genomic DNA was isolated from peripheral blood leucocytes by standard procedures using the Invisorb Spin Blood Maxi Kit . Blood samples (10 ml) treated with EDTA were processed following the manufacturers instructions. Exons 1–9 of KRT12. Therefore, PCR products of all four family members as well as 50 unrelated control subjects were submitted to restriction digestion under the following conditions. A reaction mixture containing 10 µl PCR product, 5 µl enzyme, 25 µl NEBuffer 4 , and 170 µl H2O was incubated overnight at 37 °C. In the presence of the heterozygous M129V mutation, HpyCH4III digestion resulted in fragments of 293 bp and 76 bp with the additional undigested 369 bp PCR product. The digested products were separated and visualized on a 2% agarose gel stained with ethidium bromide. Digestion analysis was additionally used to screen fifty healthy control subjects for the M129V mutation.The mutation M129V generated a HpyCH4III restriction enzyme site in exon 1b of Slit lamp examination of patient I/2 revealed multiple microcysts within the corneal epithelium of both eyes with highest density in the center of the cornea . The proKRT3 revealed no mutations in the four family members.The sequence analysis of all nine exons of KRT12 disclosed the heterozygous 409A>G transversion at the first nucleotide position of codon 129 (ATG>GTG) in the proband I/2 (NM_000223). This missense mutation was absent in the unaffected husband (I/1). Additionally, the proband and her sons harbored the single-nucleotide polymorphism (SNP) 43C>T (NM_000223). Further sequence analysis of exons 2–8 did not show any alterations.Direct forward and backward sequencing of exon 1b of band I/2 and her P) 43C>T , resultiRestriction enzyme analysis employing HpyCH4III resulted in digested fragments of 76 bp and 293 bp in the proband (I/2) and her two affected sons as shown in KRT3 and KRT12 are responsible for the development of Meesmann`s corneal dystrophy, an autosomal dominant disorder of the corneal epithelium. Until the present study, three mutations have been reported in KRT3 and 19 in KRT12, including a 27-nucleotide long insertion in exon 6. All mutations are summarized in KRT12 mutations are localized within the region encoding the helix-initiation motif. In contrast, the known mutations of KRT3 are exclusively within the helix-termination motif. In keratin proteins, the helix-initiation and –termination motifs are highly conserved regions playing an important role in normal filament assembly [All known mutations in the cornea specific genes assembly .KRT12 in a German family. Previously, Irvine and coworkers [With the predicted amino acid substitution M129V we revealed the third mutation of oworkers identifiAnother nucleotide exchange within the same codon M129T) predicting the substitution of the methionine (ATG) residue by threonine (ACG) was described independently by Cordon et al. T predict in AmeriOur index patient, an 82-year-old female, had a rather severe case of MECD with photophobia, tearing, and recurrent painful corneal erosions since adulthood. Visual acuity was highly reduced due to myopic macular degeneration. Additionally, the proband and her affected sons harboured the SNP 67C>T predicting the amino acid substitution P15S. This SNP has been previously described by Nielsen et al. in an asStill, it seems cryptic why certain mutations lead to a more or less severe phenotype. Severe cases of MECD have been reported due to mutations within the helix initiation motif ,14 as weKRT3 cause a MECD phenotype indistinguishable from KRT12 mutations. Kao et al. [KRT3/KRT12 heterodimers for corneal stability in a KRT12 knockout mouse model. Any modification that results in an impairment of the heterodimerization process will lead to corneal fragility. This could explain why mutations in any member of the heterodimer lead to almost identical phenotypes. Therefore, other individual factors may contribute to the MECD phenotype.Moreover, mutations in o et al. demonstrThe present study adds another novel mutation to the collection of mutations causing Meesmann`s corneal dystrophy.
Objective. To describe the clinical characteristics and radiological findings in two patients with subacute encephalitis associated with elevated serum voltage-gated potassium channel antibody (VGKCAb) and antithyroperoxidase (TPO) antibody. Case Reports. Case 1: 63-year-old woman was admitted for altered mental status and possible seizure activity. MRI brain showed hyperintensity in the bilateral hippocampal areas. She was positive for VGKCAb and anti-TPO antibodies. She was treated with steroids, IVIG, plasma exchange and azathioprine. After 8 months, she had marked improvement in her memory and seizures. Case 2: 61-year-old woman was admitted for video EEG monitoring of unclassified seizure and cognitive function decline. MRI of the brain showed mild hyperintensity in bilateral hippocampal areas and significant atrophy in the frontotemporal lesion. Anti-TPO antibody and VGKCAb were positive. She was treated with steroids, plasma exchange and azathioprine. After 9 months, she had marked improvement in her memory and seizures. Conclusion. Autoimmune subacute encephalitis appears to be an underdiagnosed entity. It is important to screen patients with subacute encephalitis for anti-TPO antibody and VGKCAb, particularly in the presence of seizures. Immunosuppressive therapy appears to be effective in treating this entity. Subacute encephalopathies are neurological and systemic diseases causing impairment of consciousness insidiously over weeks or months. Hashimoto's encephalitis (HE) is a neurological disorder associated with elevated levels of antithyroperoxidase (TPO) antibody, antithyroglobulin (anti-TG), antiNH2-terminal of a-enolase (NAE) antibody or anti thyrotropin antibodies. HE responds to treatment with steroids or plasma exchange and has a good prognosis , 2. Volt63-year-old, right-handed woman admitted for altered mental status and possible seizure activity. Seizure activity involved twitching of right face, a flexion motion of the elbow and stiffening of the arm, lasting around 20 seconds. She also had one episode of generalized tonic clonic seizure. She had a hypomanic state, visual hallucinations, short-term memory problems, loss of social inhibition and history of multiple falls. Past medical history was significant for untreated hypothyroidism. On examination, the patient was oriented to person only. She had receptive aphasia and perseveration. Gait was ataxic. The video EEG showed marked bifrontal and bitemporal slowing with electrographic seizures originating from the left temporal lobe. Treatment with levetiracetam resulted in initial improvement. Worsening in 3 days required the addition of valproic acid. MRI brain showed hyperintensity on T2 flair images in the bilateral hippocampal areas, right more than left . CSF shoShe was treated with synthroid 50 mcg daily and IV methylprednisone 1000 mg daily for 5 days followed by IVIG and plasma exchange with no improvement in mental status. She was discharged on prednisone and azathioprine. At 8 months follow up, she had marked improvement in her memory, seizures, and walking. VGKCAb was reduced from 6.21 to 0.86 nmol/L and antiTPO antibody had gone down from 142 to 32.4 iu/ml. Repeat MRI 8 months after the initial MRI was unchanged.61-year-old right-handed woman was admitted for video EEG monitoring of unclassified seizure. She had 6 months history of frequent, brief episodes of fearful waking, associated with stiffness and moving her right arm towards her body, grabbing her elbow and asking repeatedly “Help me. Help me. Where are they?” The patient's family reported a decline in the patient's cognitive functions over the last few months and a tendency to talk aloud to herself. She had been recently diagnosed with syndrome of inappropriate antidiuretic hormone (SIADH) which was treated with demeclocycline. On examination the patient was oriented to person only. She had word finding difficulty and mild-moderate cognitive linguistic deficits in areas of attention, memory, complex reasoning, and problem solving tasks. Gait was ataxic. Video EEG monitoring showed mild diffuse slowing but no epileptiform discharges. She was started on levetiracetam with a significant decrease in seizure frequency. MRI of the brain showed mild hyperintensity in bilateral hippocampal areas on T2 flair sequences and significant atrophy in the frontotemporal lesion . CSF anaNonparaneoplastic limbic encephalitis (LE) has been reported in association of both antiTPO antibody and VGKCAb in 2 patients by Thieben et al. and 1 paTreatment options include intravenous steroids, intravenous immunoglobulin or plasma exchange for acute presentations followed by maintenance immune therapy –7. TreatDr. Manoj Mittal and Dr. Nancy Hammond report no disclosures. Dr. Sharon G. Lynch received research support from pharmaceutical companies and National Multiple Sclerosis Society.
Esophago-gastroduodenal anastomosis with rats mimics the development of human Barrett's esophagus and esophageal adenocarcinoma by introducing mixed reflux of gastric and duodenal contents into the esophagus. However, use of this rat model for mechanistic and chemopreventive studies is limited due to lack of genetically modified rat strains. Therefore, a mouse model of esophageal adenocarcinoma is needed.p53A135V transgenic, and INK4a/Arf+/- mice of A/J strain. Some mice were also treated with omeprazole , iron , or gastrectomy plus iron. Mouse esophagi were harvested at 20, 40 or 80 weeks after surgery for histopathological analysis.We performed reflux surgery on wild-type, p53A135V mice . At week 40, metaplasia was found in wild-type mice , p53A135V mice , and wild-type mice also receiving gastrectomy and iron . Esophageal squamous cell carcinoma developed in INK4a/Arf+/- mice , and wild-type mice receiving gastrectomy and iron . Among 13 wild-type mice which were given iron from week 40 to 80, twelve (92.3%) developed squamous cell carcinoma at week 80. None of these mice developed esophageal adenocarcinoma.At week 20, we observed metaplasia in wild-type mice and p53 mutation, heterozygous loss of INK4a/Arf, antacid treatment, iron supplementation, or gastrectomy failed to promote esophageal adenocarcinoma in these mice. Further studies are needed in order to develop a mouse model of esophageal adenocarcinoma.Surgically induced gastroesophageal reflux produced esophageal squamous cell carcinoma, but not esophageal adenocarcinoma, in mice. Dominant negative Esophageal adenocarcinoma is a rising malignancy in western world in the past 30 years, and now exceeds the incidence of esophageal squamous cell carcinoma . It is gp53 gene mutation and protein accumulation were frequently detected A/J A/JAla-1NK4a/Arf . PCR genNK4a/Arf .p53A135V transgenic mice, and 24 male and 5 female INK4a/Arf+/- mice for this study in our animal facility. These mice were housed 10 per cage in plastic cages with hardwood bedding and dust covers, in a HEPA-filtered, environmentally controlled room . Animals were given lab chow before surgery. Solid food was withdrawn for one day after surgery. All mice were on AIN93M diet after surgery, except that Group E received AIN93M diet supplemented with 1,400 ppm omeprazole. All the diets were made by Research Diets, Inc. once every month and kept at 4°C until use.We bred 151 male and 62 female wild-type mice, 36 male and 42 female i.p.). Esophagogastroduodenal anastomosis was performed through an upper midline incision. Two 0.5 cm incisions were made on the esophagus and the duodenum on the anti-mesenteric border, and then were anastomosed together with accurate mucosal to mucosal opposition. Total gastrectomy was performed on some mice following the reflux procedure ; Group D ; Group E ; Group F ; and Group G . One group of wild-type mice were used as non-operated control (Group A) ; Group C was administered to mice of 4 groups through 2. Esophagus was removed, opened longitudinally, and fixed in 10% buffered formalin for 24 h, and then transferred to 80% ethanol. The formalin-fixed esophagi were Swiss-rolled, processed and embedded in paraffin. Serial sections (5 μm) were mounted onto glass slides and used for histopathological analysis.All mice were euthanized by COH&E staining was performed on slides (No. 1 and 30) for diagnosis. Metaplasia was diagnosed when mucin-producing cells were observed in the squamous epithelium of mouse esophagus as confirmed by Alcian blue staining. Squamous dysplasia was diagnosed when squamous epithelial cells lost maturation and orientation with epithelial disorganization. Esophageal squamous cell carcinoma was diagnosed when squamous epithelial cells lost their normal orientation, had a high nucleus-to-cytoplasm ratio, and were heterochromatic, and dysplastic cells broke basement membrane and invaded into lamina propria [p53A135V transgenic mice (Group C) were stained for p53 protein in the esophagus. Paraffin sections were dewaxed in xylene, and rehydrated in a gradient of ethanol to distilled water. After quenching endogenous peroxidase activity with 3% hydrogen peroxide, tissue sections were incubated in normal horse serum to minimize non-specific binding. A polyclonal p53 antibody was applied at 4°C overnight. Tissue sections were then incubated with secondary biotin conjugated antibody at room temperature for 30 minutes. An avidin-biotin peroxidase complex was then applied, and the staining was visualized with diaminobenzidine. The sections were counterstained with Mayer's hematoxylin.Tissue sections of wild-type mice (Group B) and We compared the incidence of cancer with the Fisher's exact test.Most mice 85%, 255/300) survived the surgery. The rest died of anesthesia, bleeding or unknown reasons during the surgery. After surgery, mice lost about 3 to 5 g of body weight, and then started to gain weight to a less extent compared to the non-operated control mice . Metaplasia was confirmed by Alcian blue staining as scattered mucinous cells in the middle of hyperplastic squamous cells and s Figure and 3F, p53A135V transgenic mice (Group C), and reflux plus gastrectomy with iron supplementation (Group G). Squamous cell carcinoma was found in one INK4a/Arf+/- mouse (Group C) and 3 wild-type mice treated with reflux plus gastrectomy with iron supplementation (Group G). The incidence of squamous cell carcinoma in mice treated with reflux plus gastrectomy with iron supplementation (Group G) was significantly higher than that of mice treated with reflux alone (Group B) (p < 0.05).At 40 weeks after surgery, we found 6, 2 and 1 esophageal samples with metaplasia in wild-type mice (Group B), Because we did not find any esophageal adenocarcinoma at week 40, we decided to give iron supplementation to the mice left in surgical control group (Group B). At 80 weeks after surgery, we found 12 mice with esophageal squamous cell carcinoma in 13 wild-type mice. All squamous cell carcinoma were located at the distal part of the esophagus and invaded into the muscle layer Figure . Under hUnexpectedly, none of the mice developed either esophageal adenocarcinoma or typical intestinal metaplasia as rats in our previous studies .p53A135V transgenic mice, suggesting accumulation of mutant p53 protein , iron , or gastrectomy plus iron, were given to some of these mice in order to promote disease progression. Unexpectedly, we only observed metaplasia as scattered mucinous cells, but not typical intestinal metaplasia, in a small percentage of mice. Moreover, squamous cell carcinoma, but not esophageal adenocarcinoma, was induced.This study was primarily aimed to develop a surgical model of esophageal adenocarcinoma in mice. Reflux surgery was performed on wild-type, Long-term gastroesophageal reflux in combination with iron (Group B at week 80) produced a high incidence of squamous cell carcinoma in this study. Although gastroesophageal reflux has been often associated with Barrett's esophagus and esophageal adenocarcinoma, its association with squamous cell carcinoma has also been well documented in the literature. It is known that patients after total or partial gastrectomy had increased risk of developing esophageal and laryngeal squamous cell carcinoma -35. Reflp63 knockout mice and hypomorphic sox2 mice showed metaplastic changes of morphology and gene expression [p63 or sox2 knockout mice may be more susceptible to Barrett's esophagus and esophageal adenocarcinoma after surgery. It is likely that proper combinations of genetic modifications and reflux surgery may be needed to induced Barrett's esophagus and esophageal adenocarcinoma in mouse esophagus.Scattered mucinous cells were observed in mouse esophagi after surgery, suggesting that induction of Barrett's esophagus and esophageal adenocarcinoma in mouse esophagus is still possible . Our recpression ,40, we sRodent models of esophageal adenocarcinoma have its inherent limitations. Rodents have keratinized squamous epithelium without submucosal glands in the esophagus, whereas humans have non-keratinized squamous epithelium with submucosal glands. When histological and physiological resemblance to humans is considered, a model with pigs may offer many advantages over rodent models. As humans, pigs have non-keratinized stratified squamous epithelium and submucosal glands in their esophagi . Pigs map53A135Vtransgenic and INK4a/Arf+/- A/J mice. Further studies are needed in order to develop a mouse model of esophageal adenocarcinoma.In conclusion, reflux surgery induced esophageal squamous cell carcinoma, but not esophageal adenocarcinoma, in wild-type, GERD: gastroesophageal reflux disease.The authors declare that they have no competing interests.JH performed surgery on mice, contributed to study design, histopathology and data interpretation, and drafted the manuscript with XC. BL assisted surgery and animal care, and histopathology. CSY contributed to study design and data interpretation. XC performed surgery on mice, contributed to study design, histopathology and data interpretation, and drafted the manuscript with JH. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/9/59/prepub
Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3′ untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3′ poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3′ UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs. Drosophila melanogaster, zebrafish and mammalian systems, which contributes to miRNA-mediated destabilisation of their targets miRNAs are ∼22 nucleotide (nt) sized gene regulators that have pervasive roles in development and disease Renilla luciferase (R-luc) reporter mRNA in transfected HeLa cells in vivoin vitro7GpppN cap structure and the cap-binding eukaryotic initiation factor (eIF) 4E was impaired during miRNA-mediated repression. Our observations furthermore suggested that a miRNA inhibits poly(A) tail function during translation initiation in vitro translation systems D. melanogaster cells, though in some cases at a reduced level We reported previously that a synthetic miRNA termed miCXCR4 This situation prompted us to further investigate the involvement of the poly(A) tail and its removal by deadenylation in miRNA-mediated translational repression. We report here that an established model of let-7-mediated repression in mammalian cells 3xbulge expressing a Renilla luciferase (R-luc-3xb) mRNA with three bulged let-7 sites in the 3′UTR region, together with the plasmid pGL3 expressing a Firefly luciferase mRNA (F-luc) as a transfection efficiency control. Parallel transfection of cells with the seed region-mutated plasmid pRL-3xbulgemut provided non-targeted R-luc expression levels as a reference. A schematic of the R-luc mRNAs is shown in To further characterise the role of target mRNA deadenylation in the miRNA mechanism we employed an established model of let-7-mediated repression in mammalian cells GAPDH mRNA, which we found to display an intermediate and invariant poly(A) tail length (intensity peak at ∼50 nt) throughout test (LM-PAT) hout and. As an ahout and. The lethout and. CollectHMGA2 mRNA, a confirmed let-7 target HMGA2 mRNA is present primarily in an oligo-adenylated form, while transfection of let-7 anti-miR shifts it to a longer-tailed form . Conversely, we observed a shift from longer-tailed to oligo-adenylated HMGA2 during neuronal differentiation of mouse embryonic carcinoma P19 cells tail into knock-down cells tail and its removal to translational repression by let-7. To this end we employed a direct mRNA transfection approach in vitro , Fig. S3in vitro . Repressin vitro . (Similain vitro ). To detin vitro . This rein vitro may havein vitro from plasmids that were modified to include an A62 segment downstream of the let-7 target sites to template the poly(A) tail . We also found that appending the A62 tail and 8 nt 3′ tag instead of a longer poly(A) tail to the R-luc reporter mRNAs by itself did not affect on let-7-mediated repression, which was still strong tail removal, which involved sterically blocking the 3′ end of the target mRNA's poly(A) tail. To achieve this, we prepared capped R-Luc (3xb or mut) mRNAs carrying a poly(A) tail of defined length cap& . These m62 mRNA and the ‘tail-blocker’ (tb) LNA complementary to the 3′ tag or an unrelated control LNA (ns). We found that expression of R-luc protein from the R-Luc-mut mRNA tended to be mildly elevated in the presence of tb LNA compared to transfection of non-specific control LNA or mock transfection . To demonstrate that the tb LNA prevented reporter mRNA deadenylation as intended, we incubated our R-luc mRNAs in HeLa cell extracts To block access of deadenylases to the mRNA 3′ end, we co-transfected HeLa cells with R-luc-(3xb or mut)-cap&Asfection , consistsfection . tb LNA sfection of R-LucAltogether, these results show that removal of the poly(A) tail is not essential for miRNA-mediated translational repression. Nevertheless, they also indicate that deadenylation can augment miRNA-mediated translational repression in mammalian cells.We show here using a reporter system in HeLa cells that let-7 triggers target mRNA deadenylation, which is evident before measureable translational repression and mRNA destabilisation. The presence of a poly(A) tail on target mRNA enhances repression, while blocking tail removal by deadenylation reduces the magnitude of translational repression. Nevertheless, let-7-mediated repression can be attenuated despite ongoing deadenylation as observed in cells depleted of RCK or TNRC6A. Thus deadenylation is not essential for, but can quantitatively contribute to, let-7-mediated translational repression. Our findings are also consistent with a role of deadenylation in miRNA-mediated mRNA decay. In this way, we concur with and extend similar conclusions reached in other systems as referred to throughout this paper. A distinguishing feature of the present study is its detailed description of the relationship between miRNA-mediated deadenylation and translational repression in mammalian cells.In vitro systems to study miRNA-mediated translational repression of mammalian or D. melanogaster origin also feature miRNA-mediated deadenylation D. melanogastermRNA polyadenylation control is a versatile means to regulate gene expression D. melanogaster cells that blocking translation initiation on reporter mRNAs by interfering with either 40S recruitment Deadenylation is the first step in canonical mRNA turnover D. melanogaster cells using reporter constructs designed to give rise to mRNAs either carrying the 3′ end stem-loop of histone mRNA instead of a tail, or lacking a tail due to the presence of a self-cleaving ribozyme in the 3′ UTR Alternatively, deadenylation may exclusively stimulate mRNA decay. This has been addressed in mammalian and Taken together with the extant literature, our data thus favours a model whereby mRNA deadenylation is a proximal miRNA action that not only promotes mRNA decay but also attenuates the stimulatory role of the poly(A) tail in mRNA translation, by generating an oligo-adenylated form of the mRNA that associates less with cytoplasmic PABP. Indeed, mammalian miRNA target mRNAs are enriched in Ago and GW182 immunoprecipitations, while they are underrepresented in complexes containing PABPC4 The above scenario clearly does not accommodate post-initiation effects by miRNAs, and several reports have further presented experimental evidence or theoretical arguments to link miRNA-mediated repression to late sub-steps of translation initiation 3xbulge and pRL-3xbulgemut, which encode Renilla luciferase driven from a CMV promoter, were gifts from W. Fillipowicz in vitro differ slightly in that transcription begins at the T7 promoter and the poly(A) tail is added at the Hpa1 site 7 nt preceding the SV40 poly(A) signal cleavage site. The Firefly luciferase pGL3 plasmid was obtained from Promega. The pRL-A62 constructs (62 constructs. Linearisation of this pRL-pA62 construct with EcoICRI enzyme (Promega) creates an eight nucleotide overhang after the poly(A) tail stretch. The in vitro transcribed transcripts of RL-3xb/mut-cap&A62 mRNA encode a 3′UTR of ∼107 nt, with the encoded poly(A)62 starting 20 nt downstream of the last let-7 miRNA binding site.The plasmids pRL, pRL-nstructs were engAACTATACAACCTACTACCTCA, hsa-miR-499, TTAAACATCACTGCAAGTCTTAA. LNA used with the R-luc-3xb-A62 and R-luc-mut-A62 mRNAs have 100% LNA chemistry with the following sequences: Tail blocker (tb), CTCGGTACTTTT, complementary to the tag sequence 3′ of the A62 segment; non-specific (ns) control, CGCTGTATTTTT.LNA modified anti-miR oligonucleotides were obtained from Exiqon with the following sequences: hsa-let-7a, UGAGGUAGUAGGUUGUAUAG UU and miCXCR4, UGUUAGCUGGAGUGAAAAC UUGAAAUGCUCUGGUCCGCUAGCAGAAACCCUAUGAGAUU UUSynthetic duplex miRNA sense sequences used were: le62-3xbulge and pRL-A62-3xbulgemut were linearized with EcoICRI (Promega). DNA was purified by phenol/chloroform extraction and ethanol precipitation and used as template for in vitro transcription reactions as described 260 and mRNA quality inspected using the RNA 6000 Nanochip kit on an Agilent 2100 bioanalyzer.pRL-3xbulge and pRL-3xbulgemut were linearized with Hpa1 and pRL-AHeLa cells were maintained in DMEM with 5% FCS, supplemented with glutamine and penicillin/streptomycin, as detailed in For mRNA transfections, cells were seeded in a 24-well plate and transfections were performed in triplicate at ∼60–70% confluency by adding a preincubated transfection solution (200 µl) and 300 µl of Opti-MEM I (Invitrogen) to each well. The 200 µl of transfection solution (in Opti-MEM I) contained 1 µl Lipofectamine 2000 (Invitrogen), 10 or 20 ng Renilla luciferase-encoding (R-luc) mRNA and 80 ng pGL3 (F-luc control). Cells were harvested 16 hours after transfection unless stated otherwise. For anti-miR LNA transfections , 200 µl Plasmid transfections were performed similarly to mRNA, with the 200 µl transfection solution containing 30 ng of R-luc encoding plasmid and 600 ng of pGL3 F-luc. Medium was replaced ∼8 hours after transfection and cells were harvested at times specified. Additionally, in the time-course described in 2 bacterial grade plates for 48 hours before replating on fresh bacterial grade plates with fresh media plus RA for an additional 48 hours in RA to allow aggregates to form. Aggregates were plated out onto tissue culture grade plastic in the absence of RA on Day 4. Cells were harvested at the indicated time points For These were performed using the Dual-Luciferase Reporter Assay system (Promega) in a FLUOstar Optima plate reader (BMG Labtech) as described LI-COR Biosciences).SDS-PAGE gels were transferred to Immobilon-FL membrane (Millipore). Anti-RCK and α-Tubulin antibodies (Santa Cruz) were used at 1∶1000 working dilution. Fluorescent secondary antibodies were anti-rabbit/alexa-750 and anti-mouse/alexa-680 conjugated antibodies (Invitrogen). Blot hybridisation and detection was performed using the Odyssey Infrared Imaging System and buffers according to the manufacturer's instructions (This was done using Trizol (Invitrogen) according to the manufacturer's instructions.For analysis of reporter mRNA levels , 2, 3, HRCK and TNRC6A mRNA levels were normalised to mRNA levels of the ribosomal protein RPL13a. Primer sequences are shown in For qPCR analysis of siRNA-mediated depletion efficiencies RCK and http://www.mcb.arizona.edu/parker/PROTOCOLS/protocols.htm), except that RNase digestion was for 45 min at 37°C. The cleavage oligonucleotides used in this study were RH-R-Luc CTTCAGCACGCGCTCCAC and RH-Hs GAPDH CTTGCTGGGGCTGGTGGTCC. RNA .Cleavage reactions were performed as previously described (GCG AGC TCC GCG GCC GCG TTT TTT TTT TTT. The (dT)12VN primer used in the RT to generate a size marker for the shortest possible LM-PAT product was, GCG AGC TCC GCG GCC GCG TTT TTT TTT TTT VN32P-labelled TVN-R-luc PCR product, labelled with 32P-CTP by random priming. Detection of LM-PAT products and blots was by fluorescence scans or phosphorimaging using an FLA-5100 imager and MULTIGAUGE software (Fujifilm).To measure poly(A) tail lengths of multiple individual mRNAs, we used the Ligation-Mediated Poly(A) Test (LM-PAT) assay as described AACTATACAACCTACTACCTCA, see above for U6 probe) were 32P end-labelled using Polynucleotide kinase (NEB) according to the manufacturer's instructions, and incubated with the membrane in hybridization buffer at 42°C, then washed twice with 2xSSC. Detection by phosphorimaging used an FLA-5100 imager and MULTIGAUGE software (Fujifilm).To measure let-7 expression in P19 and HeLa cells , total RSupporting Information S1Supporting Results and Methods(0.10 MB DOC)Click here for additional data file.Figure S1Endogenous miRNA targets commonly exhibit short poly(A) tails. LM-PAT assays of miRNA targets E2F5, MYO10 and VAMP3 mRNA (SH-SY5Y cells) as well as RAVER, DNAJB11 and SERBP1 (HeLa cells). ACTB mRNA served as control.(1.07 MB EPS)Click here for additional data file.Figure S2Timecourse of protein and mRNA expression in transiently transfected HeLa cells. HeLa cells were cotransfected with either pRL-3xbulge or pRL-3xbulgemut plasmid (0.66 MB EPS)Click here for additional data file.Figure S3Microfluidic assessment of in vitro transcribed mRNAs. (A) Schematic of the variant R-luc mRNAs. (B) Variant R-luc-3xb and mut mRNA, as well as parental R-luc mRNA, were transcribed in vitro and analysed by microfluidics.(0.82 MB EPS)Click here for additional data file.Figure S4The mRNA poly(A) tail contributes to translational repression by synthetic let-7a. (A) Endogenous expression of let-7 in HeLa cells and P19 cells, either undifferentiated (−) or differentiated by retinoic acid treatment (RA), was measured by northern blotting . Undifferentiated P19 cells were co-transfected with R-luc mRNA (3xb or mut), pGL3 (F-luc control) and synthetic let-7 or control miRNA followed by incubation for 16 hours. (B) R-luc activity from the R-luc-mut mRNAs transfected into P19 cells . (C) Repression by synthetic let-7a in P19 cells was calculated by dividing the normalized R-luc activity from cells co-transfected with the miCXCR4 control by the normalized R-luc activity from cells co-transfected with let-7a . Averaged results from 4-7 independent triplicate repeat experiments are shown with standard error in B and C.(1.71 MB EPS)Click here for additional data file.Figure S5Normalisation to R-luc-RL mRNA can mask the poly(A) tail dependence of miRNA-mediated repression. HeLa cells were co-transfected with R-luc mRNA (3xb or RL) and pGL3 (F-luc control) and incubated for 16 hours. (A) R-luc activity from the R-luc-RL mRNAs transfected into HeLa cells . (B) Repression by endogenous let-7 in HeLa cells. Repression was calculated as for (0.49 MB EPS)Click here for additional data file.Figure S6The tb LNA blocks let-7-mediated mRNA deadenylation in vitro. (A) Schematic of the R-luc 3xb and mut cap&A62 mRNAs indicating the site for oligonucleotide-mediated RNase H cleavage. (B) R-luc 3xb or mut cap&A62 mRNAs were incubated in cell-free translation reactions based on HeLa extract for 30–180 min as indicated. Recovered RNA was subjected to RH-R-Luc oligonucleotide-mediated cleavage by RNAse H and northern blot analysis as in (16.18 MB EPS)Click here for additional data file.Table S1Gene specific primer sequences for quantitative PCR and LM-PAT assays.(0.04 MB DOC)Click here for additional data file.
Recently, it was described alteration in levels of two dopamine signaling related proteins in schizophrenic prefrontal cortex (PFC): Neuronal Calcium Sensor-1 (NCS-1) and DARPP-32. NCS-1, which is upregulated in PFC of schizophrenics, inhibits D2 internalization. DARPP-32, which is decreased in PFC of schizophrenics, is a key downstream effector in transducing dopamine signaling. We previously demonstrated that antipsychotics do not change levels of both proteins in rat's brain. However, since NCS-1 and DARPP-32 levels are not altered in wild type rats, we treated wild type PC12 cells (PC12 WT) and PC12 cells stably overexpressing NCS-1 (PC12 Clone) with antipsychotics to investigate if NCS-1 upregulation modulates DARPP-32 expression in response to antipsychotics treatment.Schizophrenia is the major psychiatry disorder, which the exact cause remains unknown. However, it is well known that dopamine-mediated neurotransmission imbalance is associated with this pathology and the main target of antipsychotics is the dopamine receptor DWe chronically treated both PC12 WT and PC12 Clone cells with typical or atypical (Clozapine and Risperidone) antipsychotics for 14 days. Using western blot technique we observed that there is no change in NCS-1 and DARPP-32 protein levels in both PC12 WT and PC12 Clone cells after typical and atypical antipsychotic treatments.Because we observed no alteration in NCS-1 and DARPP-32 levels in both PC12 WT and Clone cells treated with typical or atypical antipsychotics, we suggest that the alteration in levels of both proteins in schizophrenic's PFC is related to psychopathology but not with antipsychotic treatment. Schizophrenia is the major psychiatry disorder with prevalence of approximately 1% of worldwide population .dopamine and cyclic adenosine 3':5'-monophosphate-regulated phosphoprotein of relative molecular mass 32,000 (DARPP-32) and upregulation of Neuronal Calcium Sensor-1 (NCS-1) expression . To address if typical or atypical antipsychotics change NCS-1 levels in PC12 WT and PC12 Clone cells, we treated them with HAL , CLO (10 μM) and RIS (20 μM) for 14 days. PC12 cells were prepared to examine NCS-1 protein expression after drug treatment and it was observed no changes in NCS-1 levels in both PC12 WT , CLO (10 μM) and RIS (20 μM) for 14 days. PC12 cells were prepared to examine DARPP-32 protein expression after drug treatment and it was observed no changes in DARPP-32 levels in both PC12 WT , 5% fetal bovine serum and 5% horse serum. Cells were cultured at 37°C in a humidified 95% air/5% CO2 incubator. The medium and drugs were replaced every 2 days and the passages were performed every seven days. PC12 cells overexpressing NCS-1 were grown in DMEM as described above with addition of G418 (400 mg/mL - Clonetch). Reagents used for cell culture were purchased from Invitrogen Corporation (USA). PC12 cells stable overexpressing NCS-1 were obtained as described by Koizumi [PC12 cells were maintained Koizumi . PC12 WTPC12 (wt and Clone) cells lysates were sonicated and centrifuged at 13,000 × g for 30 min at 4°C. Supernatants were transferred to plastic tubes, protein was quantified and extracts stored at -80°C. 50 μg of each sample was prepared for electrophoresis with sample buffer NuPAGE LDS (Invitrogen) plus 10% of β-mercaptoethanol and incubated at 70°C for 10 min. The samples were loaded into bis-Tris NuPAGE 4-12% gels (Invitrogen) and submitted to electrophoresis followed by transfering to nitrocellulose membranes . Protein loading and efficiency of blot transfer were monitored by staining with Ponceau S . The membranes were blocked for 45 min with PBS Tween 20 0.1% plus 5% non-fat milk. Membrane blots were incubated with polyclonal anti-NCS-1 antibody , polyclonal anti-DARPP-32 and monoclonal anti-actin antibody (1:5000 - MAB1521R - Chemicon) diluted in PBS Tween 20 0.1%, for 2 hours at RT. Then, membranes were washed and incubated for one hour at RT with horseradish peroxidase (HRP)-conjugated secondary antibodies, goat anti-rabbit IgG (1:20000) and goat anti-mouse IgG (1:7000) (secondary antibodies were purchased from Molecular Probes). Membranes were submitted to chemiluminescent detection with ECL Plus (Amersham Biosciences) as described by manufacturer, and visualized on ImageQuant. Densitometric analysis was performed using Scion Image Software version Beta 4.0.2 .Student t-test and one-way ANOVA. In all experiments, P values lower than 0.05 were considered to significant.All data are presented as means ± Standard Deviation of the Mean (SD). Differences among experimental groups in experiments evaluating protein expression were determined by The authors declare that they have no competing interests.BRS, BSM, ESM, MMG and DSC performed the experiment. BRS, DVFR and RPS analyzed the data. BRS, KCL, DMM and HJR wrote the manuscript. AJ developed a new research tool. MARS conceived the study. All authors read and approved the final manuscript.
High-risk human papillomavirus testing or combined screening revealed a much higher sensitivity, at the cost of a small decrease in specificity, and a higher negative predictive value for the detection of lesions ⩾CIN3 till the next screening round (5 years) than cytology alone.We prospectively evaluated the 5-year predictive values of adding high-risk human papillomavirus (hrHPV) testing to cytology for the detection of ⩾cervical intraepithelial neoplasia (CIN)3 lesions in a population-based cohort of 2810 women. At baseline, nine (0.3%) women had prevalent lesions ⩾CIN3, all being hrHPV positive. After 5 years of follow-up, four (6.5%) of the 62 hrHPV-positive women with normal cytology developed lesions ⩾CIN3, Cervical screening by cytology is known to yield a substantial proportion of both false-positive and false-negative smears. Since infection with high-risk human papillomavirus (hrHPV) is considered to be the cause of cervical carcinoma, it has been suggested that adding hrHPV testing to cervical screening might improve screening in terms of reducing false-positive and false-negative smears was enrolled from March 1995 till October 1998 by 60 general practitioners in the small district of Amstelveen, The Netherlands. In all, 194 women were excluded because of abnormal cervical cytology and/or histology during 2 years preceding the intake, 159 women because of a negative Sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of cytology, cytology and hrHPV testing combined, and hrHPV testing, for the detection of prevalent and incident lesions ⩾CIN3 were computed using two by two tables. Results are presented as percentages with 95% confidence intervals (95% CI). To determine whether any gain in test performance from the addition of the second test (hrHPV testing) was greater than that expected if the second test offered no diagnostic information, expected performance characteristics for cytology combined with a random test having the similar prevalence as hrHPV were computed .Of 2810 women for analysis at baseline, 2687 (95.6%) women had normal cytology, of whom 77 (2.9%) had a positive hrHPV test. Among 111 (4.0%) women with borderline or mild dyskaryosis (BMD), 16 (14.4%) women were hrHPV positive, as were 11 (91.7%) of 12 (0.4%) women with moderate dyskaryosis or worse (>BMD). Among the 123 (5.2%) women who had abnormal cytology, nine (7.3%) cases of prevalent lesions ⩾CIN3 were present (including three squamous cell carcinomas) , resultiThe median follow-up time until histological or cytological diagnoses was 4.6 years (range 0.1–8 years). The follow-up results of women with normal cytology at baseline are presented in vs 64.3%, respectively; P=0.065), but was less specific , and had an increased NPV with a similar PPV . High-risk human papillomavirus testing alone was more sensitive than cytology alone , more specific , and had an increased NPV and an increased PPV . Comparing the sensitivity and NPV of cytology and hrHPV combined screening to cytology with a random test, no different significance levels were revealed as when compared to cytology alone.The performance characteristics of cytology, cytology and hrHPV combined, and hrHPV testing, for the detection of lesions ⩾CIN3 are given in Combined screening revealed a much higher sensitivity, at the cost of a small decrease of specificity, and a higher NPV at the next screening interval (after 5 years) than cytology alone. Cytology alone had reasonable performance characteristics, but on adding hrHPV testing we can achieve much better performance characteristics in population-based screening.The overall detection rate by cytology of 0.3% lesions ⩾CIN3 at baseline and 0.5% after 5 years in this study is comparable to the detection rate of lesions ⩾CIN3 in cervical cancer screening in The Netherlands (P⩽0.05). This might be due to our relatively small population with a low prevalence of lesions ⩾CIN3, resulting in wide 95% CIs. However, with this low prevalence of lesions ⩾CIN3, still a borderline significance was reached (P⩽0.1). The increase in sensitivity by combined testing compared to cytology alone may be misleading because improvements in sensitivity would be expected by adding a second test, even if the second test performed randomly with respect to disease identification. For this reason, expected performance characteristics for cytology combined with a random test were computed to determine if any gain in test performance from the addition of the second test (hrHPV testing) was greater than that expected if the second test offered no diagnostic information (Our data are the first that were prospectively obtained in population-based screening, with a 5-years screening interval in women 30–60 years of age. Some methodological aspects of this study need to be discussed. The fact that histology was not obtained in all women might induce a verification bias in advantage of combined screening and hrHPV testing. However, women were followed according to current practice standards in nationwide screening with cytology after 5 years, and in this setting women with normal cytology are considered to be free of disease. As indicated by the overlapping 95% CIs, the gains in sensitivity and NPV by combined testing or hrHPV testing compared to cytology alone were not significant (ormation . By compCost-effectiveness studies and modelling studies show that cervical screening may become much more efficient in terms of decreasing numbers of false-negative and false-positive smears, if a test is used with a substantial higher sensitivity and long-term NPV than conventional cytology (The use of hrHPV testing in cervical screening could lead to several different screening strategies, including combined cytology and hrHPV testing, or primary screening by hrHPV with cytology reading only of women tested hrHPV positive. In addition, the high sensitivity and NPV of hrHPV testing opens possibilities for longer screening intervals with still acceptable rates of incipient lesions. Modelling studies and confirmation of our results in larger studies will help to clarify this discussion and to devise more efficient cervical screening strategies.
In vitro and ex vivo cellular plasmalogen deficiency models have been shown to exhibit impaired intra- and extra-cellular processing of cholesterol. Furthermore, depleted brain plasmalogens have been implicated in AD and serum plasmalogen deficiencies have been linked to AD, CVD, and cancer.Disrupted cholesterol regulation leading to increased circulating and membrane cholesterol levels is implicated in many age-related chronic diseases such as cardiovascular disease (CVD), Alzheimer's disease (AD), and cancer. Using plasmalogen deficient (NRel-4) and plasmalogen sufficient (HEK293) cells we investigated the effect of species-dependent plasmalogen restoration/augmentation on membrane cholesterol processing. The results of these studies indicate that the esterification of cholesterol is dependent upon the amount of polyunsaturated fatty acid (PUFA)-containing ethanolamine plasmalogen (PlsEtn) present in the membrane. We further elucidate that the concentration-dependent increase in esterified cholesterol observed with PUFA-PlsEtn was due to a concentration-dependent increase in sterol-O-acyltransferase-1 (SOAT1) levels, an observation not reproduced by 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition.The present study describes a novel mechanism of cholesterol regulation that is consistent with clinical and epidemiological studies of cholesterol, aging and disease. Specifically, the present study describes how selective membrane PUFA-PlsEtn enhancement can be achieved using 1-alkyl-2-PUFA glycerols and through this action reduce levels of total and free cholesterol in cells. A breakdown in cholesterol homeostasis has adverse effects at the cellular level, as well as in the context of the organism. Altered cholesterol content in cells affects membrane fluidity, which has drastic effects on cellular function, signal transduction, and intercellular communication events ,2. Elevade novo, and accounts for 25% of the total body cholesterol, wherein it exists primarily as free cholesterol in myelin and the plasma membranes of glial cells and neurons + 75%), 637 (30%), 385 (40%), 329 (15%), 311 (100%), 293 (60%), 269 (20%), 75 (60%).Obtained from C21, as light yellow oil; 62.4 mg (61.3%); TLC: Rf = 0.75 ; 1H NMR (in CDCl3): δ in ppm 0.86 , 0.94 , 1.25-1.29 , 1.51-1.55 , 1.99-2.07 , 2.15 , 2.40-2.45 , 2.77-2.83 , 3.40-3.45 , 3.53-3.64 , 3.77-3.78 , 4.98 , 5.33-5.36 ; 13C NMR (in CDCl3): δ in ppm 14.3, 14.4, 20.7, 22.7, 22.8, 25.7, 25.8 (2), 26.2, 27.4, 29.4, 29.5, 29.6 (2), 29.7, 29.9 (3), 32.1, 34.1, 64.3, 70.8, 72.0, 72.5, 127.2, 127.9, 128.0, 128.2, 128.3 (2), 128.4 (2), 128.5, 128.7, 129.7, 129.9, 130.1, 132.2, 173.1; FT-IR (cm-1) 3385 (br), 2917, 2852, 1722; HRAPCI-MS m/z: measured 653.5509 ; APCI-MS/MS m/z: 653 ([M + H]+ 70%), 635 (30%), 385 (35%), 329 (15%), 311 (100%), 293 (65%), 269 (20%), 97 (30%).Obtained from 2. CHO and NRel-4 cells were a kind gift from Dr. R.A. Zoeller (Boston University) and were cultured in F-12 medium, 10% FBS at 37°C, 5% CO2. NRel-4 cells are deficient in peroxisomal dihydroxyacetonephosphate acyltransferase or NRel-4 cells were seeded in DMEM/F-12 medium (10% FBS) on a 10 cm dish the day before the experiment. The following day, the media was replaced with fresh media containing the test compound or the solvent ethanol (0.1% V/V) as a control [2, after which they were harvested using Versene/TrypLE express . The cell pellet was washed with phosphate buffered saline , and the phospholipids were extracted and analyzed using a linear ion-trap mass spectrometer coupled to a LC system as described [The plasmalogen-deficient CHO cell line (NRel-4) was used to assay the efficacy of test compounds control . Cells wescribed .C1-10 or with ethanol as the control. Concentrations used for pravastatin, clofibrate, and troglitazone treatments were as described [Human embryonic kidney 293 (HEK293) cells and CHO/NRel-4 cells were seeded the day before the treatment. The following day, the cells were treated with the test compounds escribed -49. CellHEK293 cells were treated as described in the amyloid assay. The cell pellet was washed in PBS and lysed in RIPA buffer containing a protease inhibitor cocktail . Protein in the cell lysate was quantified using the Bio-Rad Protein Assay . The following antibodies were used for western analyses: SOAT1 and β-actin . Band intensities were calculated using ImageJ .Statistical Analysis of the data was performed using Microsoft Office Excel 2007 and JMP version 8. Multiple comparison Dunnett's tests were applied to analyze the differences between the treatments and the control.Membrane plasmalogen levels of NRel-4 cells, lacking dihydroxyacetone phosphate acyl transferase (DHAPAT), an obligate enzyme in the plasmalogen biosynthesis pathway ,51, wereUsing wild CHO and NRel-4 cells, side chain-specific PlsEtn and phosphatidylethanolamine (PtdEtn) precursors Table were evasn-3 and DHA at sn-2, PlsEtn precursors C1-3 with differing long chain ether substitutions at sn-1 revealed that these precursor compounds either partially or fully restored all ethanolamine plasmalogens with the same sn-1 alkyl ether but had no effect on PlsEtn with different sn-1 compositions restored the downstream pool of 16:0 ethanolamine plasmalogens with no effect on the 18:0 and 18:1 PlsEtn pools. Such side chain-specific restoration indicates that no rearrangement of the sn-1 moiety occurs, while the sn-2 moiety is able to undergo deacylation and subsequent reacylation with other fatty acid residues.1. Maintaining the free alcohol at s Figure . For exaC6-C10 (16:0 at sn-1 but differing at the sn-2 substituent) significantly elevate the 16:0 pool, with no effect on the 18:0 and 18:1 pools of PlsEtn . C2 on the other hand significantly augments all PlsEtns in the 18:0 pool depends on the fatty acid at l Figure .C1, C6-10, revealed that whereas DHA containing precursors can partially or fully restore all other sn-2 PlsEtn, non-DHA containing precursors cannot completely restore DHA-PlsEtn cannot restore DHA-PlsEtn deficiencies and wild-type cells (CHO) Figure . However) Figure .As demonstrated above, plasmalogen deficient cells have higher content of free cholesterol and lower amounts of esterified cholesterol in their cell membranes. To determine whether this effect was due to a general decrease in membrane PlsEtn composition or to decreased levels of specific PlsEtn, membrane PlsEtn levels in PlsEtn depleted cells (NRel-4) were selectively restored as described above and the corresponding effect on membrane cholesterol composition ascertained. The key observations were:C4 and C5) had no effect, while PlsEtn precursors with < 3 unsaturations (C7 and C8) had a mild effect on membrane cholesterol composition had a more profound effect on reducing free cholesterol Figure and a re) Figure C4 and C5) had no effect on free cholesterol and resulted in slight decreases in esterified cholesterol.2. PtdEtn precursors either elevated or had no effect on free cholesterol.3. PlsEtn precursors with sn-2 substituents containing 3 or more unsaturations either reduced free cholesterol (C1 and C9) and/or increased esterified cholesterol (C1 and C10).4. PlsEtn precursors with 5. Free DHA had a slight impact on free cholesterol (14% reduction) compared to control, while it exhibited a 24% increase in the esterified cholesterol fraction at the 20 μM concentration.6. Pravastatin treatments were most potent in reducing free cholesterol at 10 μM concentration , while the 100 μM concentration did not result in a further reduction of free cholesterol. The changes observed in the esterified cholesterol were not significant.7. Treatments with PPARα and PPARγ agonists had no impact on the cholesterol profile of HEK293 cells at the concentrations tested.C1,and of the potent HMG-CoA reductase inhibiting/total cholesterol lowering statin, pravastatin, on the basal levels of cholesterol processing enzyme SOAT1 was determined. The maximum cholesterol-lowering concentration of C1, resulted in a 50% elevation of SOAT1 levels , whereas the maximum cholesterol-lowering concentration of pravastatin had no effect on SOAT1 levels revealed that the sn-1 substituent affected rearrangement at sn-2 position due to the steric hindrance caused by 18:0 fatty acid at sn-1 position.PlsEtn deficient cells have been previously shown to have impaired HDL-mediated cholesterol efflux and impan Figure , and henC1 concentration-dependently increased membrane esterified cholesterol (Figure These structure activity relationships revealed that changes in membrane PUFA-PlsEtn levels are principally responsible for the observed cholesterol effect Figure and thatl Figure and decrl Figure . AdditioThe observed increase in cholesterol esterification is suggested to be due to elevated SOAT1, an enzyme expressed in liver cells and macrophages which is involved in cholesterol homeostasis Figure . These oIt is prudent to note that ACAT inhibition was thought to be a promising pharmaceutical target for controlling hypercholesterolemia. Several ACAT inhibitors entered clinical trials, only to emerge with disappointing results. Avasimibe and pactimibe treatment did not hamper the progression of coronary atherosclerosis ,65. On tin vitro.In summary, using a series of 1-alkyl-2-acylglycerols, we showed that membrane PlsEtn levels can be selectively restored in a PlsEtn deficient system and selectively augmented in PlsEtn normal cells in a concentration-dependent manner. Accordingly, these results represent the first report of selective plasmalogen enhancement in normal cells. The structure activity relationship study suggests that selective PUFA-PlsEtn enhancement is capable of beneficially favoring cholesterol esterification, an obligate step prior to efflux from the cell. This translates to a net reduction in the fraction of free cholesterol in cells. Plasmalogen restoration/enhancement therefore offers a novel mechanism of cholesterol reduction PLW is CEO of and owns stock in Phreedom Pharma.DBG is CEO of and owns stock in Phenomenome Discoveries Inc.RM designed and conducted experiments, prepared the manuscript. PWKA and DJ synthesized compounds used in the study. HM carried out experiments. KKS carried out statistical analyses. MAK participated in design of experiments. SR, PLW and DBG participated in design and manuscript preparation. All authors read and approved the manuscript.
Elevated intra-abdominal pressure can cause significant impairment of cardiac, pulmonary, renal, gastrointestinal, hepatic, and central nervous system function. The significant prognostic value of elevated intra-abdominal pressure has prompted many intensive care units to adopt measurement of this physiologic parameter as a routine vital sign in patients at risk. A thorough understanding of the pathophysiologic implications of elevated intra-abdominal pressure is fundamental to 1) recognizing the presence of intra-abdominal hypertension and abdominal compartment syndrome, 2) effectively resuscitating patients afflicted by these potentially life-threatening diseases, and 3) preventing the development of intra-abdominal pressure-induced end-organ dysfunction and failure. The currently accepted consensus definitions surrounding the diagnosis and treatment of intra-abdominal hypertension and abdominal compartment syndrome are presented. Although initially recognized over 150 years ago, the pathophysiologic implications of elevated intra-abdominal pressure (IAP) have essentially been rediscovered only within the past two decades . An explRecently, evidence-based consensus definitions and recommendations for the resuscitation and rehabilitation of patients with IAH and ACS have been published ,20. Cent2O significantly impaired diaphragmatic excursion leading to elevated intrathoracic pressure, respiratory failure, and death [The impact of elevated IAP upon respiratory function was first documented by Marey in 1863 and subsequently by Burt in 1870 . In 1890nd death . The thend death . The detnd death -33. Overnd death . He postnd death . The expnd death -39. Grosnd death .Although surrogate measurement of IAP via measurement of intravesicular, intragastric, and intracolonic pressure in animal models was commonplace in the 1920's and 1930's, it was Söderberg who, in 1970, first described the strong correlation between IAP and intravesicular pressure during laparoscopy in humans . The lanAn increasing body of literature has identified the significant physiologic derangements that occur as a result of elevated IAP. The effects of IAH are not limited just to the intra-abdominal organs, but rather have an impact either directly or indirectly on every organ system in the body. As a result, patients with prolonged, untreated IAH commonly manifest significant malperfusion and subsequent organ failure. Pre-existing comorbidities, such as chronic renal failure, pulmonary disease, or cardiomyopathy, play an important role in aggravating the effects of elevated IAP and may reduce the threshold of IAH that causes the clinical manifestations of ACS. The etiology for the patient's IAH is similarly of vital importance and may be determined as being either intra-abdominal, as occurs in surgical or trauma patients following damage control laparotomy, or extra-abdominal, as occurs in medical patients with sepsis or burn patients who require aggressive fluid resuscitation ,7,43-46.As originally described over 80 years ago by Emerson, rising IAP increases intrathoracic pressure through cephalad deviation of the diaphragm . IncreasParadoxically, intracardiac filling pressures such as pulmonary artery occlusion ("wedge") pressure (PAOP) and central venous pressure (CVP) typically increase with rising IAP despite the reduced venous return and cardiac output ,57,59-64IAH also reduces venous return from the lower extremities functionally obstructing inferior vena caval blood flow by two mechanisms. First, inferior vena caval pressure increases significantly in the presence of IAH and has been demonstrated to parallel changes in IAP ,33,53,56The pulmonary effects of elevated IAP have been recognized for many years ,68,72-74IAH-induced reductions in renal blood flow and function have been demonstrated in both animal and human models ,42,51,77Several mechanisms have been proposed as the etiology for IAH-induced renal dysfunction and failure. Harman et al. negated direct ureteral compression as a cause through studies utilizing ureteral stents . Other aIAH decreases glomerular filtration rate causing a rise in both blood urea nitrogen and serum creatinine and a reduction in creatinine clearance ,48,51,79Of all the organ systems, the gut appears to be one of the most sensitive to elevations in IAP. Such reductions in mesenteric blood flow may appear with an IAP of only 10 mmHg . CaldwelIn addition to reducing arterial blood flow, IAP compresses thin walled mesenteric veins promoting venous hypertension and intestinal edema. Visceral swelling further increases IAP initiating a vicious cycle which results in worsening malperfusion, bowel ischemia, decreased intramucosal pH, feeding intolerance, systemic metabolic acidosis, and significantly increased patient mortality ,50,86,87Hepatic artery, hepatic vein, and portal vein blood flow are all reduced by the presence of IAH ,54,77,912 , and decreased cerebral venous outflow [Cerebral perfusion and function are also directly affected by the presence of IAH. According to the Monroe-Kellie doctrine, the brain consists of four discrete compartments: parenchymal, vascular, osseous, and cerebrospinal fluid. An increase in the pressure within one compartment results in a reciprocal increase in the pressure within each of the other non-osseous compartments. Whereas chronic, slowly developing increases in intracranial pressure (ICP) may allow time for compensation, the acute increases in ICP characteristic of both traumatic injury and acute illness commonly result in rapidly escalating intracranial pressures. Elevations in intra-abdominal and intrathoracic pressure may also directly impact the pressures within the cranium. Coughing, defecating, emesis, and other common causes of increased intra-abdominal and intrathoracic pressure are well known to transiently increase ICP ,93,94. I outflow ,94,97,98 outflow ,97. This outflow . Intrace outflow ,98. Suge outflow . Decreas outflow ,95. Hypo outflow ,95.Although commonly overlooked, the abdominal wall is also subject to the effects of elevated IAP. Visceral edema, abdominal packs, and free intraperitoneal fluid all distend the abdomen and reduce abdominal wall compliance ,100. Abd consisting of European, Australasian, and North American surgical, trauma, and medical critical care specialists. Recognizing the lack of accepted definitions, and the resulting confusion and difficulty in comparing studies published in this area, the WSACS tasked these specialists to create evidence-based definitions for IAH and ACS. After extensively reviewing the existing literature, the authors suggested a conceptual framework for standardizing the definitions of IAH and ACS as well as a general technique for IAP monitoring based upon the current understanding of the pathophysiology of these two syndromes [In 2004, a consensus conference was convened by the World Society of the Abdominal Compartment Syndrome (WSACS) yndromes . A briefThe abdomen may be considered as a closed box with walls that are either rigid or flexible . The compliance of these walls and the volume of the organs contained within determine the pressure within the abdomen at any given time -104 IAP Analogous to the widely utilized concept of cerebral perfusion pressure, abdominal perfusion pressure (APP), defined as MAP minus IAP, has been demonstrated to be an accurate predictor of visceral perfusion and an endpoint for resuscitation ,105,106.As described above, oliguria is one of the first visible signs of IAH. Inadequate renal perfusion pressure and renal filtration gradient (FG) have been proposed as key factors in the development of IAP-induced renal failure ,108. The2O while subsequent studies using electronic pressure transducers reported IAP in mmHg (1 mmHg = 1.36 cm H2O). This led to confusion and difficulty in comparing studies. A point of further confusion has been the appropriate zero reference point for the abdomen. Changes in body position can have a significant impact upon the measured IAP. While head of bed elevation is now commonly performed to reduce the incidence of ventilator-associated pneumonia, the clinical studies that determined the threshold IAP values that lead to organ dysfunction were determined in the supine position. Further, the presence of both abdominal and bladder detrusor muscle contractions have been demonstrated to impact the accuracy of IAP measurements. Perhaps the greatest point of contention has been the proper priming-volume to be instilled into the bladder to ensure a conductive fluid column between bladder wall and transducer. Large instillation volumes, as commonly utilized in years past, have been demonstrated to result in artificial increases in IAP that could lead to inappropriate therapy. In an attempt to address these issues and ensure both the accuracy and reproducibility of IAP measurements, the WSACS has recommended that IAP be expressed in mmHg and measured at end-expiration in the complete supine position after ensuring that abdominal muscle contractions are absent and with the transducer zeroed at the level of the mid-axillary line [The sensitivity of both clinical judgement and physical examination have been demonstrated to be very poor in predicting a patient's IAP ,110. Earary line . Furtherary line .Normal IAP ranges from sub-atmospheric to zero mmHg ,113,116.2O) as follows: Grade I, 7.5–11 mmHg (10–15 cm H20); Grade II, 11–18 mmHg (15–25 cm H20); Grade III, 18–25 mmHg (25–35 cm H20); and Grade IV, > 25 mmHg (> 35 cm H20) [Pathological IAP is a continuum ranging from mild IAP elevations without clinically significant adverse effects to substantial increases in IAP with grave consequences to virtually all organ systems in the body. The exact IAP that defines IAH has long been debated. Burch et al. defined an early grading system for IAH (in cm H cm H20) . Burch s cm H20) ,118-124. cm H20) ,20. In tAmong the majority of patients, critical IAP appears to be 10–15 mmHg. It is at this pressure that reductions in microcirculatory blood flow occur and the initial signs of organ dysfunction and failure are witnessed. ACS is the natural progression of these pressure-induced end-organ changes and develops if IAH is not recognized and treated in a timely manner. Failure to recognize and appropriately treat ACS is commonly fatal while prevention and/or timely intervention is associated with marked improvements in organ function and patient survival ,125-127.In contrast to IAH, ACS is not graded, but rather considered an "all or nothing" phenomenon. The WSACS defines ACS as a sustained IAP > 20 mmHg (with or without an APP < 60 mmHg) that is associated with new organ dysfunction or failure (Appendix 1) ,20. ACS Elevated IAP commonly causes marked deficits in both regional and global perfusion that, when unrecognized, result in significant organ failure and patient morbidity and mortality. Significant progress has been made over the past decade with regard to understanding the etiology of IAH and ACS as well as implementing appropriate resuscitative therapy. Routine measurement of IAP in patients at risk is essential to both recognizing the presence of IAH/ACS and guiding effective treatment. Adoption of the proposed consensus definitions and recommendations has been demonstrated to significantly improve patient survival from IAH/ACS and will facilitate future investigation in this area.2: fraction of inspired oxygen; PEEP: positive end-expiratory pressure; ICP: intracranial pressure; PAOP: pulmonary artery occlusion pressure; CVP: central venous pressure.IAP: intra-abdominal pressure; IAH: intra-abdominal hypertension; ACS: abdominal compartment syndrome; MAP: mean arterial pressure; APP: abdominal perfusion pressure; FG: filtration gradient; GFP: glomerular filtration pressure; PTP: proximal tubular pressure; PIP: peak inspiratory pressure; FiOFinancial competing interests• Dr. Cheatham has served as a consultant for Kinetic Concepts, Inc., Wolfe-Tory Medical, Inc., and Bard Medical, Inc.Non-financial competing interests• Dr. Cheatham is a member of the World Society of the Abdominal Compartment Syndrome Executive Committee.MLC is the sole contributor to this manuscript.Abdominal distentionElevated IAPOliguria refractory to volume administrationElevated PIPHypercarbiaHypoxemia refractory to increasing FiO2 and PEEPRefractory metabolic acidosisElevated ICPLegend: These represent the most common organ dysfunctions associated with the development of severe intra-abdominal hypertension and a diagnosis of abdominal compartment syndrome.
Advanced diagnostic tools, classification systems and accordingly selected surgical approaches are essential requirements for the prevention of failure of surgical treatment of thoracolumbar fractures. The present study is designed to evaluate the contribution of classification to the choice of a surgical approach using the current fracture classification systems.We studied prospectively a group of 64 patients of an average age of 43 years, all operated on for thoracolumbar fractures during the year 2001. The AO-ASIF classification was used preoperatively with all imaging studies and magnetic resonance imaging (MRI)). When the damage was detected only in the anterior column (A type), an isolated anterior stabilization (n = 22) was preferred. If the MRI study disclosed an injury in the posterior column, a posterior approach (n = 20) using the internal fixator was chosen. Injuries involving the posterior column (B or C type) were classified additionally according to the load-sharing classification (LSC). If LSC gave six or more points, treatment was completed with an anterior fusion. The combined postero-anterior procedure was carried out 22 times. The minimum followup period was 22 months.Neither implant failure and nor significant loss of correction were observed in patients treated with anterior or combined procedures. The average loss of correction (increase of kyphosis) in simple posterior stabilization was 3.1 degree.Complex fracture classification helps in the selection of the surgical approach and helps to decrease the chances of treatment failure. Patients with multiple fractures, osteoporosis and spinal cord injury were excluded from the study. The series consisted of 22 women and 42 men with a mean age of 43 years (19-71 yrs).All patients were investigated preoperatively by plain X-ray, CT and MRI and their injury examined using the AO-ASIF classification. When the damage was detected only in the anterior column (AO-ASIF A type), an isolated anterior approach was preferred. Stabilization was carried out using an anterior angle-stable device (MACS-TL) and a spacer or tricortical bone graft. If an injury of the posterior column (bony or ligamentous) was disclosed, the posterior approach using the internal fixator was chosen. In these injuries (AO-ASIF B or C type), damage of the anterior column was classified additionally according to the load-sharing classification (LSC).The patients were divided into three groups:group I (n = 22): “A” type fractures treated with simple anterior stabilization and fusiongroup II (n = 22): “B or C” type of fractures with LSC scoring equal or higher than 6 points treated using combined posteroanterior procedure Figure –d.Figurgroup III (n = 20): “B or C” type fractures with LSC scoring less then 6 points, treated with only the posterior approachWhen applied solely without any fusion or with monosegmental fusion in combined procedures, the internal pedicular fixator was removed after an average period of 15 months. Anterior implants were not removed. Patients in all groups were followed systematically with regards to subjective, clinical and radiographic results. Followup examination was performed six and 12 weeks and six and 12 months postoperatively and then yearly. Subjective assessment was based on self-evaluation of daily activities; objective followup parameters were assessed according to Prolo´s functional and economical scale. This study exclusively compares the early postoperative and the final radiographic results (endplate angle). In light of this objective, loss of correction (increase of kyphosis), possible implant failure and the fusion rate in patients with an anterior fusion were evaluated. Fusion assessment was based on analysis of lateral plain radiographs. Patients were followed for at least 22 months after the operation; the longest followup was 38 months.Type B fractures (29 cases) were the most frequent type of fractures. Survey of diagnoses, fusion extent and LSC points in all three groups are shown in The average values of LSC scoring in fractures treated with combined procedures and with indication for mono- and bi-segmental fusion (group II) were 6.4 and 8.0 respectively. The average LSC score in fractures treated with only posterior stabilization (group III) was 4.8 . NeitherThe technology of spine fracture imaging has significantly improved in recent years. Nevertheless, high-quality X-rays are still essential in the projection of basic shape parameters of the spine and the fracture and are the most accurate tool for evaluation of treatment results. Computer tomography (CT) perfectly depicts the whole vertebra and is an important guide for surgical planning. It gives information about bone fragments within the spinal canal. So far, there is no other imaging technology that is more useful than CT to examine facet relationships. Sagittal reconstruction makes it possible to evaluate the angle of kyphosis and the shape of the spinal canal narrowing. Magnetic resonance imaging (MRI) is dominant in soft tissue imaging: intervertebral discs , vessels (thrombosis), nerve roots and mainly the spinal cord . This is a very important, noninvasive, diagnostic method for the evaluation of ligamentous injury. The AO-ASIF classification system is not applicable in preoperative decision-making without an MRI examination.11The effort to understand the principles of spine stability led to the theory of three columns initially proposed by Denis for thoracolumbar fractures,e.g., a decrease in residual fracture stability.The “comprehensive classification of thoracolumbar fractures”, also known as the AO-ASIF classification of spine fractures is based on the two column theory and divides all fractures into three categories, which are further sub divided into 55 groups, to define the various fractures of the thoracic and lumbar spine.et al.et al. reported the finding that 30% of type B fractures (AO-ASIF classification) are initially overlooked due to reliance on the AO-ASIF classification alone.11The reliability of the AO-ASIF classification was tested by Blauth The second classification system is the “Load Sharing Classification” (LSC) devised by McCormack, Karaikovic and Gaines,Preoperative analysis of bony fracture anatomy with LSC is useful in determining candidates for short segment posterior instrumentation, short segment anterior stabilization or short segment posterior stabilization and anterior fusion with strut graft. The classification does not grade ligament damage and is not related to the mechanism of injury. It is a helpful adjunctive tool that can complement but not replace other forms of classification.18The surprisingly high proportion of B-type fractures observed19The concept of surgical approach selection described in this paper is limited in several aspects. We do not use it in cases where the posterior approach is obviously the method of choice . It is also necessary to keep in mind the specific features of anterior approaches in the upper third of thoracic spine. Additionally, the anterior approach has more contraindications with respect to the patient's general condition.Complex fracture classification comprising a combination of the AO-ASIF and LSC classification methods helps to choose the surgical approach. A classification-related approach facilitates the prevention of treatment failure.
FISH assays also revealed a chromosomal inversion between Cucumis subspecies [C. sativus var. sativus L. and var. hardwickii (R.) Alef], which resulted in marker clustering on the genetic map. A quarter of the mapped markers showed relatively high polymorphism levels among 11 inbred lines of cucumber. Among the 995 markers, 49%, 26% and 22% were conserved in melon, watermelon and pumpkin, respectively. This map will facilitate whole genome sequencing, positional cloning, and molecular breeding in cucumber, and enable the integration of knowledge of gene and trait in cucurbits.The Cucurbitaceae includes important crops such as cucumber, melon, watermelon, squash and pumpkin. However, few genetic and genomic resources are available for plant improvement. Some cucurbit species such as cucumber have a narrow genetic base, which impedes construction of saturated molecular linkage maps. We report herein the development of highly polymorphic simple sequence repeat (SSR) markers originated from whole genome shotgun sequencing and the subsequent construction of a high-density genetic linkage map. This map includes 995 SSRs in seven linkage groups which spans in total 573 cM, and defines ∼680 recombination breakpoints with an average of 0.58 cM between two markers. These linkage groups were then assigned to seven corresponding chromosomes using fluorescent Cucurbitaceae family comprises about 120 genera and 800 species, including many economically important vegetable and fruit crops such as cucumber (Cucumis sativus L.), melon (C. melo L.), watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), squash and pumpkin (Cucurbita spp.) x = 14, 367 Mb; melon: 2n = 2x = 24, 480 Mb; watermelon: 2n = 2x = 22, 430 Mb, and squash and pumpkin: 2n = 2x = 40, 539 Mb) The Cucumis, cucumber is the only species with a haploid chromosome number of seven . It is cross-incompatible with other Cucumis species and consequently, cucumber has a narrow genetic basis within domesticated market types C. s. var sativus L. (cultivated) and the feral form C. s. var hardwickii (R.) Alef coexist.In the genus of in situ hybridization (FISH) Unsaturated cucumber linkage maps have been developed using morphological traits, isozymes, and molecular markers, where markers (<300 loci) were often positioned in more than seven linkage groups The availability of high-density maps in cucumber would facilitate whole genome sequencing and positional cloning, enhance MAS, and provide opportunities to investigate synteny among cucurbit species . SSRs (simple sequence repeats) or microsatellites are tandem repeats of short DNA sequences ranging in length from one to six base pair (bp), which are abundant and ubiquitous in all eukaryotic genomes C. sativus var. sativus and var. hardwickii. The integrated genetic-cytogenetic map described herein provides a platform for genetic and genomic analysis that does not currently exist in cucurbits.We present herein the development of a saturated SSR-based cucumber linkage map employing 3× Sanger shotgun sequences. We used FISH to assign linkage groups to a cytogenetic map and to define chromosomal rearrangements between 6-F8 recombinant inbred lines (RILs) derived from the inter-subspecific cross between Gy14 and PI 183967 C.s. var. hardwickii originated from India. Another population derived from an intra-subspecific cross consisted of 130 F7-F8 RILs, which was used for comparative analysis of marker clustering in the map of the inter-subspecific cross.Two mapping populations were used for linkage mapping in this study. The first one consisted of 77 FEleven cucumber inbred lines were employed for genetic diversity studies with SSR markers. These 11 lines represented six market types worldwide: ‘Chinese Long’ type , Southern China type (Baiyesan and 00956), Southwestern China type , European greenhouse type (65 G and 9110 Gt), American slicing type (Marketmore 76), and Japanese type (185). All these inbred lines were from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.Cucumis melo var. saccherinus cv.3A832 and C. melo var. chinensis cv. 4G21), two watermelon lines (Citrullus lanatus var. lanatus cv. 97103 and C. lanatus var. citroides PI 296341), and two squash lines (Cucurbita moschata Duch cv. bush#10 and C. maxima cv. Mengri) were kindly provided by Prof. Yong Xu and Dr. Jianshe Wang . These lines were used to test the cross-species transferability of SSR markers developed in cucumber.Seeds for two melon inbred lines , and then were assembled using Phrap C. abajian, http://abajian.net/sputnik/). The repeat number threshold was designated as more than five, and each putative SSR locus was defined by a SSR motif and associated up and down stream flanking sequences of 200 bp. All putative loci were compared to contig and singleton sequences by BLAST analysis at an E-value cutoff of 1e-40. Putative locus with single hit was defined as a unique locus for use in map construction. If two or more unique loci were identified in one contig, only the one with the longest repeat motif was chosen for mapping to avoid redundant mapping effort. The minimum repeat number for selected MNRs (mono-nucleotide repeats), DNRs (di-nucleotide repeats), TNRs (tri-nucleotide repeats), TTRs (tetra-nucleotide repeats), PNRs (penta-nucleotide repeats) and HNRs (hexa-nucleotide repeats) was chosen to be 20, 12, 8, 7, 6 and 6, respectively.The process of development of SSR markers employed is presented in Primer pairs were designed for these unique SSR loci using the Primer 3.0 program Taq DNA polymerase , 20 ng of forward and reverse primers, 2 mM dNTPs. Optimized PCR thermocycling incorporated a denaturation step of 5 min at 94°, followed by 35 cycles of 15 sec at 94°, 15 sec at 55°, 30 sec 72°, and a final extension at 72° for 4 min. Subsequently, 3 µl of the PCR product was employed for electrophoresis in 6% polyacrylamide gel according to Each polymerase chain reactions (PCR) was performed in a 15 µl volume containing approximately 20 ng template DNA, 1×buffer, 0.5 unit The markers with more than three missing genotype data were excluded, and the remaining marker data were used in linkage analysis with JoinMap program version 3.0 k is the total number of alleles detected for a SSR marker and Pi is the frequency of the ith allele.The efficacy of each SSR marker for germplasm discrimination among 11 genetically diverse cucumber inbred lines was estimated with polymorphism information content (PIC) using the formula www.ualberta.ca/~fyeh/).The heterozygosity for each marker based on the genotype data with the RIL mapping population was calculated with POPGEN32 software (in situ hybridization (FISH) analysis were provided by Beijing Genomics Institute, Beijing, China. The fosmid library was constructed from inbred line 9930 which was also used for whole genome sequencing. Selected fosmid clones were end-sequenced. Fosmid clone end sequences were then placed in the assembled contigs of the 3× shotgun sequences and used to screen the fosmid library to identify clones carrying genetically defined SSR sequences cucumber DNA repeats equences . SelecteDNA probes were labeled with either biotin-dUTP or digoxigenin-dUTP via nick translation and detected with anti-digoxigenin antibody coupled with Rhodamin (Roche) or anti-avidin antibody conjugated with FITC (Vector Laboratories), respectively. Chromosomes were then counterstained using 4, 6-diamidino-2-phenylindole (DAPI) in an antifade solution Vectashield , and images were captured digitally using a Sensys CCD (charge coupled device) camera attached to an Olympus BX61 epifluorescence microscope using Image-Pro Plus 6.0 software (Media Cybernetics) to capture grey scale images that were adjusted with Adobe Photoshop 6.0 software.A total of 23,800 putative SSR sequences were identified from whole-genome 3× shot-gun sequencing. The frequencies of different types of SSRs in this genome were listed in Nine hundred and ninety five SSR loci were mapped in seven linkage groups spanning 572.9 cM. In total, 678 recombination events (bins) were identified, where 311 (46%) Bins were filled by one or more markers , Fig 2. r = 0.71) and between euchromatin length vs. the number of total markers per chromosome (r = 0.82) (derived from The correlation is high between pachytene length and the number of markers per chromosome (Due to marker clustering (≥20 markers per two adjacent bins), the mapping distance (cM) or genetic recombination (Bins) on chromosomes 4, 5, and 7 was dramatically less than that detected on other four chromosomes . MoreoveC. s. var sativus line 9930× line 9110 Gt). For example, the cluster on chromosome 5 of the inter-subspecific map spanned 1.9 cM, but it was increased dramatically to 61 cM in the intra-subspecific map. Similarly, the cluster on chromosome 7 was also notably different between the intra-subspecific (18 cM) and the inter-subspecific map (0.9 cM), suggesting possible structural changes between chromosomes of Gy14 and PI 183967 that resulted in suppression of meiotic recombination. Indeed, one reversion was identified in our molecular cytogenetic analysis as described below.Chromosomal rearrangement is known to cause recombination suppression C. sativus var. hardwickii parent (PI 183967), indicating that possibly interacting allele pairs with strong effects on pollen or embryo viability or germination are located on the SDRs and that the ‘wild’ alleles confer stronger viability than the ‘domesticated’ ones.Three segregation distortion regions (SDRs) were detected on chromosomes 1, 4 and 6 . The SDRFISH analysis was used here to establish the relationships between linkage groups and chromosomes in cucumber. Three to five SSR markers located in the distal ends of each linkage group were selected to screen a fosmid library developed from inbred line 9930. To identify individual chromosome, these fosmid clones were FISH-mapped on mitotic chromosomes , which wThe discrepancy between the inter-subspecies map and the intra-subspecies map on recombination suppression strongly suggested chromosomal variations between the two subspecies. To test this hypothesis, the marker cluster on chromosome 5 containing 101 markers . Bin8–9 In this study, we described the development of highly polymorphic SSR markers using whole genome shotgun sequences leading to the construction of the first high-density genetic map in cucumber. This linkage map was then used as a reference in FISH analysis to define the first integrated genetic-cytogenetic map in this species. The integrated map will be useful in facilitating whole genome assembly, molecular breeding, and positional gene cloning in cucumber, and could act as a reference map for comparative analysis in other cucurbit genomes.According to the formulae of Lander and Waterman Since the SSR markers developed herein were created from non-repetitive regions of the genome, it might be hypothesized that these markers were associated predominantly with the euchromatic or gene-rich regions of the genome. Three lines of evidence supported this hypothesis. First, the number of mapped loci per chromosome was positively correlated with the euchromatic pachytene length of each chromosome . Second,C sativus var. hardwickii which is a native to the Sub-Himalayan region of India and is believed to be a wild feral form of cucumber var. hardwickii is a feral form of var. sativus. Although these botanical varieties are cross-compatible and var. hardwickii possesses several economically important traits ], watermelon [258 (25.9%)] and pumpkin [221 (22.2%)] . MoreoveTable S1(0.25 MB XLS)Click here for additional data file.Table S2(0.04 MB DOC)Click here for additional data file.Table S3(0.03 MB XLS)Click here for additional data file.
CCL3-like genes (CCL3L), a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001) with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10−6) among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1) the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2) stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3) CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se.Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV) at CCL3-like genes (CCL3L) plays in SIV progression rates in rhesus monkeys. Copy number variation (CNV) of these genes has previously been shown to play a role in susceptibility and progression of HIV in humans. Our results suggest that individual monkeys with lower CCL3L copy number progress more rapidly. Accounting for CCL3L CNV in rhesus vaccine trials will improve researchers' abilities to interpret survival data.Development of vaccines for HIV/AIDS is a pressing global issue. The rhesus monkey remains the primary model for testing potential human vaccines; however, little is known about similarities and differences in host genes involved in HIV/AIDS response in humans and rhesus monkeys. Understanding these similarities and/or differences should allow more efficient testing of vaccines beneficial to humans. Here we describe the role that variation in the number of copies of Rhesus macaques are the most widely used non-human-primate model of HIV/AIDS CCL3L1, a paralog of the CCL3 gene CCL3L1 is thought to have been generated through a segmental duplication of a genomically unstable region located on human chromosome 17q11-17q12 CCL3 and CCL3L1 encode chemokine ligands of CCR5, the main co-receptor used by HIV-1 for entry into host cells In humans, an important host factor for HIV susceptibility is copy number variation at CCL3-like genes, there have been a large number of studies in humans, expanding our understanding of the role of this variation in differential HIV susceptibility and progression. It has been shown that CCL3-like gene CNV plays a role in the level of chemokine production and chemotaxis CCL3-like gene CNV in HIV resistance and disease progression. In particular, findings indicate that reduced CCL3L1 copy number relative to the population median correlates with increased risk of acquiring HIV CCL3-like gene CNV and HIV/AIDS CCL3-like genes plays a role in S/HIV immunity in other primates, although it has been shown that copy number variation exists at these loci in chimpanzees After the discovery of copy number variation of CCL3-like genes show variable copy number in rhesus macaque populations and, more specifically, to study the role of the CCL3-like genes in SIV survivorship among rhesus macaques, we assayed copy number variation at these genes in a cohort of 37 Indian-origin and 20 Chinese-origin animals previously infected with SIVmac at the Tulane National Primate Research Center. Individual animals were included in our retrospective study only if the clinical results of a necropsy confirmed health complications due to simian-AIDS at the time of euthanasia or if the animal remained AIDS free for at least 18 months post-infection , and determined absolute copy numbers using two reference samples (see CCL3 and two copies of CCL3L1 (by fluorescent in-situ hybridization (FISH); CCL3L (In order to estimate ); CCL3L . The use); CCL3L .CCL3L region among animals in our study, with a range of 5 to 31 copies per diploid genome provides a significantly better fit to the data than the model (m0) without CCL3L , confirming that CCL3L CNV is contributing to the observed effect and that the effect is not likely to be explained by systematic variation at an additional allele due to population substructure. Additionally, the estimated effect size of CCL3L copy-number variation (β) on survivorship is highly comparable across subsets of the data (see β)). This observation suggests that CCL3L CNV has a similar effect across both populations, whereby each copy of CCL3L decreases the baseline risk by a constant factor of approximately exp(β) = 0.907 relative to the mean of 11 copies .We further tested the impact of population substructure by repeating our analysis using only Indian-origin rhesus macaques. or stratifying by population of origin . Additionally, we considered dividing the data into qualitative copy-number categories as identified by K-means clustering with K = 3 for the observed CCL3L CNV distribution: “low” having less than 9 CCL3L copies per diploid genome (pdg), “intermediate” having 9–14 CCL3L copies pdg, and “high” having greater than 14 CCL3L copies pdg. We also considered a two-class classification that combined the “intermediate” and “high” copy number classes into a single class. Overall, we observe a highly significant difference in survivorship between CCL3L copy classes in the combined data stratified by origin , than those designated as Chinese-origin as measured by a Mann-Whitney U test using either relative copy number estimates from rtPCR (p = 0.0088) or binned and rounded CNV calls , consistent with the average slower progression rates of Chinese vs. Indian-origin animals.Next we investigated whether differences in the distribution of CCL3L CNV affects individual level SIV progression rates in rhesus macaques. This is particularly evident in the Indian-origin rhesus macaque where lower copy numbers of CCL3L are more common, putatively leading to an overall increase in progression rates in this population. Due to the limited power in our analysis of the Chinese-only sample, we recommend further studies to confirm the role of CCL3L in this population. To our knowledge, the current study provides the first example of an association between copy number variation and disease in a non-human primate. These results broaden our understanding of the role copy number variation in disease susceptibility and point to the importance of utilizing methods which allow for detecting this type of variation in genome-wide scans of disease association.Analysis of the retrospective data provides strong support for the hypothesis that CCL3L copy number may explain a large portion of the differences in progression rates between Indian- and Chinese-origin macaques. This result is in contrast to that found in humans, where population level differences in CCL3L1 copy number did not translate into population level differences in progression CCL3L distribution between subpopulations, we have generated predictions for expected survivorship at different levels of CCL3L copy-number variation and population-of-origin designation , sooty mangabey [SM] , orangutan [PP] , chimpanzee [PT] , and humans [HS] . The rtPCR was conducted using a common primer/probe set designed for the rhesus macaque. We chose to use this primer/probe set, as genomic sequence is not available for the sooty mangabey or African green monkey. Our results confirm a previous observation CCL3L copy number in all primate species examined impact on the power of the tests. However, if for example, as copy number increases, the probability of having more pseudogenized copies also increases, this would adversely impact the power of the test.Finally, it is important to note some caveats of our work. First, the current study is based on a relatively small sample of rhesus macaques, pooled across several SIV studies. Additional data is needed to fully understand the role of CCL3L locus falls in the 1% tail of this distribution (after accounting for population substructure) and is therefore, likely a true positive. It is important to remember that there are many other host factors aside from chemokines and their receptors known to influence HIV susceptibility and pathogenesis in humans Third, we have used a candidate gene approach in our analysis of the association of genetic variation with simian-AIDS progression. Therefore, it is possible that genetic variants at other loci may account for even larger portion of the variance in survival. One possible example is variation at MHC class I genes, which has been shown to be associated with SIV progression rate in Indian-origin rhesus macaques and explains at least 48% of the variance in that study The rhesus macaques used in the retrospective analysis were all inoculated with SIVmac as part of previous SIV research programs at the Tulane National Primate Research Center. All macaques were infected with SIVmac239 or SIVmac251 with SIV inoculum given under a standard protocol and at similar mid-level dose. Doses in this range and the strain used have been previously shown not to affect the outcome of disease course All animals used in our study were euthanized under the same set of guidelines if they did not remain healthy after infection. Specifically, euthanasia was carried out if life threatening clinical conditions indicated that the life expectancy of the animal was less than 7 days. Following euthanasia, a necropsy was performed, and animals were only included in the current study if the necropsy confirmed SIV as the underlying cause of the clinical state. Animals were excluded only if they were euthanized and illness could not be confirmed to be AIDS related. Because the results of the necropsy were inconclusive with respect to the cause of the illness in these cases (SIV or not) we chose to exclude them. Conducting the analyses with these animals included as either censored data or non-censored data did not change the results of the analysis. Under these protocols, the time of euthanasia will give a reasonable approximation to both time to progression to simian-AIDS and survival time, as the presence of AIDS defining illnesses met the criterion for euthanasia. See .DNA extractions from the uninfected Chinese-origin rhesus samples were obtained from the Rhesus macaque genome consortium. The chimpanzee, orangutan, sooty mangabey, and uninfected Indian-origin rhesus macaque DNA samples were obtained from the Yerkes National Primate Research Center and the African green monkey DNA samples were obtained from the University of California Los Angeles.CCL3L gene copy number was determined using real-time quantitative PCR (rtPCR) on a 7900HT Fast Real-Time PCR System (Applied Biosystems Inc.) with the JumpStart Taq ReadyMix (SIGMA) and TaqMan probes. The PCR included 18 ng total genomic DNA. Cycling conditions were: initial denaturation at 94°C for 2 min; followed by 40 cycles of 15 sec denaturation at 94°C and 1 minute annealing/extension at 60°C. The Stat6 gene, found to be present in a single copy, per haploid genome, in rhesus macaque, chimpanzee and human reference genomes, was used as the internal control. Oligonucleotide sequences used for CCL3L were: Forward: 5′-CCAGTGCTTAACCTTCCTCC-3′, Reverse: 5′–TCAGGCACTCAGCTCCAGGT-3′, Probe: 5′-AGGCCGGCAGGTCTGTGCTGACC-3′. For Stat6, sequences were: Forward: 5′-CCAGATGCCTACCATGGTGC-3′, Reverse: 5′-CCATCTGCACAGACCACTCC-3′, Probe: 5′-CTGATTCCTCCATGAGCATGCAGCTT-3′. This primer set does not distinguish between CCL3 and the CCL3-like gene paralogs, as we did not observe sufficient fixed differences between these paralogs in rhesus macaque references genome to design a specific assay. It is also unknown whether any pseudogenized copies of CCL3L genes exist in the rhesus macaque populations. As such, we here refer to CCL3 and its paralogs as CCL3L. PCR results were analyzed using SDS v2.2.1 software package (Applied Biosystems Inc.). We performed rtPCR for each individual in triplicate and determined the normalized relative copy number by generating a standard curve and then normalizing across samples by the results of the Stat6 control gene and dividing the value obtained by one of the reference individuals.CCL3L copy number for each sample based on the rtPCR results described above, we used two reference samples: the A431 human cell line and the rhesus genome donor individual. The A431 cell line was chosen as it has previously been shown to have two copies of CCL3L1 and two copies of CCL3 pdg CCL3L using the rtPCR assay described above. To confirm the CCL3L1 copy number of the particular A431 cell line culture used here, we performed florescent in situ hybridization (FISH) of metaphase chromosomes using the human fosmid probes WIBR2-3688L07 read depth analysis CCL3L1 locus with a 95% identity threshold. We compared the average depth of WGS sequence coverage for unique (not-duplicated) sequence in 5kb windows with the depth of coverage to the CCL3L1 locus to estimate copy-number of the locus (CCL3L locus as a reference with an 88% identity threshold (results not shown). From these analyses, we predicted the CCL3L copy number for the genome donor macaque to be 6–8 copies of CCL3L pdg depending on whether the rhesus or human genome is used for alignment. The difference in estimated copy number between the alignment to the rhesus genome and that of the human genome is likely due to alignment of non-CCL3L genes. Due to this, alignment to the rhesus genome is likely a better predictor of CCL3L copy number for this individual because it is less likely to include non-CCL3-like gene paralogs.The second reference sample was the rhesus macaque genome donor sample. Copy number of the he locus . The expCCL3L copy number in each sample by comparing rtPCR results between samples and the references. Specifically, the normalized rtPCR values were averaged across the three replicates for each individual and divided by the averaged rtPCR results for one of the reference samples and multiplied by 4 (the diploid copy number of the A431 cell line including CCL3L1 and CCL3) or 7 . The resulting number was then rounded to the nearest integer value to estimate absolute copy number. In We determined the absolute CCL3L copy number for an additional rhesus macaque cell line using both rtPCR and interphase FISH (CCL3L copy number from the FISH experiment is 10.34±3.00 (mean±standard error based on 54 replicate FISH experiments). The slight discrepancy between the rtPCR and FISH is likely due to the fact that the FISH probe used contains other, known, structural variants which show higher copy number in the macaque reference genome is the base line hazard function, the ijx's for j = 1…p are the covariates for individual i, and the βj's are regression coefficients. An underlying assumption of this model is that the covariates act additively on the log of the hazard function and that the log hazard function changes linearly with the β terms. These are referred to as the proportionality assumptions. We tested this assumption using the method proposed by Grambsch and Therneau 0h(t). The reason for this is that our object of analysis is the proportional hazards among individuals that at time t are independent of 0h. For example, considering a pair of individuals i and i′, the hazard ratios are:The Cox proportional hazard model was chosen, as it is a flexible semi-parametric regression model that accounts censored data. Let 1…βp are estimated given the ranked observed failure times 1y<2y<…<ny using the partial likelihood method proposed by Cox n′ to denote the number of uncensored observations. The partial likelihood is given by:The model parameters βm0, which includes no covariates; m1, which includes only CCL3L copy number as a potential covariate; m2, which considers only population-of-origin as a factor, and m3, which considers both CCL3L copy number and population of origin. To choose among nested regression models for the SIV infected macaque survival data, we used twice the difference in log-likelihood and assessed significance using standard χ2 approximations.Four models are considered; CCL3L copy number and population-of-origin. All R scripts used for analysis and production of figures are available from the investigators upon request.The Harrington and Fleming procedure was used to assess differences among Kaplan-Meier survival curve. This method was also implemented in the survdiff function of the R survival package. All analysis labeled “stratified” were conducted by including the term strata(origin) in the right hand side of the regression equation where origin is an indicator variable of Chinese-origin . The survfit routine to generate predicted Kaplan-Meir survival curves as a function of Figure S1CCL3L .Calibration curve for rtPCR assay using A431 cell line as a standard. Since the A431 cell line has four copies of CL3L see , CCL3L c(0.81 MB TIF)Click here for additional data file.Figure S2Structure results of the retrospective individuals from the 53 microsatellite loci sorted by assumed population. Red are Indian origin animals and blue are Chinese origin animals.(1.07 MB TIF)Click here for additional data file.Figure S3PCA results for the retrospective sample. Red are Indian origin and blue are Chinese origin. (A) Box-plot of PC1 values. (B) Bi-plot of PC1 vs. PC2 showing distinct clustering of animals into proper sub-populations.(1.19 MB TIF)Click here for additional data file.Figure S4Heat plots summarizing genetic relatedness in the sample based on 53 unlinked microsattelite loci. (A) Pearson product-moment correlation of genotypic state for all individuals in the sample; (B) Queller-Goodnight r distance between pairs of individuals in the Indian-origin sample; (C) QG distances for individuals in the Chinese-origin sample.(4.89 MB TIF)Click here for additional data file.Figure S5p-value distribution from the 53 unlinked microsatellites versus that expected under a uniform distribution. (B) Histogram of the −log10 p-values from the microsatellite data with arrow showing the position of the p-value for the association with log2 CCL3L copy number and survival.(A) Quantile-Quantile plot of the empirical (1.00 MB TIF)Click here for additional data file.Figure S6CCL3L copy number applied to each population separately.Bootstrap simulations to assess power of Cox proportional hazard regression of survivorship on (0.57 MB TIF)Click here for additional data file.Figure S7CCL3L copy number and population-of-origin as covariates. Dashed lines indicate 95% prediction intervals based on application of the function survfit in the survival R package.Predicted Kaplan-Meier survival curves based on Cox Proportional hazard model of post-SIV survivorship including (0.88 MB TIF)Click here for additional data file.Table S1Results of necropsy results for 57 animals used in the retrospective study.(0.06 MB PDF)Click here for additional data file.Table S2CCL3L rtPCR assay. CH1 and CH2 are two macaque individuals of Chinese origin. IN1 and IN2 are Indian-origin macaques.Total number of polymorphic sites found per primer/probe/individual for (0.04 MB PDF)Click here for additional data file.Table S3CCL3L copy number distribution among primate species and populations.Summary statistics for (0.05 MB PDF)Click here for additional data file.Table S4Microsatellite id, number of alleles found in the retrospective sample, and heterozygosity for the 53 typed microsatellites.(0.04 MB PDF)Click here for additional data file.Text S1Additional methods describing validation of rtPCR primers and probes, analysis of microsatellite data, and power analysis.(0.05 MB DOC)Click here for additional data file.
Cerebrospinal fluid (CSF) contacts many brain regions and may mediate humoral signaling distinct from synaptic neurotransmission. However, synthesis and transport mechanisms for such signaling are not defined. The purpose of this study was to investigate whether human CSF contains discrete structures that may enable the regulation of humoral transmission.Lumbar CSF was collected prospectively from 17 participants: with no neurological or psychiatric disease, with Alzheimer's disease, multiple sclerosis, or migraine; and ventricular CSF from two cognitively healthy participants with long-standing shunts for congenital hydrocephalus. Cell-free CSF was subjected to ultracentrifugation to yield supernatants and pellets that were examined by transmission electron microscopy, shotgun protein sequencing, electrophoresis, western blotting, lipid analysis, enzymatic activity assay, and immuno-electron microscopy.Over 3,600 CSF proteins were identified from repeated shotgun sequencing of cell-free CSF from two individuals with Alzheimer's disease: 25% of these proteins are normally present in membranes. Abundant nanometer-scaled structures were observed in ultracentrifuged pellets of CSF from all 16 participants examined. The most common structures included synaptic vesicle and exosome components in 30-200 nm spheres and irregular blobs. Much less abundant nanostructures were present that derived from cellular debris. Nanostructure fractions had a unique composition compared to CSF supernatant, richer in omega-3 and phosphoinositide lipids, active prostanoid enzymes, and fibronectin.via volume transmission within the nervous system that are for slower, more diffuse, and of longer duration than synaptic transmission.Unique morphology and biochemistry features of abundant and discrete membrane-bound CSF nanostructures are described. Prostaglandin H synthase activity, essential for prostanoid production and previously unknown in CSF, is localized to nanospheres. Considering CSF bulk flow and its circulatory dynamics, we propose that these nanostructures provide signaling mechanisms Third, functional effects can be invoked via CSF: intracerebroventricular (icv) and intracisternal infusion of sodium or neuropeptides effect appetite [Physiological signaling regions ,2 and ma regions , suggest regions , unlike appetite , drinkinappetite , sleep [appetite , and paiappetite ; and icvappetite .2 [In spite of accumulating evidence for non-synaptic transmission, it is not known how the biosynthesis and transport of signals are regulated within the circulating CSF. For instance, neurotransmitters in solution would be rapidly inactivated, such as hydrolysis of acetylcholine by acetylcholinesterase in the CSF. Moreover, integral membrane proteins may not function optimally in aqueous CSF, such as the prostaglandin H synthase (PGHS) required to synthesize the sleep-inducing prostaglandin D2 . The scoHere, we demonstrate that CSF consistently has a matrix of membrane- and protein-rich nano-scaled structures with many signal transduction components bounded by lipid membranes. These structures include features of vesicles containing acetylcholine, large dense-core vesicles (LDCVs), exosomes, and spherical structures with functional prostaglandin H synthases (PGHS) -1 & -2. People in health and disease states have these structures that contribute to CSF heterogeneity, and provide unique transport and signaling capabilities throughout CSF-contacting brain surfaces.2 (PLA2) from Naja Mossambica ; cyclooxygenase activity assay and phosphatidylinositide ; electrophoresis chemicals and criterion gels ; synthetic lipid standards ; uranyl acetate, Whatman #2 filters, EPON, and 200 mesh nickel formvar/carbon coated grids ; and paraformaldehyde powder The following were purchased: Quant-iT protein assay kit ; ultrafree-MC filters and immobilon-P polyvinylidine chloride (PVDF) membrane ; 1-Step™ NBT/BCIP ; centrifuge tubes 326814 and 344057 were used in 50 ti and 50.1 sw rotors on an L8-70 ultracentrifuge ; ammonia, ultrapure HPLC grade water, parafilm, methanol, and chloroform ; luna silica normal phase HPLC column , glutaraldehyde, phosphate buffered saline (PBS), sucrose, triethanolamine, phenylmethanesulphonylfluoride (PMSF), leupeptin, bovine serum albumin (BSA), glycine, Triton X-100, bovine brain phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), and phospholipase AADP-ribosylation factor-like protein 2 (ARL2) was a generous gift from RA Kahn . 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (S5A2), secretogranin-3 (SCG3), prostaglandin G/H synthase 2 (PGHS-2) were rabbit polyclonal antibodies made by MGH & John Rush from synthetic peptides; S5A2 from CAGAGHHRFYLKMFEDYPKSRKALIPFIF; SCG3 from CAGAGKEAKEKETLITIMKTLIDFV; PTGDS from APEAQVSVQPNFQQD . The folTwenty lumbar and two ventricular CSF samples were collected prospectively for compositional studies after obtaining informed consent from 19 different participants, three of whom were sampled on two separate occasions . Twenty one mL of lumbar CSF was collected from the L2/3 or 3/4 inter-spaces, between 1 and 5 pm, with opening pressure measured in cm CSF in the recumbent position. Two ventricular CSF samples (each of approximately 250 mL) were collected by conventional external drainage at room temperature over 24 h, with pressure measured continually in cm CSF, referenced to the external auditory meatus.Selection for lumbar samples was based on participants that represent health, migraine, inflammatory, or degenerative brain disorders. Selection for ventricular samples was based on participants that had received long-term shunts for congenital hydrocephalus and had normal cognitive function. Normal (n = 3): no classifiable neurological or psychiatric disorder after detailed structured interview and clinical assessment; Multiple sclerosis (n = 2): diagnosis of clinically definite multiple sclerosis based on national criteria ; AlzheimAll CSF was prepared, aliquoted, and analyzed immediately or stored frozen within 1 h of collection. Table Concentrations of total protein (in triplicate) were determined using a microplate-based Quant-iT protein assay kit with BSA, 0-500 μg/mL as standard, as recommended by the manufacturer. For all protein shotgun sequencing (below), CSF fractions were denatured (6 M urea), reduced, amidocarboxymethylated, washed with 100 mM ammonium bicarbonate (pH 8), filtered on Viva Spin 500 , digested with trypsin overnight at 37°C, and quenched with formic acid.Liquid chromatography and mass spectrometry (LCMS) was applied to the S1 of two different samples for separation of the peptide mixture on the 10 cm PicoFrit separation column. Peptides that eluted from the reverse phase column were analyzed by a Finnigan LTQ™ linear ion trap mass spectrometer that was equipped with a nano-electrospray ion source (both Thermo Fisher Scientific). Data-dependent mass spectral acquisition (MS/MS) was enabled and allowed for the MS/MS analysis of the most intense ion in the range of 450-1600 m/z (full scan) with the following dynamic exclusion settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min.This was performed using the Proteome X Linear Ion-Trap System (Thermo Fisher Scientific). The system was fitted with a strong cation exchange column, SCX 320 μm ID × 100 mm (Thermo Fisher Scientific) and two C18 reversed-phase nanotrap columns: IntegraFrit Trap, 75 μm ID × 25 mm, Biobasic™ C18 followed by a PicoFritTM nanobore HPLC column with a 15 μm i.d. pulled tip, 75 μm ID × 10 cm Biobasic™ . 100 μg of S1 protein digest was injected onto the SCX column and peptides were eluted onto a nanotrap column by successively injecting 20 μl of NHA modified version of the Pepfinder Kit with a Surveyor HPLC, autosampler, and nanoflow solvent delivery (Thermo Fisher Scientific) was used to present the CSF sample to an LTQ ion trap mass spectrometer equipped with a nano-electrospray ion source (Thermo Fisher Scientific) and 30 μm PicoTip emitter . The Pepfinder kit was modified from its original form to contain a 5 × 0.3 mm Zorbax™ C-18 peptide trap or a 0.075 × 25 mm Biobasic C18 IntegraFrit Trap combined with a 100 μm ID × 25 cm BioBasic 18 nanobore C-18 separation column (both from New Objective). 1 - 4 μg of CSF S1 protein digest was injected onto the trap, washed, and then eluted onto and through the C-18 column with a pseudo-exponential gradient profile, from 0-80% B in ~4 hr in following gradient increments; 0.1%/min in 50 min, 0.2%/min for 50 min, 0.25%/min for 40 min, 0.33%/min for 60 min, 0.44%/min for 45 min and 4%/min for 5 min . The mass spectrometer was operated in a data-dependent MS/MS mode and dynamic exclusion was enabled. Gas-phase fractionation with three distinct scan ranges was used to maximize the number of peptides identified as described . The num® algorithm [-5 were accepted for protein analysis. Post-translational modifications were assessed using a fully automated, de novo sequencing software program, DeNovoX (Thermo Fisher Scientific). A comprehensive list of non-redundant proteins was generated from all analysis runs (n = 15), using Excel sorting and Pivot Table functions. Results were compared to existing identifications [MS/MS spectra obtained from these LCMS analysis of the S1 protein digests of sample #s 1 & 2 with Bioworks 3.2 in full tryptic and semi-tryptic search mode using the Sequest algorithm implementation of SORCERER . For unit resolution data, a precursor tolerance of 2Da was used, while 100 ppm tolerance was applied for high mass accuracy data. Positive identifications were established using the probability calculations in Bioworks 3.2, with a statistical expectation of > 10E-5. Results from tryptic and semi-tryptic searches were combined and filtered after export into Excel. A comprehensive list of non-redundant proteins was generated from all analysis runs (n = 6) using Excel sort and Pivot Table functions. Results were compared to existing identifications .S1 samples (1.5 mL) from #s 1, 2, 5, & 6 then eluted through a silica column with a chloroform/methanol/water/ammonium hydroxide gradient [Lipids were extracted from S3 fluids and P3 pellets using the method of Bligh Dyer . After rgradient . The eluPrecursor ion scans (PIS) and neutral ion loss (NIL) for different lipids were obtained using a full scan MS infusion experiment on a triple quadrupole mass spectrometer, TSQ Quantum (Thermo Fisher Scientific) operated at a spray voltage of 4500 V, sheath gas pressure of 40 units, auxiliary gas pressure of 0, capillary temperature of 225°C and collision pressure of 1.5 units. Negative ions were acquired in the profile mode with 13 different scan events after collision induced dissociation (20-24 V) of deprotonated precursor ions or the neutral loss of specific groups from lipids. Negative PIS of 196.3 (mass range 650-950), 171.14 (mass range 600-900), 240.96 (mass range 750-1200), 168.17 (mass range 600-900) were used to monitor PE, PG/PA, PI and SPM, respectively. Negative NIL of 86.99 (mass range 650-900) and 50.13 (mass range 400-1000) were used to monitor PS and PC/LPC/PAF, respectively. Lipids containing eicosanpentanoeate , arachidonate and docosahexaenoate were detected using PIS of these ions in a mass range from 600-1200. Peak intensities were integrated, processed, and mole quantities determined using ICIS and Xcalibur software (Thermo Fisher Scientific). Mole quantities were determined from standard curves obtained using known amounts of lipid standards (0-400 ng).2 at 37°C overnight in reaction buffer. The same incubation was carried out with heat-denatured enzyme, controls without enzyme. Reaction products were stored at 4°C until assayed either by LCMS or TEM with negative staining as described above.P3 samples were incubated with PLAmax kinetic microplate reader at 595 nm . PGHS activity (nmol/min/mg protein) was calculated as described by the kit manufacturer .All assay components were pre-equilibrated to the room temperature except for the PGHS-2 Standard that was kept on ice. Detection was based on measuring the peroxidase activity of cyclooxygenase by colorimetrically monitoring the appearance of oxidized N, N, N', N'-tetramethyl-p-phenylenediamine at 595 nm in 96 well plates. Either Dup-697 (PGHS-2 inhibitor) or SC-560 (PGHS-1 inhibitor) was added to inhibitor wells and both inhibitors were added into background wells. Standard, samples, and backgrounds were analyzed in duplicates. The plate was carefully shaken and after 5 min of incubation at room temperature, absorbance was read on the VPressure was normal at all lumbar (<150 cm) and ventricular (< 15 cm) CSF collections. All fluids were clear, with < 5 white blood cells per mL, and no red blood cells were seen. All samples had total protein content within the normal range of 0.1 - 0.5 g/L.Using sample #s 1 & 2 , based on specific staining at their predicted molecular weight: FINC, SNAP 23, PGHS-2, RALA, S5A2, ARL2, and SCG3. We also found that semaphorin 4D, chromogranin A & B, and the known SNARE complex proteins synaptotagmin, syntaxin, and synaptobrevin were enriched in P3, but with insufficient samples for statistical analysis.To explore the sources of these structures, we considered that the abundant spherical structures would come from the choroid plexuses and the ependymal lining of the CSF, and might be derived from similar-sized synaptic vesicles , LDCVs , or exosSince nanostructures are purified in P3 fractions, we performed iTEM to find whether the proteins identified on western blots Figure localize2 (Naja mossambica). This depleted the major phospholipid peaks as measured by mass spectrometry, and removed > 90% of the spherical structures as measured by TEM (data not shown).The morphology of the abundant spheres visible by TEM Figure suggestsp < 0.005).Liquid chromatography of S3 and P3 lipids partially resolved PI into two peaks consisting of polyunsaturated fatty acids (PUFA), (PI-peak-1), and saturated fatty acids, (PI-peak-2), Figure &9D. Thep = 0.0005).The presence of eicosanoid metabolic enzymes in the initiation of normal sleep, when it is applied via the CSF selectively to the ventral rostral brainstem [2 is an unstable intermediate in PGD2synthesis, and its local synthesis by the integral membrane protein PGHS is required for production of downstream prostaglandins. We therefore assayed PGHS activity in the CSF fractions.To test whether CSF nanostructures have functional enzymes, we selected the prostanoid pathway because our protein and lipid studies showed both their enzymes and substrates present in CSF. Furthermore, Hayaishi's group demonstrated a physiological effect for prostaglandin Drainstem , and manrainstem -37. HoweWe wanted to test PGHS activity from a large quantity of CSF because PGHS-2 staining structures were infrequent on iTEM Figure and P3 wvia the CSF. However, there is little information about the mechanisms by which such communications are regulated. A lack of structures to transport, protect, localize, and eventually transduce signals at an appropriate receptor poses major restrictions on transmission within the 150 mL of fluid. We now report details of the morphology and biochemistry of unique structures in CSF that have the potential to overcome many, if not all, of these limitations.A variety of experiments, as outlined in the background, support volume transmission between brain regions CSF proteomic studies have recently revealed many proteins that are substantially different from those found in plasma ,38-41. OWhen classes of CSF protein components from published data were compared based on GO component categories, around 40% are membrane proteins . ZougmanOur TEM data demonstrates small particles on filtered CSF. The presence of these nano-sized particles from filtration provides evidence that these structures are not formed during the ultracentrifugation procedure. The presence of identical nanostructures in CSF from two participants that were collected and prepared freshly as compared to stored CSF, demonstrates that the nanostructures are not a product of sample storage.Past reports of CSF sub-cellular structures have been interpreted as resulting from blebbing, apocrine secretion, apoptosis events, or cellular debris ,42-45 an2 caused dissolution of the TEM nanostructures, and depleted major P3 phospholipids as determined by LCMS, demonstrate that lipid membranes enclose these spherical structures. These membranes provide an appropriate environment for some of the abundant transmembrane proteins found in our shotgun sequencing experiments of CSF , persons with intermittent disability (migraine), and persons with serious brain pathology of either an inflammatory (multiple sclerosis) or degenerative disease (Alzheimer's disease) suggests strongly that these structures are ubiquitous in CSF. Though the unknown functions of these nanostructures require much further study, our data hints at multiple roles.The different electrophoretic and western blots of P3s in this nanostructure-rich fraction are evidence that the P3 fraction from CSF contains biochemically distinct components. The FINC immunostain is greatly enriched in P3 fractions that mainly contain the varied blobs and strands. The known functions of FINC as an adhesive extracellular molecule involved in neurite development support Our data Figure also sho2, are present for synthesis of the obligatory prostanoid intermediate, PGH Figures &8A and Figures in CSF. Figures . These r Figures and lipa & -2 forvia CSF. For example, topical acetylcholine has been consistently shown to dilate cerebral vessels [Membrane-bound nanospheres with acetylcholine provide potential protection from acetylcholinesterase in CSF, thus enabling a humoral mode for vesicle neurotransmission vessels , raising9 and 1012 nanospheres are produced per day, comprised of 5 μg total protein. These relatively small amounts of CSF nanostructures may have physiological roles since the readily releasable synaptic vesicle pool size ranges from as low as 5 to as many as 5,000 per single synapse, as reviewed by Südhof [For both healthy as well as those with brain disorders we estimate, assuming production and turn over at the same rate as the fluid, between 10y Südhof . While trd ventricle of about 5 mm/sec [rd ventricle to move to receptors in the medial hypothalamic wall of the same ventricle. For a simple circulation of pre-synthesized signal within this small space, we estimate transmission speed will be in the order of a second. At the other extreme, such as nanostructures originating in a ventricle, migrating to a more distant subarachnoid location, and requiring signal synthesis, we estimate speeds ranging from minutes to several hours, since total CSF exchanges 3-5 times a day and is stagnant in some regions [Brain extracellular fluid is known to diffuse ions, dopamine, proteins, and 35 nm particles ,56 in sp5 mm/sec ,58. Figu regions ,58. Bulk regions -7,53,59.Human lumbar and ventricular CSF samples demonstrate abundant membranous CSF structures, 30-200 nm in size from both healthy and sick participants. Compared to the supernatant, these structures have unique protein and lipid compositions, contain acetylcholine, and have complete prostanoid pathways from membrane phospholipids to specific receptors. Variation in CSF nanostructures may be informative in both health and disease studies, but they require enrichment since they are diluted by more abundant fluid components. We anticipate that further study of these biochemically and morphologically unique CSF nanostructures will identify their roles in modulating brain functions and dysfunctions.2: prostaglandin D2; PGH2: prostaglandin H2; PGHS: prostaglandin H synthase; PI: phosphatidylinositide; PIS: precursor ion scan; PLA2: phospholipase A2; PMSF: phenylmethanesulphonylfluoride; PS: phosphatidylserine; PTGDS: prostaglandin D synthase; PUFA: polyunsaturated fatty acid: PVDF: polyvinylidine difluoride; RALA: Ras-related protein ral-A; S1: CSF 3,000 g supernatant; S2: CSF 17,000 g supernatant; S3: CSF 200,000 g supernatant; S5A2: 3-oxo-5-alpha-steroid 4-dehydrogenase 2; SCG3: secretogranin 3; SE-1: statistical expectation value; SNAP-23: synaptosomal-associated protein 23; SNARE: soluble N-ethylmaleimide-sensitive fusion attachment protein receptor; SPM: sphingomyelin; TEM: transmission electron microscopy (iTEM: immuno-TEM).AA: arachidonic acid; ARL2: ADP-ribosylation factor-like protein 2; BSA: bovine serum albumin; CM: ceramide; CS: cerebroside sulfate: CSF: cerebrospinal fluid; DHA: docosahexaenoate; EPA: eicosanpentanoeate; FINC: fibronectin; GO: gene ontology; LCMS: liquid chromatography mass spectrometry; LDCV: large dense core vesicle; LPC: lysophosphatidylcholine; NIL neutral ion loss; P1: CSF 3,000 g pellet; P2: CSF 17,000 g pellet; P3: CSF 200,000 g pellet; PA: phosphatidic acid; PAF: platelet-activating factor; PBS phosphate buffered saline; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; PGDThe authors declare that they have no competing interests.MGH conceived and designed both the human subjects and laboratory aspects of the project, recruited and diagnosed study participants, collected some of the CSF, carried out some of the shotgun protein sequencing experiments, performed the ultracentrifugation and sample preparations for biochemical and electron microscopy studies (TEM and iTEM), analyzed all data, and drafted the manuscript. ANF participated in design of all experiments, carried out the lipid analyses, and participated in manuscript revisions. EO carried out electrophoresis, western blots, developed and carried out the enzyme activity assays. PL carried out the early ultrafiltration of nanostructures and participated in the electron microscopy and analysis of these filters. RPC and GM recruited and diagnosed study participants, collected CSF, and participated in manuscript revisions. JNC helped design and supervise all electron microscopy experiments. RGB and AFH helped design and carry out most of the protein shotgun sequencing experiments, and participated in manuscript revisions. All authors read and approved the final manuscript.CSF proteins from repeated analyses of two independent samples. CSF proteins identified by Uniprot number, name, and GO component category, from 15 shotgun sequencing analyses of sample #s 1 & 2 in Table Click here for fileCSF proteins from the P3-enriched pellet and the S3 supernatant. CSF proteins identified from a single, high resolution shotgun sequencing run of two sets of P3 and S3 samples from one participant collected on two independent occasions (sample #s 3 & 3' in Table Click here for file
The complex interplay between B-cell lymphoma 2 (Bcl-2) family proteins constitutes a crucial checkpoint in apoptosis. Its detailed molecular mechanism remains controversial. Our former modeling studies have selected the ‘Direct Activation Model’ as a better explanation for experimental observations. In this paper, we continue to extend this model by adding interactions according to updating experimental findings.Through mathematical simulation we found bistability, a kind of switch, can arise from a positive (double negative) feedback in the Bcl-2 interaction network established by anti-apoptotic group of Bcl-2 family proteins. Moreover, Bax/Bak auto-activation as an independent positive feedback can enforce the bistability, and make it more robust to parameter variations. By ensemble stochastic modeling, we also elucidated how intrinsic noise can change ultrasensitive switches into gradual responses. Our modeling result agrees well with recent experimental data where bimodal Bax activation distributions in cell population were found.Along with the growing experimental evidences, our studies successfully elucidate the switch mechanism embedded in the Bcl-2 interaction network and provide insights into pharmacological manipulation of Bcl-2 apoptotic switch as further cancer therapies. Apoptosis is a highly regulated cell suicide program in response to cell stress, damage or conflicting division signals It has been widely embraced that the intricate interplay between three groups of the Bcl-2 family proteins determines the commitment of MOMP and subsequent apoptosis Although growing evidence demonstrates Bcl-2 family functions as an upstream life/death switch Now, quickly accumulated experimental evidence in this field can help update our understandings for the regulation of Bcl-2 network. As reviewed by Galonek and Hardwick In this study, we extended the Direct Activation Model by incorporating these newly emerged experimental findings. We mainly focused on their influence on the dynamic patterns in the extending models. Our analysis demonstrates that two independent positive feedback loops in the Bcl-2 network contribute to an inherent switch to govern apoptosis decision. Bistability can arise from a positive (double negative) feedback provided by the interaction between anti-apoptotics and activated Bax/Bak. Moreover, Bax/Bak auto-activation as another positive feedback can enforce this switch and make it more robust with respect to parameter variations. We also use ensemble stochastic modeling to elucidate the influence of intrinsic noise on this bistable switch. Our result is well in accord with certain experimental data of flow-cytometry analysis, where bimodal Bax activation distributions in cell population are reported. In all, our studies successfully extend our understanding of the switch mechanism beyond the apoptosis decision and provide insights into pharmacological manipulation of Bcl-2 family members as further cancer therapies.We constructed our models of Bcl-2 apoptotic switch mainly based on the previously described Direct Model [14]. To1, c2 … cn). The reaction rates are dependent on these concentrations and on biochemical parameters such as binding constants and dissociation constants. To describe the temporal behavior, a set of ordinary differential equations is generated in terms of the following general equation:a/dtdc represents the concentration changing rate of molecule a. bJ denotes the rate of reaction b, and abv denotes the stoichiometric matrix linking the reaction rates of bJ with the affected molecule a. Here the reaction scheme, kinetic equations as well as model parameters of the Direct Model II are represented in 2 = J4 = J8 = J9 = JAcBaxBcl2 = 0,J see 9J = 0, see Here we bring forward a set of models realizing mass action kinetics implemented as ODEs, which is based on the outline of the Direct Model, Direct Model I and Direct Model II as shown in in silico simulations, the percentage of existence of bistability was monitored. The generation of Latin hypercubes and the determination of bistability are implemented in Matlab. For each set of kinetic parameters, we search for the existence of a bistability by the “coming up and going down” method described previously Similar to A Bax-activation module has been identified as a bistable switch in our previous work without considering protein production and degradation We presented two models of increasing complexity to demonstrate how bistable behaviors of Bax activation emerge from the intricate interactions of Bcl-2 family proteins. From We next focused on the mechanism that leads to bistability of Bax activation. It is apparent that the inhibition of Bcl2 brings about ‘inhibitor ultrasensitivity’. That means the adding Activator is first inhibited till their amount exceed the inhibition effect of Bcl2. Also, we suggested that the interactions between AcBax and Bcl2 can form a positive (double negative) feedback. AcBax can bind Bcl2, and thereby sequesters Bcl2 away from the Activator. Thus more Bax can be activated. It is well known that positive feedback incorporated with non-linear elements can lead to bistability Direct Model II which isBax auto-activation can also enforce model's robustness of the bistability. Here, we used a Monte Carlo based method to evaluate the robustness of bistable behavior of the Bcl-2 network to parameter variations. Latin Hypercube Sampling is used to generate 2000 random sets of all the parameters of the model with the variation of 40% (+/−20%) relative to the reference parameter values. From From the bifurcation analysis of these models, we demonstrate the potential molecular mechanism of the bistability arising from hierarchic interactions of the Bcl-2 network. A positive (double negative) feedback formed by activated Bax-Bcl-2 interactions could be an internal reason of the emergence of this bio-switch. Another possible positive feedback loop describing Bax auto-activation can enforce such behavior and make the bistable range wider and more robust with respect to parameter variations.Most bistable models only maintain their switch-like behavior in a limited parameter range. These systems can hardly be considered ‘biologically bistable’ as they may not behave in a bistable manner in the face of biological uncertainties, such as intrinsic noise and parameter variations The time course of Bax activation in response to stimuli is simulated and the results indicate that the Bax activation switch is a robust bistable system since noise-induced transitions are rare unless the stimulus is very near the bifurcation point , which further proves the crucial therapeutic role of Bcl-2 apoptotic switch. Our studies here also provide insights into pharmacological manipulation of Bcl-2 family members as cancer therapies. Here the central issue is how to switch the bistable system of Bcl-2 network to on-state and kill tumor cells by manipulating Bcl-2 family proteins. Several BH-3 mimetics have already been designed to sequester anti-apoptotics and trigger apoptosis in tumor cells In all, through extending the models of Bcl-2 apoptotic switch based on updating experimental findings, we successfully identified two independent positive feedbacks which contribute to the switch mechanism in Bax-activation.Figure S1Bifurcation diagram of Bax activation as a function of the degradation rate of Bax. Steady states of Activated Bax/Bak (AcBax) are plotted as a function of the degradation rate of Bax.(0.02 MB PDF)Click here for additional data file.Figure S2Influences of the intrinsic noise on the transition of the Bcl-2 apoptotic switch. Possibilities of transition from resting state to on state of Bax activation by changing production rate of Activator under deterministic simulation (solid line) and stochastic simulation (dashed line). For each given value of production rate of Activator, 10000 independent runs are taken for stochastic simulation.(0.02 MB PDF)Click here for additional data file.Table S1Reaction scheme of the Direct Model.(0.06 MB PDF)Click here for additional data file.Table S2Ordinary Differential Equations of the Direct Model.(0.04 MB PDF)Click here for additional data file.Table S3Reaction scheme of the Direct Model I.(0.06 MB PDF)Click here for additional data file.Table S4Ordinary Differential Equations of the Direct Model I.(0.04 MB PDF)Click here for additional data file.
Diligent and correct laboratory diagnosis and up-front identification of risk factors for progression to severe disease are the basis for optimal management of malaria.® instrument.Febrile children presenting to the Medical Research Unit at the Albert Schweitzer Hospital (HAS) in Lambaréné, Gabon, were assessed for malaria. Giemsa-stained thick films for qualitative and quantitative diagnosis and enumeration of malaria pigment, or haemozoin (Hz)-containing leukocytes (PCL) were performed, and full blood counts (FBC) were generated with a Cell Dyn 3000® instrument detected significantly more patients with PCL (p < 0.01). Both PCM and PCN numbers were higher in severe versus non-severe malaria yet reached statistical significance only for PCN . Of note was the presence of another, so far ill-defined pigment-containing group of phagocytic cells, identified by laser-flow cytometry as lymphocyte-like gated events, and predominantly found in children with malaria-associated anaemia.Compared to standard light microscopy of Giemsa-stained thick films, diagnosis by platelet count only, by malaria pigment-containing monocytes (PCM) only, or by pigment-containing granulocytes (PCN) only yielded sensitivities/specificities of 92%/93%; 96%/96%; and 85%/96%, respectively. The platelet count was significantly lower in children with malaria compared to those without (p < 0.001), and values showed little overlap between groups. Compared to microscopy, scatter flow cytometry as applied in the Cell-Dyn 3000In the age group examined in the Lambaréné area, platelets are an excellent adjuvant tool to diagnose malaria. Pigment-containing leukocytes (PCL) are more readily detected by automated scatter flow cytometry than by microscopy. Automated Hz detection by an instrument as used here is a reliable diagnostic tool and correlates with disease severity. However, clinical usefulness as a prognostic tool is limited due to an overlap of PCL numbers recorded in severe versus non-severe malaria. However, this is possibly because of the instrument detection algorithm was not geared towards this task, and data lost during processing; and thus adjusting the instrument's algorithm may allow to establish a meaningful cut-off value. Malaria is known to cause several changes in full blood count (FBC) parameters, of which the most prominent are anaemia and thrombocytopaenia . However®, Abbott, Santa Clara, California) allows the automated detection of Hz-containing cells. The instrument has been shown to be useful in the diagnosis of malaria [®) has been installed in a remote malaria-endemic area in Central Africa . The objective of this study was to investigate the haematological parameters in children with malaria as well as the importance and potential usefulness of the automated detection of PCL.Haemozoin (Hz), the end product of the detoxification of haem, is phagozytosed by monocytes and granulocytes. Some studies have reported a link between Hz and dyserythopoiesis and anaemia -6. Using malaria -15. One ® (CD3000) instrument .The study took place at the HAS, Lambaréné, Gabon, in 2003 and 2004. The area is mainly tropical rainforest with holoendemic malaria ,17. Bloo® instruments generate a five-part differential white blood cell count, using scatter flow cytometric principles based on the manufacturer's patented multi-angle-polarized-scatter-separation (M.A.P.S.S.®) [® instruments and the transformation of the image, the maximum resolution of the analysed bitmap image was 140 × 140 pixels, rather than the 256 × 256 channels.The Cell-DynP.S.S.®) as descrP.S.S.®) ,23. The P.S.S.®) . As the P.S.S.®) . Due to In the granularity/lobularity plot, two different areas were defined to classify monocytes as pigment-containing monocytes (PCM), shown in Figure A higher degree of depolarization may be caused by either a higher amount of phagozytosed Hz or larger Hz crystals. Thus, Hz-containing monocytes and granulocytes with higher depolarization values contain possibly more Hz and thus could be correlated with severity. To test this hypothesis, two "severity indices" with the intention to weigh the degree of depolarization were constructed: (i) the sum of the y-axis values of Hz-containing monocytes and granulocytes and (ii) the sum of the y-axis values after mathematical transformations . These indices were then used to determine a meaningful cut-off value that would allow to distinguish between severe and non-severe malaria.During the analysis of the granularity/lobularity plot it was noted that blue coloured events, that usually represent lymphocytes, showed depolarization Figure . A horizData was analysed using SPSS 14.0 software. The student t-test for unpaired samples with unequal variance was used to test quantitative data and the chi-square test to test qualitative data.During the study period a total of 368 children were included, of which 152 had falciparum malaria and 216 children were either healthy or had other diseases. More than 88% of malaria occurred in children older than one year, while 99.1% of all children without malaria were below one year of age. The children in the malaria group were significantly older than the children without malaria . Of the 152 children with malaria, 48 had severe malaria, classified as 15 cases with severe anaemia, 13 cases with hyperparasitaemia, three cases with hypoglycaemia and 17 children with cerebral malaria. In three cases, children had both cerebral malaria and severe anaemia, which were included in the cerebral malaria group. The mean age of children with severe malaria (3.8 years) and non-severe malaria (3.4 years) was not significantly different (p = 0.27).The FBC results are shown in Table Sixtyfive percent of children with malaria had < 150,000 thrombocytes/μL. Given the little overlap of the platelet counts between the malaria and non-malaria groups we computed a Receiver-Operator-Characteristic (ROC) curve Figure . The ROC® instrument. Concerning Hz-containing monocytes, no significant difference was observed between the two defining lines the sensitivity was 97% and the specificity of 93%.The other parameters that were analysed for their usefulness to diagnose malaria were the presence of Hz-containing leukocytes detected by the Cell-Dyn 3000s Figure yielded s Figure . When th® instrument which showed that the analyser detected significantly more patients harbouring PCL (p < 0.01) (Table The detection of PCL by microscopy was compared with results from the Cell-Dyn1) Table . This wa1) Table . FurtherThe results for the automated detection of Hz-containing leukocytes in severe and non-severe malaria are shown in Figure The number of Hz-containing leukocytes showed a wide distribution with an overlap between the non-severe and severe malaria groups Figure . This diConcerning the presence of depolarizing blue coloured events (lymphocytes), they were found in 77 of 152 children with malaria (51%). The mean number of these events in the 77 children was 4.2 (range: 3 – 24). Although they appeared to be evenly distributed between the non-severe and severe malaria groups, they seemed to be more frequent in children with anaemia, with 11/14 FBC results yielding depolarizing blue coloured events.This study underpins the value of thrombocytopenia for malaria in the investigated age groups and in the setting of a Central African rainforest area and provides more insight into the usefulness of PCL analysis as a prognostic marker in malaria. It furthers the knowledge about the utilization of scatter flow cytometry for this purpose, which may be applied in future in low-cost, robust devices needed for other applications such as CD4+ cell identification.Concerning the FBC parameters, most of the values showed a significant difference between the malaria and non-malaria groups. As the children with malaria were significantly older (mean: 3.7 years) than the ones who had no malaria (mean: 0.6 years), these results may in great part simply reflect the different reference values in both age groups . This efA notable observation was not only the significantly different platelet count between the malaria and non-malaria groups, but even more so, that almost no overlap of values was observed. However, it could be argued again that this observation reflects rather the different age in both groups. Interestingly, most textbooks give only one set of reference values for all age groups, which implies the same or very similiar values for children of different ages . On the Given the frequently-used cut-off value of 150,000 platelets/μL, this cut-off would have identified 66% of malarious children, while almost none of the children without malaria had such a value (specificity 99%). These values are in keeping with other reports and confirm the usefulness of a platelet count as marker of acute malaria. In fact, ROC curve analysis indicates that this parameter showed a high degree of accuracy Figure ; and eve® instrument has a large potential for the rapid and accurate detection and enumeration of PCL as it identified more PCL-positive samples than microscopy , or were lost during the "screen shot" transformation that resulted in a decrease from 256 × 256 channels to a 140 × 140 pixel image. In fact, the number of coloured pixels that represent leukocytes were analysed and compared to the total number of leukocytes in a subset of FBC results. It was found that the granularity/lobularity plot events that are highly depolarising. However, lymphocytes do not phagozytose and consequently, these cells cannot be lymphocytes, but must be phagocytic cells with cell characteristics that are similar to lymphocytes are more readily detected by automated laser-flowcytometry than by microscopy. Mechanical Hz detection by an instrument as used here is a reliable diagnostic tool and correlates with disease severity. However, clinical usefulness as a prognostic tool is limited due to an overlap of PCL numbers recorded in severe versus non-severe malaria; possibly because of a detection algorithm not geared towards this task, and data lost during processing. Newly described 'lymphocyte-like' gated events warrant further examination and should be analysed by flow cytometry to establish their nature and role in the pathophysiology in malaria.FBC: full blood count; Hb: haemoglobin; Hz: haemozoin or malaria pigment; IPTi-SP: Intermittent Preventive Treatment in infants of malaria with sulfadoxine-pyrimethamine; PCL: pigment-containing leukocyte(s); PCM: pigment-containing monocyte(s); PCN: pigment-containing neutrophil(s) or granulocytes; WBC: white blood cell countTH and MPG designed the study, collected and analysed data and prepared the manuscript.ML helped designing the study, collected and analysed data and contributed to the manuscript's final version.MP and SO helped collecting the data and contributed to the manuscript's final version.BL contributed to study design, data analysis and the manuscript's final version.PGK contributed to the study design and to the manuscript's final version.
Endoplasmic reticulum (ER) stress elicits the unfolded protein response (UPR), initially aimed at coping with the stress, but triggering cell death upon further stress. ER stress induces the C/EBP-® variant Liver-enriched Activating Protein (LAP), followed by the dominant-negative variant, Liver Inhibitory Protein (LIP). However, the distinct role of LAP and LIP in ER stress is unknown. We found that the kinetics of the ER stress-induced expression of LIP overlapped with that of the cell death in mouse B16 melanoma cells. Furthermore, inducible over-expression of LIP augmented ER stress-triggered cell death whereas over-expression of LAP attenuated cell death. Similar results were obtained in human 293T cells. Limited vasculature in tumors triggers hypoxia, nutrient shortage and accumulation of toxic metabolites, all of which eliciting continuous ER stress. We found that LAP promoted and LIP inhibited B16 melanoma tumor progression without affecting angiogenesis or accelerating the cell cycle. Rather, LAP attenuated, whereas LIP augmented tumor ER stress. We therefore suggest that C/EBP-® regulates the transition from the protective to the death–promoting phase of the UPR. We further suggest that the over-expression of LAP observed in many solid tumors promotes tumor progression by attenuating ER stress–triggered tumor cell death. The endoplasmic reticulum is the site of post-translational processing of secreted and cytoplasmic membrane proteins. The ER is also the site of membrane biogenesis and helps maintain intracellular calcium ion and lipid homeostasis. Accumulation of client proteins or misfolded proteins, overload of free cholesterol, perturbations in calcium ion homeostasis, oxidative stress and xenobiotic toxins rapidly induce ER stress, which triggers an evolutionarily conserved cellular response, termed the Unfolded Protein Response (UPR) C/EBP-β-deficient mice are less sensitive to ER stress–mediated cell death ER stress induces the expression of the transcription factor C/EBP-β ER stress has been associated with many diseases, where it leads to cell death To study the role of C/EBP-β LAP and LIP in ER stress and in tumor progression, we utilized B16 melanoma clones that inducibly over-express the dominant-negative LIP, thereby inhibiting endogenous LAP activity, as well as other clones that inducibly over-express LAP. Our findings suggest that C/EBP-β has a key role in regulating the transition from protective to death promoting UPR; LAP attenuates and LIP augments cell death, and LAP contributes to tumor progression by attenuating ER stress and subsequent cell death.Figure S1). Similarly, Clone F10.9-4 expressed only LIP, which is the dominant-negative form of LAP . Initially, we triggered ER stress in un-induced cells and compared the progression of cell death and the expression of endogenous LAP and LIP. Cell detachment and death started 4–6 h after addition of tunicamycin, as determined quantitatively by staining of the remaining attached cells with Crystal Violet . Basal LAP and LIP were undetectable by immunoblotting. As previously reported To study the role of LAP to LIP ratio in ER stress-triggered cell death, we used the specific inducers of ER stress tunicamycin (Tm) and thapsigargin (Tg) and murine B16 melanoma clones that inducibly express either LAP or LIP P<0.009, N = 4), as well as at all later time points . No cell death was seen upon LIP expression in the absence of ER stress (data not shown). Over-expression of LAP had no significant effect in the first 4 h, but significantly attenuated cell death at all other time points . These observations demonstrated that LAP attenuates and LIP augments ER stress-triggered cell death. Since both LAP and LIP were induced past the first 5 h, we evaluated their combined effect on cell viability by siRNA knockdown. Silencing of both LAP and LIP expression significantly augmented the tunicamycin-triggered death of F10.9-3 cells as determined by viability staining with Neutral Red , whereas over-expression of LAP significantly attenuated cell death . Similarly, the attenuation of cell death was dose–dependent and apparent upon induction of LAP with ≥100 ng/ml doxycycline .Microscopic observation of Crystal Violet–stained cultures further demonstrated that LIP over-expression augmented thapsigargin or tunicamycin–triggered cell death at 24 h . Rather, tunicamycin triggered cell rounding and detachment from the plate. The detached cells were dead or dying, as determined by propidium iodide staining . Some of the tunicamycin-treated cells remained attached to the plate and although they were propidium iodide negative, they appeared to be dead, as they were condensed and rounded . Unlike control cells, the LAP–over expressing F10.9-3 cells survived the tunicamycin treatment, they remained attached to the plate and appeared viable . Furthermore, they propagated normally after removal of the ER stress triggers (data not shown). Based upon these observations we concluded that the ER stress-induced LAP attenuated cell death, whereas LIP augmented cell death, probably by inhibiting or re-directing the pro-survival activity of LAP.Microscopic evaluation of Annexin V staining of tunicamycin-challenged cultures revealed lack of apoptotic cells within 24 h of treatment rendered the cells significantly more resistant to tunycamycin-triggered cell death . In contrast, over-expression of LIP in human 293T cells by transfection with an expression vector encoding only the LIP open reading frame rendered the cells significantly more sensitive to tunycamycin-triggered cell death .To determine how general is the role of C/EBP-β in ER stress, we studied the impact of C/EBP-β over-expression and knockout in additional cell types. Over-expression of LAP in human 293T cells by transfection with an expression vector designed to encode LAP only . The silencing of C/EBP-β increased the sensitivity of HeLa cells to ER stress–triggered cell death , leading to a statistically significant decrease in the cell counts . Over-expression of LAP in human HeLa cells by transfection with pcLAP rendered the cells significantly more resistant to tunicamycin-triggered cell death . In contrast with all other experiments, over-expression of LIP in HeLa cells by pcLIP also rendered the cells more resistant by 13% to tunicamycin-triggered cell death . Yet, all other results suggest a general role for C/EBP-β in modulating ER stress–triggered cell death.Knockdown of C/EBP-β in human HeLa cells by siRNAs specific to To study the mechanism by which LAP and LIP regulate the cell fate upon ER stress, we first determined the impact of LAP over-expression on the cell cycle. As previously reported Figure S3A) or cleaved caspase 3 in the cell extracts . In contrast, over-expression of LAP significantly increased the tumor mass by fourfold over a 5 day period in the drinking water. Palpable tumors were detected in the mice after 8 days and differences in tumor mass were apparent by day 13. Therefore tumors in one group of mice were isolated and examined at day 8 and at day 13 in a second group of mice. The inhibition of endogenous LAP by over-expression of LIP led to a significant attenuation of tumor growth as determined at day 13 . In accordance with our results in vitro . Hence, the increase in the tumor mass upon induction of LAP must have been due to reduction in cell death, which apparently overcame the anti-proliferative effect of LAP. Indeed, necrotic areas were abundant in the tumor sections and HERPUD , whereas over-expression of LAP reduced the expression of these markers , we were able to determine the specific impact of LAP and LIP on the cell fate under ER stress in vitro and in tumors in vivo. Induction of LIP results in suppression of endogenous LAP activity. Induction of LAP, on the other hand, mimics the over-expression seen in many tumors.The reported induction of C/EBP-β by ER stress , &S2) correlated with the observed fourfold increase in the size of LAP-expressing tumors during days 8-13 and the mirror image fourfold decrease in size of LIP-expressing tumors we demonstrated the general role of LAP and LIP in modulating the resistance to ER stress-triggered cell death. A previous study demonstrated that C/EBP-β null mice (lacking both LAP and LIP) are completely refractory to generation of skin tumors upon exposure to a carcinogen Our finding that LAP attenuated whereas LIP augmented ER stress–triggered cell death in vitro , 2 &S2 CHOP expression in rat cells &CHOP. Thus, LIP may relieve the transcription inhibition of LAP by forming a futile LAP-LIP heterodimer and thereby triggering cell death. Alternatively or additionally, such a LAP-LIP heterodimer may trigger cell death in a manner analogous to the proposed action of the LAP-CHOP heterodimer Our findings suggest that changes in the LAP-LIP ratio control the transition from the early, protective UPR to its late, death–promoting phase. Others The Weizmann Institute Animal Care and Use Committee approved all studies in mice, according to institutional guidelines and Israeli law.2 incubators at 37°C. Expression of LAP and LIP was induced in the F10.9-3 and F10.9-4 cells, respectively by doxycycline unless otherwise stated. ER stress was elicited by treatment with tunicamycin or thapsigargin unless otherwise stated. Antibodies to C/EBP-β (C-19) and CHOP (B-3) were purchased from Santa Cruz Biotechnology ; the TRIB3 antibody (ST1032) was from Calbiochem ; the Ki-67 antibody (clone TEC-3) was from DakoCytomation ; the cleaved caspase 3 antibody (Asp 175) was from Cell Signaling ; the HERPUD antibody was from Protein Tech Group , the rat anti-mouse CD31 (PECAM-1) and BiP/GRP78 antibodies were from BD Pharmingen, , the HMGB1 antibody (H9539) was from Sigma-Aldrich and the Cy3-conjugated streptavidin, biotin-SP–conjugated AffiniPure donkey anti-rabbit IgG and biotin-SP–conjugated goat anti-mouse IgG antibodies were from Jackson ImmunoResearch . All other reagents were purchased from Sigma-Aldrich .The murine B16 melanoma clones, F10.9-3 and F10.9-4, were derived from the parental cell line B16-F10 (ATCC CRL-6475) and were kindly provided by M. Revel and J. Chebath (Weizmann Institute of Science) 5 cells mixed with 100 µl Matrigel). Doxycycline (1 mg/ml) was added to the drinking water for the induction of LAP and LIP. Mice (10 per group) were euthanized at day 8 or 13, and tumors were resected, weighed, photographed and either fixed in 10% formaldehyde or frozen before sections were prepared. The significance of differences in the tumor mass was determined by Student's t-test of independent samples. The paraffin sections were immunostained with antibodies to Ki-67, cleaved caspase 3, TRIB3 and HERPUD, followed by incubation with biotin-conjugated antibodies and Cy3-conjugated streptavidin. Cell nuclei in tumor slices and in cultures were stained with Hoechst 33258 . For CD31 immunostaining, tumor tissues were embedded in OCT and were frozen at −80°C for cryosectioning. After fixation in 100% acetone , slides were immunostained as described above.C57/BL6 mice were inoculated subcutaneously with F10.9-3 or F10.9-4 cells nuclei. The fraction of cleaved caspase 3 cells was similarly determined. The staining intensity of TRIB3 and HERPUD was determined by the histogram function of The Photoshop program (red channel) and normalized to the staining intensity of the nuclei (blue channel) in the same field. The significance of differences in cell counts or in the staining intensity was determined by Student's t-test on the indicated number of replicates.siRNA pools (ON-TARGETplus), directed against murine and human C/EBP-β mRNA were purchased from Dharmacon RNAi Technologies . The knockdown of the transcripts was performed in 6-well plates with DharmaFECT 1 reagent, according to the manufacturer's protocol. Cells were seeded in media without antibiotics for the knockdown experiments.t-test of the independent samples. Each independent experiment consisted of 9 replicates. Apoptosis was evaluated by staining with the rh annexin-V/FITC kit . Cell cycle analysis was performed by collecting the cultured cells, fixing in 70% ethanol on ice, treatment with RNase A , staining with propidium iodide and flow cytometry.To evaluate the extent of cell death, cultures were stained first with Hoechst 33258 washed and stained with propidium iodide and then visualized by fluorescence microscopy. To evaluate cell morphology, cultures were fixed and stained with 5% crystal violet in 66% methanol. Total cell counts were determined by counting manually, by using the ImageJ program (NIH) or by reading the crystal violet stained cultures in 96 well plates with an ELISA reader at 570 nm. Alternatively, staining intensity was determined in photographs by the histogram function of Photoshop. For quantitative measurement of viable cells, cultures in 96 well plates were incubated with Neutral Red , washed 3X with PBS, re-suspended in lysis buffer (0.1 ml) and OD was measured with an ELISA reader at 540 nm. A calibration curve was prepared by seeding known amounts of cells onto 96-well plates. After 6 h the cells were stained with Neutral Red and the OD was read as described above. For each experiment, statistical analyses were performed on the indicated number of independent experiments using Student's Cells were washed three times with ice-cold phosphate buffered saline (PBS). Cell pellets were resuspended in two packed cell volume of lysis buffer , 0.5 mM dithiothreitol (DTT), 25% glycerol). The resuspended pellets were then frozen and thawed twice and kept on ice for 20 min. The lysates were centrifuged and the supernatants containing the cellular proteins were collected, frozen in liquid nitrogen and stored at −80°C. Protein concentration was determined by a BCA Protein assay reagent kit using bovine serum albumin as a standard. Protein samples were boiled in SDS-PAGE sample buffer containing 25 mM DTT, and the supernatants were resolved by gradient SDS-PAGE (7.5–15% acrylamide). Proteins were then transferred onto a nitrocellulose membrane, which was incubated with the indicated antibodies. Second antibody conjugates were visualized by the Super Signal Detection Kit (Pierce).CTGCAACGCCTGGTGGTGTGCTGGAATTCC and the reverse oligo 5′ GGCCTGCGCCGCCGCACGGGAAGCCCGCCGCCA. Codon 199 (ATG) was then point-mutated as above with the forward oligo 5′ CTGGCGGCGGGCTTCCCGTGCGGCGGCGCAGGC and the reverse oligo 5′ GGCCTGCGCCGCCGCACGGGAAGCCCGCCGCCA to prevent expression of LIP. The LIP ORF (codons 199–346) was inserted into pcDNA4 to generate pcLIP, which expresses only the human LIP isoform of C/EBP-β.To construct a mammalian expression vector that expresses only LAP (pcLAP), the complete human C/EBP-β ORF was inserted into pcDNA4 . Codon 1 (LAP*-initiating ATG) was point-mutated to CTG using Pfu Turbo DNA polymerase , the forward oligo 5′ Figure S15/well) were cultured in 6-well plates with or without doxycycline (Doxy). (A) RT-PCR of C/EBP-β mRNA from F10-9.3 cells. (B) GFP expression in F10 9.3 cells. Bar = 0.5 mm. (C) Dose response of LAP expression in F10 9.3 cells following 24 h treatment with increasing concentrations of doxycycline, as determined by immunoblotting. (D) Dose response of LIP expression in F10 9.4 cells, as described in (C). The gels and blots are representative results of three replicate experiments.Inducible over-expression of LAP and LIP in murine B16 F10.9 clones. B16 F10.9 cells (4×10(1.00 MB TIF)Click here for additional data file.Figure S24/well) were cultured in 96-well plates with the indicated concentration of doxycycline for 24 h. Diluent (Ctrl), tunicamycin (Tm) or thapsigargin (Tg) were then added. After an additional 24 h the plates were stained with crystal violet and photographed. The photograph is representative of five replicate experiments. (B) F10.9-3 cells (1.5×104/well) were cultured in 96-well plates with the indicated concentration of doxycycline for 24 h. Diluent (Ctrl), tunicamycin (Tm) or thapsigargin (Tg) was then added. After 24 h the plates were stained with crystal violet and photographed under a light microscope. Control cells were also photographed by fluorescent microscopy (top panel) to evaluate the extent of GFP and C/EBP-β induction by doxycycline. The photograph is a representative of five replicate experiments. Bar = 0.5 mm.LIP augments and LAP attenuates ER stress-triggered cell death in a dose-dependent manner. (A) F10.9-4 cells (2×10(9.92 MB TIF)Click here for additional data file.Figure S3Survival and death of F10.9-3 cells under ER stress. (A) F10.9-3 cells were seeded in 8 well microscopy chambers and after 24 h challenged either with tunicamycin or with TNF-α (100 ng/ml) plus IFN-γ (1000 IU/ml) for 24 h. The cells were stained for apoptosis by the rh annexin-V/FITC kit followed by Hoechst 33258. The treatment with TNF-α plus IFN-γ served as a positive control for apoptosis, as determined by the annexin V/FITC staining. The photographs are representatives of four replicate studies. Bars are 0.2 mm. Insets: larger magnification to show cellular staining details. Bar = 0.05 mm. (B) F10.9-3 cells in 6-well plates were challenged with tunicamycin . The detached cells were collected, plated and stained with propidium iodide and counter-stained with Hoechst 33258. The photograph is a representative of three replicate experiments. Bar = 0.2 mm. (C) Control (Ctrl) and doxycycline-treated F10.9-3 cells (LAP) were challenged with tunicamycin (Tm) or Thapsigargin (Tg) as described in (4.02 MB TIF)Click here for additional data file.Figure S45/well) were cultured in 6-well plates for 24 h and then transfected with a control siRNA or C/EBP-β-specific siRNA. After 24 h, RNA was isolated and analyzed by RT-PCR (left panels), and cell extracts were prepared and analyzed by immunoblotting with antibodies directed to the common C-terminus of C/EBP-β (right panels). The gels and blots are representatives of three replicate experiments. (D) HeLa cells were seeded and transfected as described in (C). After 36 h, diluent (Ctrl) or Tunicamycin (Tm) were added and after an additional 31 h the cultures were stained with Crystal Violet and photographed. The photographs are representatives of four replicate experiments. Bar = 0.5 mm. (E) Cell counts following the treatments described in (D). The data are average of counting four fields. (F) Human HeLa cells were transfected with 1 µg of either control pcDNA4 vector, pcLAP vector (mutated to encode LAP only), or pcLIP vector (encoding LIP only). After 24 h the cells were seeded in 96 well plates and after additional 24 h diluent or tunicamycin (4 µg/ml) were added. After 48 h cell viability was determined by Neutral Red staining. Percent viability was determined by the ratio of tunicamycin to diluent-treated cells. Data are mean±SD of six replicates.Effects of LAP and LIP knockdown or over-expression on cell survival upon ER stress. (A) Human 293T cells in 6 well plates were transfected with 1 µg of control pcDNA4 (middle lane), pcLAP (left lane), or pcLIP (right lane). After 24 h cell extracts (20 µg protein) were immunoblotted with antibodies directed to the common C-terminus of C/EBP-β. N.S. is non-specific bands. The blot is a representative of three replicates. (B) Human 293T cells were transfected as in E and after 24 h were trypsin-digested and seeded in poly(D-lysine)-coated 96 well plates . After additional 24 h the cells were either untreated (Ctrl) or challenged with tunicamycin (2 µg/ml). The plates were left for 24 h and the number of viable cells was determined by the Neutral Red assay. Percent survival represent the ratio of tunicamicyn-treated to diluent-treated cells. Data are mean±SD of four replicates. Similar transfection efficiencies were obtained with the three vectors and cell counts were very similar in the control cultures not treated with tunicamycin (Ctrl). (C) HeLa cells (2×10(2.03 MB TIF)Click here for additional data file.
Thrombin-anti-thrombin III complex (14.2 ± 4.3 ng/ml on PREOP and 26.0 ± 4.1 ng/ml on POD 7(mean ± SE)) and D-dimer (335 ± 96 ng/ml on PREOP and 1859 ± 258 ng/ml on POD 7 (mean ±SE)) increased in the early postoperative stage. The level of 6-keto-prostaglandin F1α increased afterthe operations (from 13.2 ± 1.8 pg/ml on PREOP to 37.8 ± 12.8 pg/ml on POD 7 (mean ± SE)). Thelevel of thromboxane B-2 decreased at first, and then gradually increased and returned to itspreoperative level on POD 7 ). Superoxide dismutase activity increased at first, and thengradually decreased, postoperatively ). That is, biodefensive reactions which protect patients againstthe shift to disseminated intravascular coagulation (DIC) were inferred with by the increase in antiplateletaggregation, despite the activation of coagulation and fibrinolytic mechanisms after hepaticresection.A survey of the blood of twenty-two patients who had undergone hepatic resection was performed.Serum levels of
Cardiac involvement in malignant lymphoma is one of the least investigated subjects in oncology. This article reports a case of cardiac involvement in Hodgkin's lymphoma which presented as heart failure.We report the case of an 8-year-old Afghan girl with Hodgkin's lymphoma. The disease presented with systemic signs and symptoms, including abdominal distension, weakness, pallor, chills, fever, generalized edema, hepatosplenomegaly and generalized lymphadenopathy, as well as signs of heart failure. Test results showed a rare form of heart metastasis.We report a case of Hodgkin's lymphoma with metastasis to the heart, detected premortem. Although the involvement of the heart in a malignancy is relatively common, premortem detection is unusual and only few studies have reported it in the literature. Cardiaclymphoma ,4. Some lymphoma , arrhythlymphoma . Echocarlymphoma ,8, but iet al. explained the pathogenesis of cardiac involvement in lymphoma in their 2006 study [Bashir 06 study . One sixThe diagnostic standard in these conditions includes acquiring the patient's history and performing physical examinations, chest radiography, contrast-enhanced computed tomography (CT) of the neck, chest, abdomen, and pelvis, and gallium scan or positron emission tomography. Patients with suspected bone involvement usually undergo bone scans, while patients in the advanced stages of the disease undergo bone marrow biopsies. Baseline studies consist of a complete blood count, blood chemistry analyses, kidney and liver function tests, echocardiography, electrocardiography, and pulmonary function tests .Low-dose radiation plus multi-agent chemotherapy for pediatric Hodgkin's disease was adopted after a pioneering study reported using 15 to 25 Gy and six cycles of mechlorethamine, vincristine, procarbazine, and prednisone chemotherapy for children . ChemothPrimary presentations of Hodgkin's lymphoma have been reported many times, but heart failure in Hodgkin's lymphoma has not yet been reported in the literature. This case report describes a rare case of cardiac involvement in Hodgkin's lymphoma which presented as heart failure and was diagnosed through echocardiography findings.An 8-year-old Afghan girl presented with a two-month history of edema, abdominal distension, weakness, pallor, chills, fever, anorexia, and weight loss. Her medical history was not remarkable. Physical examinations showed severe mucosal and conjunctival pallor, periorbital and sacral edemas, and abdominal distension. She also presented with tender mobile lymph nodes in her right neck (5 × 5 mm), bilateral inguinal area (0.5 cm × 0.5 cm) and left axillary (0.7 cm × 0.7 cm), as well as marked hepatosplenomegaly and ascites with shifting dullness. Systolic murmurs (II/III) of the heart and lungs were apparent.In this patient, Hodgkin's lymphoma had metastasized to the myocardial tissue. The tumor involved all the cardiac tissue and the septum. Metastasis must have occurred via the blood vessels because it involved the cardiac tissue itself as well as the lymph nodes. Other hematopoetic areas such as the liver, spleen and bone marrow were also involved.The symptoms of lymphadenopathy included enlargement of the lymph nodes, in particular the para-aortic lymph nodes. There were symptoms of cardiac failure in the form of tachycardia, cardiomegaly, gallop rhythm, tachypnea, weak pulse, and hypotension.In examining the patient, we used all diagnostic standards except positron emission tomography, because the patient had already been diagnosed with lymphoma and metastasis. There were no signs of Hodgkin's lymphoma in the bone marrow and bone aspiration test results.Laboratory findings included severe anemia with moderate anisopoikilocytosis, hemoglobin level of 3.2 , erythrocyte sedimentation rate of 50 , and positive C-reactive protein. Polymerase chain reaction for tuberculosis, blood culture, urine culture, hydatid antibody, Coombs Wright and 2 ME, direct Coombs, bone marrow culture, and blood smears for malaria and borrelia were all negative. Our patient's G6PD level was also normal.There were several findings that led to the identification of appropriate treatment for our patient. In abdominal sonography, her liver was found to be enlarged with heterogenic echo. Marked hepatosplenomegaly (spans = 17 cm) and two round hypoechoic areas in the hepatic portal space due to adenopathy were seen. Her biliary gall bladder had no stones and its wall had an increased thickness. An abdominal CT scan (with and without contrast) showed severe hepatosplenomegaly, a hypodense area in the liver that might be a small hemangioma or cyst, considerable para-aortic adenopathy, and dilated small bowel loops with thickened walls. Her spleen was enlarged to a diameter of 13 cm and had homogenous echo. Her internal and external biliary tract liver were of normal diameter. There were some circular hypoechoic masses in our patient's portohepatic region, which indicated lymphadenopathy in this area. Her para-aortic region could not been observed because of the abdominal gas. Her intestinal loops were dilated in the pelvic region and were full of liquid.Her kidneys had normal secretions and appeared normal. No other abnormalities were seen. Her lungs were clear and of normal size, as shown on contrast chest X-ray. The chest X-ray also showed cardiomegaly. A CT scan of our patient's chest showed multiple lymph adenopathies in the paratracheal and subcarinal regions.Our patient's lung parenchymas were reported to be normal in a thorax CT scan with contrast. There were no effects of impressed masses, parenchymal nodules or abnormal infiltration. The vessels and bronchus seemed normal. Multiple lymph nodes were seen in the para-aorta, subcarina, lungs or esophagus.Anemia due to tumor involved all parts of the hematopoetic areas such as the liver, bone marrow and spleen, and also due to cardiac deficiency and endocarditis. Cardiac failure could occur after a bacterial endocarditis and the tumor development. There was also lymphadenopathy, pericardial effusion, fever, tremor, and edema.In treating our patient, acute symptoms such as severe anemia, infection, electrolyte and biomedical imbalances and hypoglycemia were encountered. Once these had been treated, we addressed the Hodgkin's lymphoma.2, Bleomycin 10 mg/m2, and vincristine 6 mg/m2, as well as 375 mg of dimethyl, triazeno, imidazole and carboxamide (DTIC) as infusion.Our patient was given a high-protein and high-calorie diet, and treatment for tuberculosis was started. Gentamycin, penicillin, and vancomycin were prescribed because of the presenting endocarditis. After stabling the patient's condition, 14 sessions of chemotherapy were started. Chemotherapy included intravenous Adriamycin (doxorubicin) 25 mg/mAfter one year, no evidence of the disease were reported in a thorax CT scan with injection contrast or in abdominal and pelvic CT scans with oral and injection contrast. There was also no evidence of abnormal opacity in the lung parenchyma. The pathologic area was not seen either in the thorax bone structure or the adjacent soft tissue. There were no symptoms of either pleural fluid or pleural thickness. There was also no evidence of the anterior, medial of posterior mediastinal masses. The major vessels appeared normal. Small aortocaval and thorocaval lymph nodes were observed. A myeloma with a maximum diameter of 1 cm at the left kidney was detected. The size and density of our patient's kidneys were normal. The urine tract was not obstructed. The hilar areas were normal in both kidneys. The bronchus had normal calibers. The gall bladder and the internal and external liver biliary tracts were also normal. The spleen was of normal size and had systematic margins and homogenous density. The attenuation valve was also normal.After completing the treatment, our patient was discharged. Her parents were told that she should attend our clinic for follow up every three months. All the symptoms of the disease have now disappeared and the girl is living an ordinary life. She does not have symptoms of lymphadenopathy, splenomegaly or any other problems.Although the involvement of the heart in malignancies is relatively common, premortem detection is unusual and only a few studies have reported this subject in the literature. We report a case of Hodgkin's lymphoma which presented with systemic signs and symptoms including abdominal distension, weakness, pallor, chills, fever, generalized edema, hepatosplenomegaly and generalized lymphadenopathy, as well as signs of heart failure. Echocardiography revealed pericardial effusion, left ventricular hypertrophy, and lucent myocardial lesions. A right cervical lymph node biopsy established the diagnosis of nodular sclerosing Hodgkin's lymphoma with involvement of the bone marrow at biopsy. After 14 chemotherapy sessions, systemic and cardiac abnormalities had improved. To the best of our knowledge, this is the first reported case of Hodgkin's lymphoma accompanied by cardiac metastasis and heart failure.Echocardiograph findings have shown that pericardial effusion is the most common abnormality in cardiac metastasis. The early detection of metastatic cardiac involvement can be beneficial for the patients because it can lead to careful monitoring, better management of morbidity and decreased mortality.Studies have shown that patients with lymphoma associated with cardiac involvement can be treated successfully.Written informed consent was obtained from the patient's next-of-kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.ZA and MK gathered and interpreted data on the patient. NDN and SM assisted in gathering and organizing the data. They were also major contributors in writing the manuscript. All authors read and approved the final manuscript.
The effects of weather on West Nile virus (WNV) mosquito populations in the United States have been widely reported, but few studies assess their overall impact on transmission to humans.We investigated meteorologic conditions associated with reported human WNV cases in the United States.We conducted a case–crossover study to assess 16,298 human WNV cases reported to the Centers for Disease Control and Prevention from 2001 to 2005. The primary outcome measures were the incidence rate ratio of disease occurrence associated with mean weekly maximum temperature, cumulative weekly temperature, mean weekly dew point temperature, cumulative weekly precipitation, and the presence of ≥ 1 day of heavy rainfall (≥ 50 mm) during the month prior to symptom onset.Increasing weekly maximum temperature and weekly cumulative temperature were similarly and significantly associated with a 35–83% higher incidence of reported WNV infection over the next month. An increase in mean weekly dew point temperature was significantly associated with a 9–38% higher incidence over the subsequent 3 weeks. The presence of at least 1 day of heavy rainfall within a week was associated with a 29–66% higher incidence during the same week and over the subsequent 2 weeks. A 20-mm increase in cumulative weekly precipitation was significantly associated with a 4–8% increase in incidence of reported WNV infection over the subsequent 2 weeks.Warmer temperatures, elevated humidity, and heavy precipitation increased the rate of human WNV infection in the United States independent of season and each others’ effects. West Nile virus (WNV) is a globally distributed, mosquito-borne flavivirus that caused 3,510 known cases and 109 deaths in the United States in 2007 . The WNVLike malaria in tropical Africa and St. In contrast, the effects of precipitation on WNV and other U.S. mosquito-borne disease transmission remain controversial . Broad rThe Intergovernmental Panel on Climate Change projectsWe obtained dates of symptom onset, age, and county location for human cases during 2001–2005 from the CDC using their case definition . A case We obtained daily data on ambient temperature, dew point temperature, and precipitation from the National Oceanic and Atmospheric Administration (NOAA) National Climatic Data Center . Only daA degree-day is a measure of cumulative daily temperature within a temperature band. We calculated degree-days using the single-sine method , which mWe performed a geographic analysis to match meteorologic data with individual cases. Specifically, we created a map of all case-positive counties and superimposed the location of all U.S. first-order weather stations based on their latitude and longitude. In counties with multiple weather stations, we calculated a daily average across all stations. Counties lacking weather stations were paired with the closest station to the county’s geometric centroid using ArcGIS 9.2 .We used the case–crossover study design to evaluate the association between meteorologic variables and WNV case occurrence. The case– crossover study design was specifically developed to study the effect of transient exposures on the risk of acute events . In thisa priori to use maximum daily temperature as the primary temperature measure because previous studies demonstrated an association with maximum values (r = 0.95) and minimum daily temperatures (r = 0.73). We also modeled cumulative temperature using the single-sine method as estimates of incidence rate ratios and 95% confidence intervals (CIs) associated with meteorologic variables. Using a distributed lag model , both unWe assessed 16,298 human WNV cases during 2001–2005 across 17 states. Cases ranged from < 1 to 99 years of age, with a median age of 49 years, and spanned the entire calendar year . Three hIn a model controlling for dew point temperature and precipitation, mean weekly maximum temperature was positively and significantly associated with the incidence of reported WNV infection during the same week and in the subsequent 3 weeks . SpecifiIn a model controlling for mean weekly maximum temperature and cumulative weekly precipitation, mean weekly dew point temperature was significantly associated with the incidence of reported WNV infection 1–3 weeks later, but not during the same week .In a model adjusting for mean weekly maximum temperature and mean dew point temperature, a 20-mm increase in cumulative weekly precipitation was associated with a 4–8% increase in incidence of reported WNV infection 1–2 weeks later . CumulatOne or more days per week of heavy precipitation (defined as ≥ 50 mm in a single day) was associated with a 33% higher incidence of reported WNV infection during the same week, and the incidence remained elevated in the subsequent 2 weeks . LowerinIn this study we examined the influence of temperature, humidity, and precipitation on the incidence of reported WNV infections among 16,298 reported human cases. We found positive associations with increasing temperature over each of the 4 weeks prior to symptom onset. In addition, heavy but not low levels of rainfall were significantly and positively associated with the incidence of reported WNV infections over the same week, while both were significantly and positively associated with WNV infections over the subsequent 2 weeks. Although insignificant during the case week, elevated weekly dew point temperature progressively increased the incidence of reported WNV infections over the ensuing 3 weeks.Culex is approximately 8–12 days, and the EIP under laboratory conditions ranges from 4 to 12 days at 30°C and > 28 days at 18°C . All of these measures of temperature yielded a similar pattern of incidence rate ratios during each of the 4 weeks, with the maximum rate occurring at a lag of 1 week. On days when the temperature did not drop below our minimum threshold of 14°C, the degree-days calculation approximates the mean temperature above the threshold. The fact that the association was not universally stronger with accumulated degree-days than with mean weekly maximum temperature suggests that conditions below the specific threshold of 14°C did not significantly attenuate the rate of WNV transmission, as would be predicted based on experimental data in the laboratory.The strongest effects of both light and heavy precipitation appear to transpire 1–2 weeks after the rainfall, during a period when they might expand mosquito populations and influence mosquito host-seeking. We found that the definition of “heavy” precipitation significantly influenced the results, with the strongest associations observed using a definition of ≥ 50 mm precipitation in a single day. This finding may imply that large, single-day rainfalls influence transmission in a distinct manner from cumulative precipitation. In turn, the fact that heavy but not light rainfall was positively associated with incidence over the same week suggests that a downpour may increase human–mosquito interaction and the likelihood of an infectious bite. Prior research suggests that heavy rainfall can stimulate episodes of disease transmission by increasing near-surface humidity, which stimulates mosquitoes to oviposit and seek hosts . HoweverThis study has important strengths and limitations that merit discussion. First, we had data only on the county of residence of each case, leading to possible misclassification of exposure due to heterogeneity in weather conditions within counties. Errors in reported dates of symptom onset may also contribute to exposure misclassification. However, both of these sources would be expected to bias our effect estimates toward the null. Second, the relationship between meteorologic conditions and vectorborne disease risk are enormously complex , invokinBecause the impacts of major meteorologic phenomenon such as global warming on temperature and precipitation are varied and occur over long time periods, the effects of climate shifts could not be evaluated in this study, which is designed to assess the effect of meteorologic variations on a shorter time scale. Still, our results carry implications for future studies of major climatic trends. Although many factors influence WNV incidence, this study indicates that increasing temperature, humidity, and rainfall across the United States independently and transiently elevates WNV incidence over the subsequent month regardless of season and location. This knowledge may complement traditional surveillance techniques and suggests, if weather trends continue, that the human impact of this and related diseases may increase in coming decades.Warmer temperatures, elevated humidity, and heavy precipitation increased the relative rate of human WNV infection in the United States independent of season and each others’ effects.
The technological development of telemedicine has performed important progress, assuming a diagnostic relief role inside of the processes. Among the fields in fast evolution, telepathology is placed among those of greater interest. Up to some years ago, telepathology allowed us to observe at a distance and in real time, histological or cytological slides through the Internet, using a motorized microscope (dynamic telepathology). Currently, telepathology has completed an important step in ahead being possible to digitize completely a slide and to store it. This allows observation of the whole surface of histological or cytological slides remotely with a customary PC, without human intervention . The described systems have exclusive characteristics, so that a "hybrid system" supporting both technologies, turns out to be the best solution applicable in a wide range program. In order to realize the theoretical aspects previously described, we report an organizational model practicable and applicable to a territory in which three hospitals operate. An essential prerequisite in order to arrange an efficient telepathology system turns out to be one structured data transmission network, equipped with elevated guaranteed bandwidth, and one consolidated experience in the registration and management of digital images. The acquisition of a telepathology system designed for a district territory that comprises three hospitals managed from two different health administrations (Adria and Rovigo) is inserted in a wide plan of local development of telemedicine. The challenge has been planned from the two administrations and fully financed by the Cariparo Foundation.In order to achieve the implementation of this telepathology system there was a need for hospitals technology structures innovation. In particular: physical and logical structures for data transport, hardware modernization, software development, processes reorganization.This technological innovation process is essential for developing and offering to citizens high quality and reliable health services. In particular, a telepathology system needs high hardware and network performance for processing, managing and archiving digital slide images.The main aims, thanks to the introduction of telepathology, can be summarized as follows:• Increase of service quality offered to patients thanks to qualified second opinions;• Sharing and discussion of interesting pathological cases for increasing professional ability;• Creation of a permanent and available scientific repository.We can distinguish three types of telepathology: static telepathology, dynamic telepathology, virtual slide telepathology. In this paper, we analyze the last two for describing how we can implement a "hybrid telepathology system" that takes advantage of the peculiarities of each one. The dynamic telepathology allows exploration, in real time, of the whole slide surface thanks to a robotized remote controlled microscope; this also permits us to change magnification, focus and digitize the interesting portion. The disadvantages regard the necessary presence of a technician for positioning the desired slide within the microscope and the discontinuous digitization in terms of area and magnification. In virtual slide telepathology, the slide is completely digitized and stored in a repository; this permits a single or multiple user consultation, in every time and without human intervention. The virtual slide allows the exploration of the whole slide surface with different magnification. There are many types of devices that can be used for slide digitization and the right option depends on workload and necessary scan rate . VirtualThere are mainly two types of second opinions: second opinions in real time on frozen section during surgery (intraoperative consultation) and second opinions on histological or cytological slides which require a complex interpretation. Each type of second opinion has specific characteristics that are directly correlated with previous discussion. In the first second opinion type the analyses are conducted on thick sample or with irregular surface ; for this reason the automatic scanner device can't focus correctly on the whole slide surface but with a motorized microscope (dynamic telepathology) it is possible to focalize each sample surface point. Therefore, the dynamic telepathology represents an important tool for second opinion during intraoperative consultation, especially for the presence on the territory of two hospitals with surgery but without a resident pathologist. Regarding histological and cytological slides, the tissue samples are treated for obtaining thin sections; this deletes the trouble regarding focus due to irregular surface, as previously described. In this case the use of virtual slide telepathology is favorable because it allows the visualization of the morphological picture at any time, remotely with a customary PC, without human intervention. This allows one to obtain a second opinion on complex cases from an expert of personal choice and to perform an external quality control.Based on previous considerations and for obtaining an effective telepathology system, the best choice is a "hybrid system" composed of motorized microscopes, with remote control, and a scanner for slide digitization, in order to achieve the best characteristics from each system without respective disadvantages. This choice has been applied to Rovigo province health structures, in particular Rovigo, Adria and Trecenta hospitals. For storage purposes it has been used a NAS (Network Attached Storage) device (50 TB capacity – 1 Gb/s transfer rate). The previously described system is completely integrated with the CPOE (Computerized Physician Order Entry) based Hospital information System.Thanks to complete slides digitization and the use of Image Server with high computational performances, it will be possible to apply filters to acquired images or to apply algorithms for calculating interesting quantities (e.g.: the cellular membrane distribution and continuity). These techniques adequately developed, tested and standardized will be the base for Computer Aided Diagnosis (CAD) introduction.The implementation of a telepathology "hybrid system" adequately projected and integrated with the Hospital information System, leads to an increase in the service quality offered by the Pathological Anatomy department, principally thanks to: (a) qualified and real time second opinions on frozen section during surgery or histological-cytological consultation on slides which requires a complex interpretation; (b) continuous education process thanks to the sharing of interesting virtual slides and creation of a permanent scientific archive.The design and implementation of a telepathology system must be based on real operative demands, supported by a technological structure that can guarantee a reliable and efficient service and projected using hospital experience on digital images acquisition and storage.
The cytoxicity of both intercalating (m-AMSA) and non-intercalating topoisomerase II-targeting drugs is thought to occur via trapping DNA topoisomerase II on DNA in the form of cleavable complexes. First, analysis of cleavable complexes (detected as DNA double-strand breaks) by pulsed-field gel electrophoresis confirmed the correlation between cleavable complex formation and cytotoxicity of three topoisomerase-targeting drugs in HeLa S3 cells (the order of effects being VM26 > m-AMSA > VP16). In contrast to many antineoplastic agents, hyperthermic treatments were found to protect cells against the toxicity of all three topoisomerase II drugs. Hyperthermia treatment does not alter drug accumulation but reduces the ability of the drug-topoisomerase II complex to form the cleavable complexes. Nuclear protein aggregation induced by heat at the sites of topoisomerase II-DNA interaction may explain such an effect. In thermotolerant cells, the toxic effects of VP16 but not m-AMSA were reduced. For both drugs, however, the status of thermotolerance did not affect cleavable complex formation by the drugs. Thus, protection against VP-16 toxicity seems not to be associated with heat-induced activation of the P-gp 170 pump or altered topoisomerase II-DNA interactions. Rather, a protective (heat shock protein mediated?) mechanism against non-intercalating topoisomerase II drugs seems to occur at a stage after DNA-drug interaction. Finally, heat treatment before topoisomerase II drug treatment reduced toxicity and cleavable complex formation in thermotolerant cells to about the same extent as in non-tolerant cells, consistent with the presumption of nuclear protein aggregation being responsible for this effect.
Kepler system. Calibrated polypeptide spot abundances (volumes)were compared to assesses qualitative and quantitative changes of the spot volumes. Amongover 300 proteins evaluated at GD (gestation day) 13,15, and 17, there were sets of proteinsthat increased and others that decreased in intensity. We could in addition recognize proteinsthat were completely absent at GD 13 and/or 15 and that appeared thereafter togradually increase in intensity. Conversely, various polypeptide spots present at early stages(at GD 13 and 15) disappear later (at GD 17 or at birth). Among the proteins that increasein intensity prevail molecules with masses less than 35 kD, whereas a considerable portionof those that decrease in intensity are characterized by masses above 60 kD. Spots reportedin this communication were not defined beyond tagging them with numbers, which is aprerequisite to follow them up in the proteinpaedia developed in our laboratory. The nextstep will be to retrieve the coding sequences from the existing partitioned cDNA library(BW 5147) as well as from thymocyte subtraction libraries. We predict that among thosepolypeptides with varying intensity, important regulatory proteins in thymus developmentwill be found.Driven by our long-standing interest in identifying proteins of the immune system and incharacterizing processes involved in lymphocyte differentiation, we studied protein expressionin biosynthetically labeled fetal and newborn thymus by 2D gel electrophoresis.Autoradiographs of the gels were scanned with a densitometer and image analysis wasperformed using the
Increased muscle loading results in the phosphorylation of the 70 kDa ribosomal S6 kinase (p70S6k), and this event is strongly correlated with the degree of muscle adaptation following resistance exercise. Whether insulin resistance or the comorbidities associated with this disorder may affect the ability of skeletal muscle to activate p70S6k signaling following an exercise stimulus remains unclear. Here, we compare the contraction-induced activation of p70S6k signaling in the plantaris muscles of lean and insulin resistant obese Zucker rats following a single bout of increased contractile loading. Compared to lean animals, the basal phosphorylation of p70S6k , Akt , and mTOR was higher in obese animals. Contraction increased the phosphorylation of p70S6k (Thr389), Akt (Ser473), and mTOR (Ser2448) in both models however the magnitude and kinetics of activation differed between models. These results suggest that contraction-induced activation of p70S6k signaling is altered in the muscle of the insulin resistant obese Zucker rat. Insulin resistance is an important health concern that is rapidly increasing worldwide. If allowed to precede unchecked, insulin resistance often leads to increased morbidity and mortality. Anaerobic exercise has recently been recognized to be of potential benefit for the treatment of this condition –4, as prRecent in vitro and in vivo studies have suggested that increased muscle loading correlates with increases in the rates of accumulation and synthesis of muscle protein –14. ThisThe purpose of this study was to determine whether exercise-induced p70S6k signaling is altered in the skeletal muscle of the insulin resistant obese Zucker rat. We hypothesized that contraction-induced p70S6k signaling would differ between normal and insulin resistant muscle. To test this hypothesis, the activation of p70S6k and its pathway related proteins mTOR, Akt, and PTEN were assessed in the plantaris muscles of normal and insulin resistance rats either immediately after or during the recovery phase of an acute bout of high-frequency electrical stimulation. Our findings suggest that insulin resistance or the co-morbidities associated with this disorder may affect how muscle regulates contractile-induced signaling.n = 12) male Lean Normal Zucker (LNZ) and young male Obese Syndrome-X Zucker (OSXZ) rats were obtained from the Charles River Laboratories. Rats were housed two to a cage in an AAALAC approved vivarium. Housing conditions consisted of a 12 H: 12 H dark-light cycle and temperature was maintained at 22° ± 2°C. Animals were provided food and water ad libitum and allowed to recover from shipment for at least two weeks before experimentation. During this time the animals were carefully observed and weighed weekly to ensure none exhibited signs of failure to thrive, such as precipitous weight loss, disinterest in the environment, or unexpected gait alterations.All procedures were performed as outlined in the Guide for the Care and Use of Laboratory Animals as approved by the Council of the American Physiological Society and the institutional animal use review board. Young (#9332), PTEN (#9552), Thr389 (#9206) and Ser 421 / Thr 424 (#9204) phosphorylated p70S6K, Thr308 (#9275) and Ser473 (#9271) phosphorylated Akt, Ser2448 phosphorylated mTOR (#2971), Ser 9 phosphorylated GSK-3β (#9336), Ser 380/Thr 382/383 phosphorylated PTEN (#9554), Mouse IgG, and Rabbit IgG antibodies were purchased from Cell Signaling Technology . Enhanced chemiluminescence (ECL) western blotting detection reagent was from Amersham Biosciences . Restore western blot stripping buffer was obtained from Pierce and 3T3 cell lysates were from Santa Cruz Biotechnology . All other chemicals were purchased from Sigma or Fisher Scientific .Anti-p70S6k (#9202), Akt (#9272), mTOR (#2972), glycogen synthase kinase-3The high-frequency electrical stimulation (HFES) model has been previously described and was μL protease inhibitor cocktail, and 1 mM sodium vanadate to inhibit phosphatase activity, and then centrifuged for 10 minutes at 2000 g to pellet particulate matter. This process was repeated twice and the supernants combined for protein concentration determination using the Bradford method . Samples were diluted to a concentration of 3 μg/μL in SDS loading buffer, boiled for 5 minutes, and 60 μg of protein were separated using 10% SDS-PAGE gels. Transfer of protein onto nitrocellulose membranes, verification of transfer, and determination of equal loading between lanes and membranes was determined as outlined previously [β-actin by densitometry using a flatbed scanner (Epson Perfection 3200 PHOTO) and Imaging software (AlphaEaseFC). Molecular weight markers were used as molecular mass standards and NIH 3T3 cell lysates were included as positive controls. To allow direct comparisons to be made between the concentration levels of different signaling molecules, immunoblots were stripped and reprobed with Restore western blot stripping buffer as detailed by the manufacturer .Plantaris muscles were pulverized in liquid nitrogen using a mortar and pestle until a fine powder was obtained. After washing with ice cold PBS, pellets were lysed on ice in for 15 minutes in T-PER lysis buffer composed of T-PER reagent (2 mL/g tissue weight) , 1 mM ethylene-diamine tetraacetic acid , 1 mM ethylene-glycol tetraacetic acid , 1 mM magnesium chloride, phenylmethane sulfonylfluoride (PMSF), 1 eviously . ProteinP < .05.Results are presented as mean ± SEM. Data were analyzed by using the Sigma Stat 3.0 statistical program. The effects of insulin resistance on protein phosphorylation were analyzed using a two-way ANOVA followed by the Student-Newman-Keuls post-hoc testing when appropriate. Differences were considered significant at P < .05). Plantaris muscle mass in the obese Zucker rat expressed as a percentage of body weight was 35.9% less than that (0.41 ± 0.01 versus 1.14 ± 0.04 mg/gm) seen in the lean Zucker rat (P < .05). Average body mass of the obese Zucker rats was 82.0% higher (597 ± 21.7 gm versus 328 ± 12.2 gm) than their lean counterparts (β were ~10% and ~12% higher in the obese Zucker plantaris compared to muscles obtained from the lean Zucker (P < .05) and p70S6k (Thr421/Ser424) was 37.2% and 101.4% higher, respectively, in the obese Zucker plantaris (P < .05) (β (Ser 9), and PTEN (Ser 380/Thr 382/383) was 63.0%, 25.1%, 17.5% and 31.4% higher, respectively, with insulin resistance (P < .05) . There w < .05) . As p70S < .05) . Similar Figures .β in exercised plantaris muscles was determined at 0, 1, and 3 hours after a bout of HFES and compared with control (unstimulated) muscles. Contraction-induced phosphorylation of these molecules was compared between lean and obese Zucker rats. In the case of each molecule examined, significant differences existed between lean and obese Zucker rat models . In the obese Zucker, HFES increased p70S6k (Thr389) phosphorylation 79.9%, 43.6% and 104.5% and the phosphorylation of p70S6k (Thr 421/Ser 424) by 158.9%, 127.8%, and 238.6% at 0, 1, and 3 hours post exercise, respectively, (P < .05) (P < .05) (P < .05) . In the P < .05) whereas P < .05) . P < .05) (P < .05) by 59.9% and 40.8% immediately after and 1 hour post exercise while Akt (Ser473) was increased by 109.9%, 23.7%, and 105.9% at 0, 1, and 3 hours post exercise, respectively, (P < .05) . Convers < .05) . β (Ser 9) phosphorylation in the lean animals by 36.9%, 30.4%, and 23.4% at 0, 1, and 3 hours post exercise (P < .05) (β (Ser 9) phosphorylation did not change with contraction in the obese plantaris. In lean animals, PTEN (Ser380/Thr382/383) phosphorylation was increased 23.1% at 3 hours post exercise while conversely, in the obese Zucker animals, PTEN phosphorylation decreased by 18.3%, 23.0%, and 13.1% at 0, 1 and 3 hours post exercise, respectively (P < .05) . GSK-3β P < .05) .fa/fa) rat is a well-established animal model of skeletal muscle insulin resistance that is characterized by marked hyperinsulinemia, glucose intolerance, dyslipidemia, and central adiposity. Recent data has suggested that normal and insulin resistant muscle may differ in their adaptation to an exercise regimen [The obese Zucker of the p70S6k . ActivatSimilar to previous studies, increased contractile activity appears to be a strong stimulus to increase the phosphorylation level of mTOR in nondiabetic muscle , 38, howThe precise influence of insulin resistance on Akt regulation in muscle contraction remains unclear. Akt is a critical signaling mediator of cellular growth and metabolism in skeletal muscle , that isβ. Similar to our findings concerning Akt at Thr 308 phosphorylation, we failed to find any increase in GSK-3β (Ser 9) phosphorylation with HFES in the obese Zucker plantaris. The significance of this finding is currently not known, however it is thought that the Akt-dependent phosphorylation of GSK-3β leads to the activation of pathways promoting protein synthesis via a decreased inhibition of the translational initiation factor eIF2B [To explore Akt-related signaling further, we next investigated the regulation of Akt substrate GSK-3or eIF2B . Taken tor eIF2B . Given tIn summary, our data are consistent with the notion that the insulin resistance may alter contractile signal transduction pathways in skeletal muscle. Whether this finding alone is able to explain differences in how insulin resistant muscle respond to exercise regimen is not known and beyond the scope of this study. Likewise, whether the differences we observe between models is due to insulin resistance or to the existence of other abnormalities associated with the obese phenotype or other characteristics of the obese Zucker rat model is currently unclear. Given the multitude of factors thought to be involved in regulating how skeletal muscle “senses” and “responds” to increased loading stimuli it will
In the crystal structure, molecules are linked into a three-dimensional network by intermolecular O—H⋯O and N—H⋯O hydrogen bonds.In the title compound, C Å b = 10.0598 (12) Å c = 17.667 (2) Å β = 93.702 (2)°V = 1668.3 (3) Å3 Z = 4 Kα radiationMo −1 μ = 0.11 mmT = 298 K 0.23 × 0.20 × 0.20 mm Bruker APEXII CCD area-detector diffractometerSADABS; Sheldrick, 2004T min = 0.976, T max = 0.979Absorption correction: multi-scan (9554 measured reflections3606 independent reflectionsI > 2σ(I)2530 reflections with R int = 0.022 R[F 2 > 2σ(F 2)] = 0.041 wR(F 2) = 0.111 S = 1.05 3606 reflections239 parameters4 restraintsH atoms treated by a mixture of independent and constrained refinementmax = 0.14 e Å−3 Δρmin = −0.18 e Å−3 Δρ APEX2 used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008SHELXTL (Sheldrick, 2008SHELXTL.Data collection: 10.1107/S1600536809033236/ci2887sup1.cif Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809033236/ci2887Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:
In a previous study of a hippocampal neuron model, we demonstrated that its dynamics could be of both Type 1 and Type 2, depending on ion channel density. In the present study we analyse the effect of varying channel density on threshold firing frequency on two well-studied axon membranes, namely the frog myelinated axon and the squid giant axon. Moreover, we analyse the hippocampal neuron model in more detail. The models are all based on voltage-clamp studies, thus comprising experimentally measurable parameters. The choice of analysing effects of channel density modifications is due to their physiological and pharmacological relevance. We show, using bifurcation analysis, that both axon models display exclusively Type 2 dynamics, independently of ion channel density. Nevertheless, both models have a region in the channel-density plane characterized by an N-shaped steady-state current-voltage relationship . In summary, our results suggest that the hippocampal soma and the two axon membranes represent two distinct kinds of membranes; membranes with a channel-density dependent switching between Type 1 and 2 dynamics, and membranes with a channel-density independent dynamics. The difference between the two membrane types suggests functional differences, compatible with a more flexible role of the soma membrane than that of the axon membrane.The threshold firing frequency of a neuron is a characterizing feature of its dynamical behaviour, in turn determining its role in the oscillatory activity of the brain. Two main types of dynamics have been identified in brain neurons. Type 1 dynamics (regular spiking) shows a continuous relationship between frequency and stimulation current ( All activity of the brain is manifested in electrical oscillatory patterns, shaped by the firing dynamics of the many neurons forming the brain networks. The underlying mechanisms of the firing pattern in the single neurons are still not fully understood. The distribution and identity of different channel types have been suggested as critical factors. We have suggested that the density of channels in the membrane is a fundamental complementary mechanism. In a hippocampal soma membrane model study we have shown that altering the ion channel densities can cause the membrane to switch between two qualitatively different firing patterns. Here we extend the analysis to two axon membranes. Unexpectedly, both show that channel density alterations do not cause switches between different firing behaviours. We believe that this is an important property of axon membranes, explaining their limited flexibility. Carcinus maenasstimf-I relationship, whereas Class 2 axons start firing abruptly with a relatively high frequency at threshold, yielding a discontinuous stimf-I relationship.It is now more than 60 years since Alan Hodgkin categorized the firing behaviour in his classical study of isolated axons from the crab stimf-I relationships, respectively. This classification takes the threshold dynamics of the regular and fast spiking neurons, and that of the Class 1 and 2 axons, into account, but not all behavioural aspects of these classes On the basis of a similar categorization mammalian cortical neurons have also been separated into main classes The intricate interactions between the many factors involved in the dynamical regulation of neuronal firing are poorly understood stimI space and C1 (saddle node) is more complex than previously described. When more bifurcation parameters are changed (in our case channel densities and stimulation current) a more intricate loss of stability occurs (e.g. bifurcations with a co-dimension 2) Xenopus laevisLoligo forbesiThus, to obtain a better understanding of the processes we reanalysed the hippocampal neuron model in more detail. Furthermore, we extended the analysis to two other well-described excitable membranes, i.e., the myelinated axon of Rattus norvegicus. The myelinated axon model used is basically the same as that developed by Frankenhaeuser and Huxley Xenopus laevis) Loligo forbesi.The three membrane models analysed here are all based on voltage-clamp data, and on the formalism originally developed by Hodgkin and Huxley CI) and a three-component ionic current (ionicI), consisting of a Na current (NaI), a delayed rectifier K current (KI), and a leak current (leakI). It should be noted that in all three models the description of the K currents are based on experimentally measured currents, which cannot be regarded as homogeneous, but are most likely the sum of currents passing through several types of voltage-gated K channels. The descriptions of the currents differ slightly between the three membrane types. For instance, the Na and K currents of the squid axon are described using the conductance concept, while the corresponding currents of the myelinated axon and the hippocampal somatic membrane use the permeability concept, developed by Goldman All the models assume that the membrane current consists of a capacitive current , charging the capacitor, and the ionic current ionicI. Thus,MC is the membrane capacitance. To obtain the time-course of V(t), we solve this differential equation numerically, using the expressions for ionicI presented in the section of sV), i.e. the potentials at which the system is in a stationary state and, consequently, the time derivatives of all variables are zero. This was done by solving the following equation for different values of stimI, ssI is ionicI at steady state. The stability of the system in the neighbourhood of the stationary potentials was examined by a linearization procedure as described in ss-VI curve for the model to produce Type 1 dynamics when entering the oscillatory regime. This is due to the nature of a saddle-node bifurcation on an invariant circle (SNIC), requiring an stimI interval at which Equation 2 yields three solutions , with area C1 representing the subregion associated with oscillations. As mentioned above, most of this region is associated with Type 1 threshold dynamics. A more detailed analysis reveals, however, that for density values close to the border of the B area the model demonstrates Type 2 dynamics (s1V) becomes unstable as indicated in the bifurcation diagram of In our previous examination of the hippocampal model see , we defidynamics . This caIt should be noted that in addition to the C1b region, there is another C1 subregion, a narrow strip along the borders to the B, A1 and C2 regions for ss-VI curves (region C1b) becomes evident when the corresponding stimI−ss-VI region), with thick continuous lines. The thin continuous line marks points associated with Andronov-Hopf bifurcation dynamics and the hatched line depicts the double-limit cycle bifurcation. The Andronov-Hopf bifurcation line intersects the three-solution region and collides with the saddle-node bifurcation line in a Bodganov-Takens bifurcation point s1V and s3V. Thus, at these permeabilities and stimulation currents the threshold dynamics is due to subcritical Andronov-Hopf bifurcations and not to saddle-node bifurcations, and the model will show typical Type 2 behaviour with a minimum non-zero threshold frequency.The explanation for the deviant (i.e. Type 2) threshold dynamics in the region associated with non-monotonic Xenopus leavisLoligo forbesiHow general is our present description of neuronal models? And how does the density of channels influence the threshold dynamics and firing patterns in other models? To address these issues, two well-described excitable membranes, i.e., the node in myelinated axons of stimI−ss-VI relationship. However, unlike the narrow Andronov-Hopf region C1b of the hippocampal neuron model, the corresponding region of the myelinated axon model covers the whole C1 area. Consequently, saddle-node bifurcation dynamics is missing, explaining the absence of Type 1 dynamics in the myelinated axon model. It can also be noted that the double-limit cycle bifurcation occurs very close to the Andronov-Hopf bifurcation, why great care is needed to distinguish them numerically. That this kind of change can occur very close to each other in a parameter space is a known feature in these kinds of models A stability analysis revealed the mechanisms involved. The findings suggest less dynamic flexibility in this axon membrane than in the hippocampal neuron model discussed above. Since the hippocampal neuron model is based on measurements of the soma membrane properties, the comparison between the myelinated axon and the hippocampal model mainly concerns a comparison between axonal and soma membranes. To get further information about the functional relevance of the found differences between axon and soma membranes we next analysed the classical squid giant axon membrane, using the description given by Hodgkin and Huxley Calculations showed that the giant squid axon model in similarity with the myelinated axon did not show saddle-node or Type 1 dynamics . In contaxon see . The firstimI−The reason for the absence of saddle-node dynamics is evident in the Carcinus maenasThe manner in which interactions between the ionic currents in a neuron determine the pattern and dynamics of firing is a multifaceted problem, having many ramifications within theoretical systems biology In the present analysis we show that corresponding channel density alterations in two well-studied axon models (the amphibian myelinated axon and the squid giant axon) cannot change the firing dynamics, exclusively being of Type 2. This suggests that the hippocampal soma and the two axon membranes represent two distinct types of membrane with respect to the excitability pattern; one more flexible that can switch channel-density dependently between Type 1 and Type 2 dynamics and one, less flexible, that exclusively shows Type 2 dynamics (represented by the membranes of the two axons and here denoted M2).ss-VI relationship. In the present analysis we show that this requirement is not sufficient. In all three models we find regions in the ss-VI relationship, that are associated with Type 2 dynamics (C1b regions). In the hippocampal neuron model the region consists of a narrow band, and in the axon models they cover the whole C1 region, and thus, the axonal models lack Type 1 threshold dynamics ? Type 1 and 2 dynamics per se most likely have different functional roles; Type 1 dynamics being required for low frequency firing and Type 2 being essential for doublet spiking induced switchability between Type 1 and Type 2 dynamics see e.g. . Such flIn contrast to the flexible or plastic soma membrane, the axon membranes form passive information transport chains, requiring reliable triggering mechanisms and, therefore, Type 2 dynamics. It should be pointed out, however, that the main value of the axon type dynamics most likely relates to its action when associated with a trigger zone, which likely is the case of the distal process of the dorsal root ganglion. Features associated with Type 2 dynamics, such as subthreshold oscillations and doublet spiking have been postulated to play an important role in pain modulation Xenopus laevis) and one cephalopod (Loligo forbesi), cannot be generalized to axons from all animal phyla. As mentioned above, certain axons from the arthropod Carcinus maenas display Type 1 dynamics Clearly, this discussion, based on an analysis of axons from one amphibian and Type 2 dynamics (fast spiking), with Type 1 in majority Contrary to the majority of pyramidal cells, mammalian cortical interneurons mainly display Type 2 dynamics (fast spiking). This suggests that the membranes of their trigger zones are either of M1/2 or of M2 type. In the latter case the trigger zone could be assumed to be located in the axon proper; i.e. in the first node of Ranvier or in an initial segment that is more functionally axon-like than that of the pyramidal cells. A way to experimentally separate between these two hypotheses (whether the trigger zone in interneurons is of M1/2 or M2 type) could be to analyse the dynamics after blocking K channels. Such a test is also under way.As pointed out previously V, m, h, n) may then take place on a two-dimensional surface in the four-dimensional variable space. Hence, a model with reduction of variables was calculated by solving the following equation (derived from Equation 1) numerically:For the squid axon model we use the original expressions by Hodgkin and Huxley m, h and n) are in all three models described by their time derivatives:iα and iβ denote rate functions. For the hippocampal neuron model they are defined as follows:The activation and inactivation parameters at steady state together with Equation 3. The time derivates of the gating parameters are zero at stationary potentials, and hence the stationary values of the parameters (denoted ∞m etc) become:sV):sV. However, if the Na channel density ,h∞(V),n∞(V)V,m), when the stimulation current (stimI) and the permeability or conductance parameters yielding an approximate time evolution of the system. Hence any perturbation δr around the equilibrium point r* can be written asic depends on initial conditions and ir is the associated eigenvector. Two of the eigenvalues are in the present system always real and negative. Consequently the remaining two eigenvalues determine the character of the sV , one can rewrite the equation, using Euler's formula, asb/2π correlates with the firing frequency.If Mathematica 6.0.2 on a 64-bit IBM compatible computer. All values are given in SI-units.All computations were done in custom software written in Figure S1ss-VI relationship is non-monotonic (N-shaped) for values of higher than 166 µm/s.Firing frequency as a function of Na channel density in a rat axon model. The firing frequency at threshold is plotted against Na permeability constant. For Na permeabilities less than 59µm/s the axon model does not show any repetitive firing. The curve is calculated from the equations described in (0.54 MB EPS)Click here for additional data file.Figure S22. The search was performed using custom written software in Mathematica 6.0.2. Out of about 100.000 trials, eight parameter combinations yielded chaos like firing. When a similar search, equally extensive , was performed with an hippocampal model comprising only one extra channel (Na or K), no positive results were found. The results seem to suggest that at least four channels (two Na and two K channels), and thus a seven-dimensional system, are required to give neuron models of the type studied here chaotic dynamics.Chaotic firing in a modified hippocampal neuron model. Time course and phase-plot of a modified hippocampal neuron model, including an extra Na and an extra K channel. The figure depicts data from an extensive search for parameter values that give the model chaos like dynamics . The extra channes were assumed to be described by the same kinetics as the hippocampal channels, i.e. by equations 4, 5 and 6 in the (1.63 MB EPS)Click here for additional data file.Text S1A model of the myelinated rat axon(0.07 MB PDF)Click here for additional data file.
R) in standardized liquid chromatography–UV–mass spectrometry of 67 mycotoxins based on molecular descriptors calculated from the optimized 3D structures. By applying missing value, zero and multicollinearity tests with a cutoff value of 0.95, and genetic algorithm method of variable selection, the most relevant descriptors were selected to build QSPR models. MLR and SVMs methods were employed to build QSPR models. The robustness of the QSPR models was characterized by the statistical validation and applicability domain (AD). The prediction results from the MLR and SVM models are in good agreement with the experimental values. The correlation and predictability measure by r2 and q2 are 0.931 and 0.932, repectively, for SVM and 0.923 and 0.915, respectively, for MLR. The applicability domain of the model was investigated using William’s plot. The effects of different descriptors on the retention times are described.In the present work, support vector machines (SVMs) and multiple linear regression (MLR) techniques were used for quantitative structure–property relationship (QSPR) studies of retention time (t Growth of fungi on animal hosts produces diseases collectively known as mycoses, while dietary, respiratory, dermal, and other exposures to toxic fungal metabolites produce diseases collectively called mycotoxicoses. Mycotoxicoses are examples of “poisoning by natural means” and thus are analogous to the pathologies caused by exposure to pesticides or heavy metal residues. The symptoms of mycotoxicosis depend on the type of mycotoxin; the amount and duration of the exposure; the age, health, and sex of the exposed individual; and many poorly understood synergistic effects involving genetics, dietary status, and interactions with other toxic insults. Thus, the severity of mycotoxin poisoning can be compounded by factors such as vitamin deficiency, caloric deprivation, alcohol abuse, and infectious disease status. In turn, mycotoxicoses can heighten vulnerability to microbial diseases, worsen the effects of malnutrition, and interact synergistically with other toxins mass spectrometry (MS). Nearly all methods use water–acetonitrile gradient elution on reversed-phase Cescribed .However, only a few reports have investigated the quantitative correlation between the molecular parameters and the property of retention time of mycotoxins . The comAfter the calculation of molecular descriptors, many different chemometrics methods, such as multiple linear regression (MLR), partial least squares regression (PLS), different types of artificial neural networks (ANN), genetic algorithms (GAs), and support vector machine (SVM) can be employed to derive correlation models between the molecular structures and properties. As a new and powerful modeling tool, support vector machine (SVM) has gained much interest in pattern recognition and function approximation applications recently. In bioinformatics, SVMs have been successfully used to solve classification and correlation problems. SVMs have also been applied in chemistry, for example, the prediction of retention index of protein , and oth54 descriptors were calculated by the ChemOffice software. By applying missing value, zeroand multicollinearity tests with a cutoff value of 0.95 and variable selection by genetic algorithm, the number of descriptors was reduced to 22. The stepwise regression routine was used to develop the linear model for the prediction of the retention time of mycotoxins using calculated structural descriptors. The best linear model contained four molecular descriptors. The regression coefficients of the descriptors, Mean effect and variable inflation factors (VIF) are listed in R, whereas negative values indicate that the greater the value of the descriptor, the lower the value of tR. In other words, increasing the electronic energy (ElcE), dipole length (DPLL)and Lowest Unoccupied Molecular Orbital energy (LUMO) will decrease tR, and the increase in the C logP increases the extent of tR of the compounds.Positive values in the regression coefficients show that the indicated descriptors contribute positively to the value of tR of the compound. The mean effect of a descriptor is the product of its mean and the regression coefficient in the MLR model and [r2 − r02′/r2] are less than 0.1 [r02 and r02′ are correlation coefficient of regression between the predicted and experimental property of compounds in the test set and vice versa without using y-intercept.where and 1.15 . The valed value). r02 and2, where r2 is the multiplecorrelation coefficient of one descriptor’s effect regressed on the remaining molecular descriptors. If the VIF value is larger than 10, information of the descriptor could be hidden by correlation of descriptors [To further check the inter-correlation of descriptors variance inflation factor (VIF) analysis was performed. The VIF value is calculated from 1/1 − rcriptors .R of a diverse set of mycotoxins from the molecular structure alone. We have compared two linear models, MLR and SVM, with the data set. The obtained results show that both MLR and SVM methods could model the relationship between tR and their electronic and thermodynamic descriptors; on the same sets of descriptors, using SVM based produced a better model with a better predictive ability than the MLR model. SVM exhibit the better overall performance due to embodying the structural risk minimization principle and some advantages over the other techniques of converging to the global optimum and not to a local optimum. By performing model validation, it can be concluded that the presented model is a valid model and can be effectively used to predict the tR of mycotoxins with an accuracy approximating the accuracy of experimental tR determination. Moreover, the mechanism of the model was interpreted, and the applicability domain of the model was defined. It can be reasonably concluded that the proposed model would be expected to predict tR for new organic compounds or for other organic compounds for which experimental values are unknown. Additionally, the presented method could also identify and provide some insight into what structural features are related to the tR property of organic compounds.In recent years, attention has been paid to QSAR/QSPR methods as an interesting complement, or even as an expensive, time consuming alternative, to laboratory data. In this paper, new QSPR models have been developed for predicting the t
Subcellular localization of Kaiso in cell lines and in normal and cancerous human tissues revealed that its expression is not restricted to the nucleus. In the present study we monitored Kaiso's subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody. We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular complex. We analyzed the functions of different domains of Kaiso by visualizing the subcellular distribution of GFP-tagged Kaiso fragments throughout the cell cycle. Our results indicate that two domains are responsible for targeting Kaiso to the centrosomes and microtubules. The first domain, designated SA1 for spindle-associated domain 1, is located in the center of the Kaiso protein and localizes at the spindle microtubules and centrosomes; the second domain, SA2, is an evolutionarily conserved domain situated just before the zinc finger domain and might be responsible for localizing Kaiso towards the centrosomal region. Constructs containing both SA domains and Kaiso's aminoterminal BTB/POZ domain triggered the formation of abnormal centrosomes. We also observed that overexpression of longer or full-length Kaiso constructs led to mitotic cell arrest and frequent cell death. Knockdown of Kaiso accelerated cell proliferation. Our data reveal a new target for Kaiso at the centrosomes and spindle microtubules during mitosis. They also strongly imply that Kaiso's function as a transcriptional regulator might be linked to the control of the cell cycle and to cell proliferation in cancer.Kaiso is a BTB/POZ zinc finger protein known as a transcriptional repressor. It was originally identified through its Notably, Kaiso was also described as a transcriptional activator of the human and mouse Rapsyn promoter Kaiso is a member of the BTB/POZ protein family The role of Kaiso in embryogenesis is still unclear. Kim et al. in vitroHelicobacter pylori infection counteracts Kaiso's repression of the tumor initiating protein matrix metalloproteinase (MMP7) mmp-7 (Matrylisin) in colonic epithelial cells CDKN2A in colon cancer, and in this way grants cancer cells a survival advantage Kaiso's importance in cancer development has also not been fully elucidated. Kaiso's discovery as a nuclear DNA-associating protein and as a binding partner of the well described membrane protein p120ctn inspired researchers to speculate that a Kaiso-p120ctn complex might play an important role in cancer Most researchers try to elucidate a role for Kaiso in the nucleus. However, this protein is often not detected in the nuclei of different human tissues, dense cell cultures, and three-dimensional cultures In this study we strengthened the idea of Kaiso's possible importance in cell dynamics, particularly during cell division. Using different specific antibodies and also different experimental conditions, we showed that Kaiso localizes to cytoplasmic microtubules and centrosomes during interphase and concentrates at the centrosomes and spindle microtubules during mitosis. The spindle is essential for normal cell division in eukaryotic organisms and is composed of a highly dynamic array of microtubules nucleating from two centrosomes and extending to the chromosomes We demonstrate here the rather unexpected presence of Kaiso at microtubules and centrosomes, mediated by two conserved domains within the protein. We show an association of Kaiso with the PCM constituent pericentrin. Overexpression of some GFP-Kaiso fragments caused abnormal centrosomes and even cell death. Knockdown of Kaiso accelerated cell proliferation. Our present findings reveal that Kaiso is a new component of the centrosomes and the mitotic spindle, where it might play a role in centrosomal duplication or segregation, cell cycle control, and/or microtubule stabilization.Cell lines of human origin were HT29, SW48, SK-LMS-1, MCF-7, MCF-10A, HEK293, HeLa, and MDA-MB-435. Cell lines originating from other species were Ptk-2, L929, HL-1 and MDCK cells. MCF-7 cells were obtained from Prof. Dr. M. Mareel . Other cell lines were purchased from the American Cell Type Culture collection , with the exception of MCF-10A and HL-1. MCF-10A cells were provided by Dr. J. Brugge and grown in a specially supplemented medium as described Kaiso was detected by using monoclonal antibodies 6F, 12H, 2G and 12G, as well as a rabbit polyclonal antibody (pAb R) raised against a purified 6x-histidine-tagged Kaiso fragment consisting of amino acids (AA) 1–499 Different GFP-tagged Kaiso fragments were constructed by the Gateway cloning technology (Invitrogen). Starting from the eukaryotic expression plasmid pcDNA3-mKaiso , we performed PCRs with primers containing attB1 and attB2 sequences in order to introduce the Kaiso fragments in the entry vector pDONR207 (Invitrogen). We used the following mouse Kaiso fragments cloned in a derivative of pEGFP-C2 we adapted for Gateway cloning: K1 (aa 1–158), K2 (aa 12–175), K3 (aa 35–501), K4 (aa 64–317), K4x (aa 64–207), K4y (aa 64–225), K4z (aa 195–317), K4a (aa 213–264), K4b (aa 213–287), K4v (aa 213–317), K5 (aa 1–671), K6 (aa 86–490), K7 (aa 1–501), K8 (aa 115–509), and K9 (aa 343–501).HeLa and HEK293 cells were synchronized by a double thymidine block Cells were rinsed briefly with PBS and fixed either with 4% paraformaldehyde in PBS for 25 min at room temperature, or with methanol for 15 min at −20°C . Immunostaining was performed as described Cells used in time-lapse microscopy were grown on an eight-well Lab Tek II chamber glass slide . Time-lapse microscopy of synchronized transfected cells was carried out using the Leica AS MDW live cell imaging system (Leica Microsystems). This system includes a DM IRE2 microscope equipped with an HCX PL APO 63x/1.3 glycerin-corrected 37°C objective and a 12-bit Coolsnap HQ Camera. The microscope is surrounded by an incubation chamber maintained at 37°C. One hour before imaging, the culture medium was replaced with L15 phosphate buffered medium (Invitrogen), and the cells were placed on the microscope stage to prevent focus drift due to differences in temperature between the Lab Tek chamber and the objective.DIC and fluorescence images were obtained every 3 min for 5.5 hours. The GFP fluorescence images were monitored by using a monochromator system with a 75-W Xenon lamp for excitation, in combination with a standard B/G/R filter cube. Images were processed into movies using the Leica AS MDW software.6 cells were transfected with 2 µg pEGFP-HsCentrin-1 plasmid HeLa cells were stably transfected by using Amaxa Nucleofector Technology . 1×10GAATTCGAACGCTGACGTCATCAA-3′5′-CTGCAG and as reverse primer TCTCTTGAATTTAGTAAGACTCTGGTATGGGGATCTGTGGTCTCATACAGAACTTATAA-3′5′-AAA. The Kaiso target sequence is indicated in bold; part of the H1 promoter is underlined; the loop sequence is indicated in italics. A thousand-fold dilution of this PCR product was used as a template for a second PCR reaction, with the same forward primer and as a reverse primer ATCGATTTCCAAAAAATACCAGAGTCTTACTAAATCTCTTGAATTTA-3′5′-CC . The PCR program consisted of initial denaturation at 94°C for 3 min, followed by 20 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 55 sec, elongation at 72°C for 55 sec, and a final elongation at 72°C for 7 min. This product was subjected to agarose gel electrophoresis, followed by gel extraction (Qiagen), EcoRI/ClaI digestion, purification (Qiagen) and ligation into EcoRI/ClaI digested, dephosphorylated pLVTH vector . After transformation of competent DH5α bacteria with the ligation product, ampicillin-resistant colonies were screened by PCR using primers complementary to sequences of the pLVTH vector surrounding the insert. All constructs were verified by sequencing. They encode transcripts comprising Kaiso-specific shRNA and GFP mRNA. Production of lentiviral particles and transduction of SK-LMS-1 cells was essentially as described before For stable knockdown of Kaiso by shRNA, target sequences were cloned in the vector pLV-TH 5′-AAGCTTTATCGTTTACATCCAT-3′ and reverse primer 5′-ATACCCAATACCATCATCCTT-3′. Kaiso was normalized to three housekeeping genes: hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase I (HPRT) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ).RNA was isolated using the RNeasy mini kit (Qiagen) and quality (260/280 nm ratio) and quantity were determined using a Nanodrop® ND-1000 system . For analysis, 400 ng of total RNA was subjected to complimentary DNA (cDNA) synthesis using the iScript cDNA synthesis kit in a volume of 20 µl. Quantitative PCR was performed using the LightCycler 480 system (Applied Biosystems) for 10 ng of complementary DNA with SYBR Green Master mix (Applied Biosystems). For expression of Kaiso we used forward primer Cells were seeded at 5000 cells/well in 96-well plates and grown for 1 to 14 days. Each test was performed in six replicates. Briefly, cells were fixed by incubation with 50% trichloroacetic acid at 4°C for 1 h or overnight followed by 5 washes with distilled water. Cells were stained by the addition of 1% acetic acid containing 0.4% (w/v) SRB (Sigma) solution to the culture medium at room temperature; after 30 min, plates were washed with 1% acetic acid and air-dried. After the addition of 10 mM Tris-HCl, pH 10.5, to dissolve the SRB bound to cellular proteins, absorbance at 595 nm, proportional to the number of cells attached to the culture plate, was measured by spectrophotometry.6 cells were labeled using the PKH26 Red Fluorescent Cell Linker Kit according to the manufacturer's instructions. Duplicates of labeled cells were plated in six-well plates at a dilution of 1/2.5 and harvested 24 and 48 h later for analysis. After detachment, washing and counting, cells were stained with 20 µg Hoechst33342 (Sigma-Aldrich)/ml for 60 min at 37°C, and with 2 nM SytoxRed for 10 min at 37°C, after which the cells were analyzed. Dead cells were excluded on the basis of SytoxRed positivity. Transduced cells with strong shKAISO expression were distinguished from less efficiently transduced cells on the basis of GFP expression. Cell proliferation in GFP+ and GFP–cells was determined by calculating the mean fluorescence intensity (MFI) ratio for PKH26 at 24 h and 48 h.Proliferation rate and cell cycle status were analyzed for SK-LMS-1_shKAISO cells by monitoring equal dilutions of fluorescently labeled membrane lipids in dividing cells and nuclear Hoechst33342 staining, using a triple-laser LSR-II flow cytometer and FACSDiva software for analysis . 2×10For co-immunoprecipitation of endogenous proteins, cell lysates were prepared in a CoIP solubilization buffer containing 145 mM NaCl, 5 mM EDTA, 2 mM EGTA, 10 mM Tris-HCl pH 7.4, 1% Triton X-100, and a protease inhibitor cocktail (Roche). Lysates were centrifuged and a sample containing 1.25 mg protein was precleared with 25 µl Dynabeads protein G (Invitrogen), and incubated for 4 h or overnight with 3 µg of antibody S1337. 50 µl Dynabeads protein G were then added for 1 h, after which the beads were washed five times with the DynaMag-2 and CoIP solubilization buffer. Washed beads were combined with sample loading buffer and boiled for 5 min. Samples were analyzed by SDS-PAGE and western blotting. Proteins were visualized using the ECL detection system (Amersham).The subcellular distribution of Kaiso was followed throughout the cell cycle by immunostaining. Human cell lines SK-LMS-1 and HEK293 were studied with the six Kaiso antibodies described in “Evidence for specific association of Kaiso with the centrosomes was strengthened by the following observations. Confocal fluorescence images were obtained for HeLa cells expressing GFP-centrin during G2/M and metaphase. A Kaiso-specific signal was invariably observed in close proximity of the tiny GFP-centrin spots in the centrosomes . CentrinTo further confirm our data obtained by the double stainings above and to determine which domains of Kaiso are required for targeting to microtubules and centrosomes, we carried out expression and functional analysis of several GFP-tagged Kaiso fragments . We examDuring interphase, cells transfected with plasmids encoding GFP-tagged K3, K6, K7, K8 or K9 fragments, or full-length Kaiso (K5), displayed strong Kaiso expression in the nucleus. This is exemplified in the upper row of At mitosis, the GFP-tagged protein encoded by the full-length Kaiso construct was distributed at the spindle material. Although we do not know which amino acid residues of Kaiso are physically associated with the spindle material, we designated the sequence AA 213–264 as the SA1-domain (for spindle-associated domain 1), and AA 343–501 as the SA2-domain . In summhttp://www.dmbr.ugent.be/ext/public/publications/Soubry_2009/GFP_FL-Kaiso.avi) as a Suppl. Time-lapse microscopy was used to follow HEK293 cells transfected with full-length GFP-tagged Kaiso (construct K5) after cell cycle synchronization. Premitotic cell cycle arrest was observed in 95% of the cells. Indeed, mitotic cells were rarely observed and high (hi) expression of GFP were resorted, yielding cell populations with robust (labeled +) or weak (labeled –) Kaiso knockdown. For instance, strongly reduced Kaiso mRNA and KaisWe then addressed the functional implications of Kaiso knockdown. Abnormalities in mitotic spindle formation, centrosomal division or PCM composition appeared not to be increased by the knockdown (data not shown). However, in a cell proliferation assay, cell populations with efficient Kaiso knockdown showed significantly increased growth rate and saturation density . It was in vivo is very limited In this study we found a novel localization for the transcription factor Kaiso, suggestive of a new function. We examined the expression patterns of the Kaiso protein and several fragments thereof during the cell cycle and found that Kaiso is present not only in the nucleus, but also at the centrosomes and the mitotic spindle. Localization of Kaiso outside the nucleus of cultivated cells has been referred to only very briefly Immunofluorescent colocalization revealed that Kaiso is less focally expressed in the centrosomes than centrin. Evidence for association of Kaiso with the PCM was strengthened by our finding of pericentrin and ©-tubulin in a molecular complex with Kaiso. PCM is a structurally intricate protein complex consisting of a lattice of large coiled coil proteins, including pericentrin and AKAP-450 e.g. K4a, K4b, K4v and K4z) concentrated at the centrosomes, spindle microtubuli and midbody of mitotic cells. In contrast, fragments such as K4, containing also CD1, a conserved acidic region just before SA1, did not localize at the centrosomes. We assume that in some protein constructs CD1 causes conformational changes in such a way that SA1 is kept off the centrosomes. SA2-containing fragments showed remarkably strong expression at the centrosomes during mitosis (e.g. K9), but not at the spindle microtubules. The differences seen between constructs containing SA1 and SA2 might indicate that they bind differently to the mitotic spindle components: either transported along the microtubules by motor proteins (in case of SA1), or by binding directly to centrosomal components (in case of SA2). A hypothetical model of the binding mechanisms and possible functions of Kaiso in mitosis is presented in From our transient transfection experiments we identified at least two domains within Kaiso that appear to be responsible for its localization at the mitotic spindle and/or centrosomes, and designated them SA1 and SA2 (for spindle-associated-1 and -2). We also observed that transfection with particular constructs can result in abnormal centrosome shapes and aberrant mitotic cleavage. Kaiso fragments containing the SA1 domain are insulator zinc finger proteins and mutual binding partners, associating with chromatin during interphase and with centrosomes in mitotic cells and other vertebrates . RefSeq sequences were obtained from the Entrez Gene database (0.18 MB PDF)Click here for additional data file.Figure S2The GFP-tagged Kaiso fragment K4x causes formation of protrusions during interphase. HEK293 cells are transfected with construct GFP-K4x. DNA was stained with DAPI, and cells were imaged with a Zeiss Axiophot microscope (100× objective lens).(0.18 MB PDF)Click here for additional data file.Figure S3The GFP-tagged Kaiso fragment K4z localizes at spindle material during mitosis. During metaphase (upper row) and anaphase (lower row) of transfected HEK293 cells, the Kaiso fragment K4z concentrates at the centrosomes (arrowheads) and the minus ends of the spindle microtubules (arrows). DNA was stained with DAPI and cells were imaged with a Leica DM IRE2 microscope (63× objective lens).(1.59 MB PDF)Click here for additional data file.Figure S4The GFP-tagged Kaiso fragments K4a and K4b localize at the spindle material during mitosis. In transfected HEK293 cells the K4a (upper row) and K4b (lower row) Kaiso fragments both localize at the spindle microtubules (arrows) and at the centrosomes (arrowheads). DNA was stained with DAPI and cells were imaged with a Zeiss Axiophot microscope (100× objective lens).(0.70 MB PDF)Click here for additional data file.Movie S1(3.41 MB AVI)Click here for additional data file.
While high-dimensional molecular data such as microarray gene expression data have been used for disease outcome prediction or diagnosis purposes for about ten years in biomedical research, the question of the additional predictive value of such data given that classical predictors are already available has long been under-considered in the bioinformatics literature.We suggest an intuitive permutation-based testing procedure for assessing the additional predictive value of high-dimensional molecular data. Our method combines two well-known statistical tools: logistic regression and boosting regression. We give clear advice for the choice of the only method parameter (the number of boosting iterations). In simulations, our novel approach is found to have very good power in different settings, e.g. few strong predictors or many weak predictors. For illustrative purpose, it is applied to the two publicly available cancer data sets.Our simple and computationally efficient approach can be used to globally assess the additional predictive power of a large number of candidate predictors given that a few clinical covariates or a known prognostic index are already available. It is implemented in the R package "globalboosttest" which is publicly available from R-forge and will be sent to the CRAN as soon as possible. While high-dimensional molecular data such as microarray gene expression data have been used for disease outcome prediction or diagnosis purposes for about ten years in biomeThis issue can be summarized as follows. For a given prediction problem (for example tumor subtype diagnosis or long-term outcome prediction), we consider two types of predictors. On the one hand, conventional clinical covariates such as, e.g. age, sex, disease duration or tumor stage are available as potential predictors. They have often been extensively investigated and validated in previous studies. On the other hand, we have molecular predictors which are generally much more difficult to measure and collect than conventional clinical predictors, and not yet well-established. In the context of translational biomedical research, investigators are interested in the additional predictive value of such predictors over classical clinical covariates.A particular challenge from the statistical point of view is that these molecular predictors are often high-dimensional, which potentially leads to overfitting problems and overoptimistic conclusions on their additional predictive power ,3. The qThe formulation "additional predictive value compared to classical clinical predictors" is ambiguous because it actually encompasses two distinct scenarii. In the first scenario, the prediction model based on clinical covariates is given (for instance from a previous publication) and can be directly applied to the considered data set. Such models are usually denoted as "risk score" or "index" in the medical literature and often use a very small number of predictors, such that they are widely applicable in further studies. However, clinicians often want to develop their own clinical score using their own data (second scenario) because it is expected to yield higher accuracy for their particular patient collective, or because they want to predict a different outcome or use different predictors. These two scenarii are different from the statistical point of view: in the first scenario the prediction rule based on clinical covariates is fixed, while it has to be constructed from the data in the second scenario.In this article, we present a method for testing the additional predictive value of high-dimensional data that fulfills the following prerequisites:Prerequisite 1: The additional predictive value is assessed within a hypothesis testing framework where the null hypothesis corresponds to "no additional predictive value".• Prerequisite 2: The focus is on the additional predictive value, i.e. the model selection procedure for the high-dimensional data takes the clinical covariates into account.• Prerequisite 3: The method can address the two scenarii described above .• not to construct a combined prediction rule based on clinical and high-dimensional data: the focus is on the testing aspect.Note that our aim is In the last few years, a couple of methods fulfilling one of these three prerequisites have been proposed to handle this problem. In the context of class prediction, the pre-validation procedure proposed by Efron and Tibshirani ,5 consisτ2. The null-hypothesis that all regression coefficients are zero can then be reformulated as τ2 = 0. In their second paper on this subject, the same authors suggest a variant of this test that adjusts for additional covariates in the context of survival analysis [(see 'Methods' Section for an explanation of the notation) is multiplied by a small shrinkage factor ν. Instead, CoxBoost does not multiply by ν but penalizes the update through a penalty matrix in the loss function. Like the CoxBoost approach, our method fulfills prerequisite 2. To address prerequisite 1, we suggest a simple permutation-based testing procedure. The resulting novel approach thus fulfills the two first prerequisites. Moreover, we suggest a variant for addressing the application of a risk score fitted previously using other data (prerequisite 3).Motivated by the strong advantages of the CoxBoost approach, we suggest an alternative simple two-stage approach which also uses a boosting algorithm, but in a different scheme which is more appropriate for the testing purposes considered here. Our approach combines a standard generalized linear model for modeling the clinical covariates (step 1) with a boosting algorithm for modeling the additional predictive value of high-dimensional molecular data (step 2). The differences between our approach and the CoxBoost approach are as fIn the next section, we briefly review the methods involved in the first step (logistic regression) and second step (boosting with componentwise linear least squares), and we describe the combined two-step procedure as well as the permutation test.Z1,..., Zq)⊤ with n independent realizations zi = ⊤, for i = 1,..., n. Similarly, the random vector of molecular covariates is denoted as ⊤ (with p >n) with n realizations xi = ⊤, for i = 1,..., n. The response variable is denoted as Y and coded as Y ∈ {-1, 1}, with realizations y1,..., yn.In the following, we consider a random vector of clinical covariates ⊤, one obtains the so-called linear predictor aswhere from which the predicted probability X1,..., Xp. The AdaBoost algorithm was originally developed by Freund and Schapire as a machine learning tool, see [In this section, we give a short general overview of boosting as reviewed by Bühlmann and Hothorn , and expool, see for an eool, see then devρ(·) is a loss function which will be discussed in this section. Friedman, Hastie and Tibshirani [f(·) as sketched below [where bshirani formulated below .m = 0.1. Initialize m by 1. Compute the negative gradient ρ and evaluate it at xi), for each observation i = 1,..., n:2. Increase u1,..., un to x1,...,xn using a so-called base procedure (which will be discussed later in this section):3. Fit the ν ≤ 1 is a step-length factor (see below), that is, proceed along an estimate of the negative gradient vector.4. Update m = mstop for some stopping iteration mstop.5. Iterate steps 2 to 4 until Note that the offset term is simply the best constant model (without taking the covariates into account) and, therefore, the algorithm starts at the center of the unconditional distribution of the response for fitting the conditional distributions.Y is binary), it is usual to use the so-called log-likelihood loss functionIn the context of binary class prediction and boosting (step 2)'.Meanwhile, componentwise linear least squares can be considered as one of the standard base procedures for boosting. We choose it as a base procedure for the second step of our two-stage analysis scheme. A major advantage of componentwise linear least squares as a base procedure in the context of our two-stage approach is that the final estimated function In this section, we show how logistic regression and boosting as described in the two above sections can be combined into a two-step procedure. We first present the procedure for the case when the model with clinical covariates has to be estimated from the data and then address the other scenario (application of a fixed risk score known from a previous study).Z1,..., Zq, yielding estimates 1.1 Fit a logistic regression model as outlined in the Section 'Logistic regression' to the clinical covariates i = 1,..., n.1.2 Compute the linear predictor mstop, which is discussed in the Section 'The choice of mstop'.This step involves one method parameter, the number of boosting iterations ρlog-lik and componentwise linear least squares as a base procedure with mstop boosting iterations, as implemented in the R package mboost [X1,..., Xp. Note that, in practice, many of these coefficients are zero.2.1 Define the offset function e mboost ,18. Deri2.2 Compute the resulting linear predictor as2.3 Compute the predicted probabilities from the linear predictor as A small negative binomial log-likelihood indicates good model fit. Note that we could have used another goodness criterion in place of the negative binomial log-likelihood. However, the binomial log-likelihood is especially appropriate, since it is the criterion optimized by the boosting procedure. To assess the additional predictive value of the molecular data, we suggest to compare ℓ to the negative binomial log-likelihood obtained from permuted data, as outlined in the Section 'Permutation-based testing procedure'.In the situation where a risk score is already available (e.g. from a previous publication), step 1 can be skipped. The linear predictor Alternatively, our method can also be used to globally assess the molecular variables independently of any clinical covariates. This would be done by ignoring the first step (logistic regression) of our method and simply setting the offset to the value of the intercept.X1,..., Xp have no additional predictive power given the clinical covariates. The considered model is given asWe consider the null-hypothesis that the variables and the null-hypothesis is formally stated asX1,..., Xp only. More precisely, we replace x1,...,xn by xσ(1),...,xσ(n), where σ is a random permutation of , while the clinical covariates zi are not permuted. The two-step procedure is applied and the negative binomial log-likelihood ℓ is computed again for this permuted data set. The whole procedure is repeated a large number of times B, yielding the negative binomial log-likelihoods ℓ1,...,ℓB. The permutation p-value is then obtained asWe suggest to test this null-hypothesis using a permutation procedure by permuting 1 denotes the indicator function.where In the case of a fixed risk score as discussed at the end of the Section 'Combining logistic regression (step 1) and boosting (step 2)', the underlying model is slightly different and can be formulated asR(.) denote the fixed risk score function based on the clinical covariates Z1,..., Zq. Note that, if R(.) is simply a linear function of Z1,..., Zq, this version of the test will rather lead to rejection of the null-hypothesis than the first version with coefficients β1,..., βq estimated from the data.where mstop would yield an overcomplex model overfitting the training data, while a too small mstop would yield a too sparse model that do not fully exploit the available predicting information. In practice, the number of boosting iterations can be selected using an AIC-like criterion or by minimization of the out-of-sample negative binomial likelihood within a bootstrap procedure [mstop, the obtained regression model overfits the training data set, but the differences between permuted and non-permuted data, on which the test is based, do not seem to strongly depend on the number of boosting steps.When boosting is used for building a prediction model, the choice of the number of boosting iterations is crucial. A too large rocedure . In contμX = 5, p* = 1) and b) 200 very weakly informative molecular variables , all the other molecular variables and clinical covariates being irrelevant for the prediction problem. The second setting can be considered as an extreme case, since there are often less than 200 informative variables in practice, and relevant between-group shifts are often larger than μX = 0.2. In these settings, we compute the negative binomial log-likelihood ℓ as well as its permuted versions ℓ1,...,ℓB for a grid of mstop values ranging from 10 to 2000. The resulting curves are displayed in Figure X variables) decreases with increasing mstop more rapidly than the curves of the permuted data sets until a certain value of mstop. After this value, all curves are approximately parallel. Hence, further increasing mstop would not change the test result much. This is because, roughly speaking, the newly added components do not improve the model anymore - even with the original non-permuted variables.To illustrate this, we follow the simulation scheme described in the 'Results' section and consider two extreme cases: a) one strongly informative molecular variable Core(TM)2 CPU T7200 2.00 GHz). Note that the permutation-based procedure can be parallelized very easily, since the permutations are independent of each other.n of observations is set to n = 100, the number p of molecular predictors to p = 1000 and the number q of clinical predictors to q = 5. The binary variable Y is drawn from a Bernoulli distribution with probability of success 0.5. The p* relevant molecular variables follow the conditional distribution Xj\|(Y = 1) ~ μX, 1) and Xj\|(Y = -1) ~ j = 1,..., p. The other molecular variables Xp+1,..., Xp simply follow a standard normal distribution. Similarly, the clinical covariates are drawn from the conditional normal distribution Zj\|(Y = 1) ~ μZ, 1) and Zj\|(Y = - 1) ~ j = 1,..., q.In all settings, the number μZ = 0) and uncorrelated variables X1,..., Xp, Z1,..., Zq, and consider the six following cases:We first consider the case of non-informative clinical covariates (p* = 0 (no informative molecular variables), for comparison(null) p* = 5 and μX = 0.5: few relevant variables, weak between-group shift(a) p* = 5 and μX = 0.8: few relevant variables, strong between-group shift(b) p* = 50 and μX = 0.3: many relevant variables, very weak between-group shift(c) p* = 50 and μX = 0.5: many relevant variables, weak between-group shift(d) p* = 200 and μX = 0.3: very many relevant variables, very weak between-group shift(e) additional predictive value of high-dimensional data, we also consider the following special setting (f): both the q = 5 clinical covariates and the p* = 5 relevant molecular predictors are highly predictive (μZ = μX = 1), but in the first case they are mutually uncorrelated (f.1), while we have X1 = Z1,..., X5 = Z5 in the second case (f.2).To show that our method focuses on the For each setting, 100 simulated data sets are generated. The two following methods are applied to each data set for each setting:mstop = 100, 500, 1000 and AIC-optimized mstop with B = 200 permutation iterationsA. Our method for globaltest package [B. Goeman's global test with adj package mstop seems to be minimal in all settings except in setting (f.1), where mstop = 1000 has a noticeably better power. Hence, this simulation study confirms that, as outlined in the Section 'The choice of mstop', the choice of mstop is not of crucial importance in most cases, and that mstop should rather be large. Secondly, our method shows high power in very different difficult situations such as a small number of strong predictors or a large number of very weak predictors. In all the examined settings, its power was in average better than the power of the standard globaltest - at the price of an increased computational expense. The power difference between our approach and the global test is especially striking in the case of a small number of strong predictors (b). Another interesting result is that the p-values of the global test are not uniformly distributed in the null case. Note, however, that we do not consider the permutation-based global test in this comparison study. Its results may be different with respect to the null-distribution of the p-values. Thirdly, our method finds additional predictive value in setting (f.1) but does not in setting (f.2) , thus fulfilling prerequisite 1.Figure ALL [We first analyze the ALL data set included in the Bioconductor package ALL . The ALLALL . The datDENMARKLAB [The second example data set considered in this paper is the van't Veer breast cancer data set . The datNMARKLAB , which wmstop = 100, 500, 1000 and AIC-optimized mstop) to both data sets. Additionally, we also apply the global test without adjustment and our method without first step for comparison. The results are given in Table mstop for the original data sets (black) and for the permuted data sets (grey). It can be seen from Table mstop may yield noticeably different results, in contrast to what was observed in simulations. In this context, the AIC-based choice of mstop is helpful.We apply the global test with adjustment for the clinical covariates and our new approach or all hypotheses are accepted (too weak predictors). Following [Our simulation and real data studies were performed with the values ollowing , the aimL1-penalized regression. Indeed, the Lasso and boosting regression can be seen as two sparse regularized regression methods addressing the same problem in a different way. If L1-penalized regression is applied in the second step, the penalty applied to the L1 norm plays the role of the number mstop of boosting steps as a complexity parameter. In principle, many other regularized regression techniques based on the logistic model may be used in the second step of our procedure, such as, e.g., L2-penalized regression. An extensive comparison study would go beyond the scope of this paper. However, a preliminary study using an arbitrary value for the penalty parameter indicates that similar performance can be obtained using L1-penalized regression as implemented in glmpath (data not shown).As suggested by a reviewer, other regularized regression techniques could be used in place of boosting regression in the second step of our procedure, for example, mstop = 100 or mstop = 500 are expected to perform reasonably well in any case, there are no universal standard values for the penalty parameter in L1-penalized regression. Most importantly, the range of the penalty values considered by glmpath depends on the data. Thus, it may be difficult in practice to find a penalty value common to all permutations and the p-value cannot be simply calculated. On the whole, the choice of the complexity parameter seems to be more delicate in methods with direct penalization than in the context of boosting regression.Beside the high computational expense, an important problem of this approach is the choice of the penalty parameter. Whereas standard values of the number of boosting steps like additional predictive value of high-dimensional data in the sense that the clinical covariates are involved in the model as a fixed offset. We provide clear advice for choosing the parameters involved in the procedure. The shrinkage factor ν should be set to the standard default value ν = 0.1 as recommended in previous publications [B of permutations should be set as high as computationally feasible . The most delicate parameter is the number of boosting iterations mstop. Note, however, that the choice of mstop is not as crucial as in the context of prediction. The AIC-based procedure provides a reliable objective criterion. Except for the computational expense, there is almost no inconvenience to set the maximal number of boosting steps to a very large value, for instance We propose a simple boosting-based permutation procedure for testing the additional predictive value of high-dimensional data. Our approach shows good power in very different situations, even when a very small proportion of predictors are informative or when the signal in each informative predictors is very weak. Unlike approaches like pre-validation , it asseications . The nummboost package [Note that our methodology can be easily generalized to a wide range of more complex regression problems such as survival analysis or non-linear regression. These problems can all be handled within the boosting regression framework using the package ,18. Henc package , the com package .ALB drafted the paper and performed the analyses. Both authors developed the method and contributed to the manuscript. TH implemented the boosting procedure applied here. Both authors read and approved the final manuscript.R script for the analysis of the ALL data. This R file includes the R script for reproducing our analyses of the ALL data.Click here for fileR script for the simulations. This R file includes the R script for reproducing the simulation study.Click here for fileR function to generate the simulated data sets. This R file includes the function used in our simulations to 1) generate the simulated data sets, 2) compute the p-value with our new method.Click here for file
Measurement of disease-related impairment and distress is central to diagnostic, therapeutic, and health policy considerations for eating disorders across diverse populations. This study evaluates psychometric properties of a translated and adapted version of the Clinical Impairment Assessment (CIA) in an ethnic Fijian population.N = 215). We calculated Cronbach's α to assess the internal consistency, examined the association between indicators of eating disorder symptom severity and the CIA to assess construct and criterion validity, and compared the strength of relation between the CIA and measures of disordered eating versus with measures of generalized distress.The adapted CIA was administered to ethnic Fijian adolescent schoolgirls . Both construct and criterion validity were supported by the data, and regression models indicated that the CIA predicts eating disorder severity, even when controlling for generalized distress and psychopathology.The adapted CIA has excellent psychometric properties in this Fijian study population. Findings suggest that the CIA can be successfully adapted for use in a non-Western study population and that at least some associated distress and impairment transcends cultural differences. © 2009 by Wiley Periodicals, Inc. Int J Eat Disord, 2010 Eating disorders have well-established public health impact in North American and European populations as evidenced by their association with both high mortality and high suicide rates.6A number of disease-specific measures have been developed to assess quality of life and impairment for individuals with eating disorders. Three instruments—the Clinical Impairment Assessment (CIA),The primary aim of this study was to evaluate reliability and validity of a measure of distress and impairment for eating disorders, the CIA, which we adapted and translated for use in an ethnic Fijian adolescent population.The study was conducted in two adjoining provinces on the main island in the Republic of Fiji, a lower middle income country undergoing rapid social change. Ethnic Fijians comprise a small-scale indigenous society—traditionally with a subsistence agriculture economy—making up approximately half of Fiji’s population. The gradual economic transition to a wage-based economy accompanied by widespread exposure to Western-based mass media has stimulated consumption of Western food and electronic products.N = 215) were a subset of a larger study population of ethnic Fijian schoolgirls, ages 15–20 enrolled in the 12 secondary schools registered in Fiji’s Ministry of Education for this administrative area when the study was designed. In the latter study, participants responded to a battery of self-report assessments and were weighed and measured. Procedures for translating and adapting the self-report assessment battery and administering it to the larger study sample are described elsewhere.16Study participants as well as 10%–15% of respondents within each school site who did not screen positive (‘‘asymptomatic respondents’’). Asymptomatic respondents were randomly selected for participation within each site among study participants present in school at Time 2.2The primary outcome reported here, eating disorder specific distress and impairment, was assessed by a version of the CIA, adapted as an interview schedule for this study population. The CIA is self-report measure designed to be used as a companion instrument to the EDE-Q. A total of 16 items are scored on a 4-point Likert scale . These responses correspond to numeric values 0 to 3, which are summed to calculate a global score as long as at least 12 of the items have responses.17The CIA was adapted for use in this study population as follows from a previous 22-item version obtained from its authors. Four items that referenced ‘‘work’’ were rephrased to reference school or schoolwork instead after consultation with an author of the CIA . An additional two items presented a culture-specific content problem. Item 11 that referenced ‘‘sex life’’ was omitted based on ethnographic work by the first author suggesting it could be culturally insensitive in a face-to-face interview and item 16 that referred to feeling ‘‘guilty’’ was rephrased to ask about feeling ‘‘like you had done something wrong’’ because guilt has a jural—but not affective—meaning in the local cultural idiom. The 21 remaining items and their response options were then translated into the vernacular language by the first author, edited by a bilingual native speaker, back-translated into English by another bilingual speaker, and then reviewed to ascertain that versions were comparable and translatable with respect to conceptual content. Study staff administered the CIA orally to the participants in their choice of either English or the vernacular language following an EDE interview. Respondents could have questions posed or rephrased in the standard Fijian language for clarification if necessary. Although we had collected data for the 21-item version as described earlier, for this analysis we discarded items not in the current version of the CIA and scored the remaining items by summing numeric responses for each of 16 total items—as long as at least 12 items were present—in order to facilitate comparison of our results with other study populations. Questionnaires with missing items were prorated through mean replacement to achieve the full range of scores. The retained items for this study analysis are the same as those in the current version of the CIA except for the minor wording changes as described earlier for 2 items.We chose several self-report assessments relating to disordered eating and body satisfaction to examine their expected correlations with distress and impairment due to eating disorder symptoms. We also selected several proxies for generalized distress to examine their correlation with the CIA.The EDE-Q, adapted for this study population, was used to assess the presence and severity of disordered eating symptoms in this study.The BFS directs respondents to view 9 female adult figure drawings that range from very thin to obese body size and select the figure that they perceive matches their own body shape as well as the one that matches the body shape they feel is ideal. A discrepancy score can be derived by subtracting the numeric value of the ideal shape from the self-perceived shape value. Scores close to 0 reflect little or no discrepancy; positive values reflect perception of being larger than ideal whereas negative values reflect perception of being smaller or thinner than ideal.The FBSQ is a 6-item self-report measure assessing body dissatisfaction; five items have a Likert format with four response options and one has a forced choice yes/no response option. A previous version of this questionnaire has shown acceptable internal consistency reliability in a similar study population.The BESAA is a 23-item self-report questionnaire measuring dimensions of body esteem including feelings about general appearance and bodies.The CES-D is a 20-item self-report questionnaire designed to measure depression in the general population.The GSHS is a self-report assessment of health related behaviors in school-going adolescents developed by the WHO in collaboration with the U.S. Centers for Disease Control. The GSHS is comprised of modules relating to health risk behaviors that can be adapted to the needs assessment and local cultural context across global settings.Weight was measured on the day of the self-report assessment on an electronic scale with a digital readout in gradations of 0.2 kg. Respondents were asked to remove shoes, sweaters or jackets, and heavy jewelry prior to being weighed in a lightweight school uniform. Measured weight was adjusted by sub-tracting 0.5 kg to yield an estimate corrected for the average contribution of the clothing. Height was measured to the nearest millimeter (mm) in bare or stocking feet with a portable stadiometer, but rounded to the nearest centimeter for the majority of observations in this analysis. BMI was calculated based on the adjusted weight and height for each respondent as kilograms/mCharacteristics of the study population were summarized by assessing frequencies and means of demographic covariates, including age, BMI, urban/rural location of school, school form (grade), EDE-Q scores, and CIA scores. Performance of the CIA in this study population was assessed in four domains: (1) feasibility; (2) internal consistency reliability (for both the Fijian and English language versions); (3) criterion validity with regard to participants’ group classification on known measures; and (4) construct validity.We assessed feasibility by evaluating the proportion of measures that were ultimately scoreable after accounting for missing data. We then calculated a Cronbach’s a coefficient, a summary measure of the shared variation among all the CIA items, to evaluate the internal consistency reliability of the CIA total score for both English and Fijian language versions of the interview.Next, we evaluated criterion validity by dividing the sample into asymptomatic versus symptomatic groups on nine different indices of eating disorder psychopathology, and conducting a series of t-tests comparing CIA scores across groups. We hypothesized that symptomatic participants would exhibit higher CIA scores, indicative of greater functional impairment and distress, than asymptomatic participants in the following nine comparisons:Respondents with self-reported presence of vomiting or laxative abuse versus those who reported noneRespondents with self-reported presence of herbal purgative use versus those who reported noneRespondents with self-reported presence of binge eating versus those who reported noneRespondents with self-reported presence of fasting versus those who reported noneRespondents with global EDE-Q scores > = 4 versus scores \4Respondents with a discrepancy score > = 2 on the Body Figure Scale versus \2Respondents with a BMI at or above the 95th percentile based on WHO guidelines versus those below the 95th percentileRespondents with a BMI at or below the 5th percentile based on WHO guidelines versus those above the 5th percentileRespondents who screened in for an interview based on eating disorders symptoms, low weight, or high EDE-Q global scores versus those chosen randomly who did not meet screening criteriaLastly, to assess construct validity, we calculated Pearson correlation coefficients to determine the extent to which CIA scores were correlated with measures of both eating pathology as well as proxies for generalized psychopathology and distress . Moreover, we tested the hypothesis that eating disorder psychopathology would be predictive of CIA scores independently from generalized measures of distress not specifically related to disordered eating. We did this by fitting three linear regression models predicting mean CIA scores. In the first model, we included global EDE-Q only; in the second model, we added participant demographic covariates ; finally, in the third model we added measures of generalized psychopathology and/or distress (CES-D and GSHS items).The administration and evaluation of the CIA was a component of a study protocol that was approved by the Fiji National Research Ethical Review Committee (FN-RERC), the Partners Healthcare Human Subjects Committee, and the Harvard Medical School Committee on Human Studies. Parental informed consent and youth assent were obtained for all study participants.Of 183 symptomatic study participants selected for an interview, 178 were present in school on the day that interviews were conducted; of these, all completed a CIA interview. Of 37 asymptomatic respondents randomly selected and present in school, all completed a CIA interview. The mean age of the sample was 16.80 years (SD = 1.10 years), and the mean BMI was 24.28 kg/mAll 215 respondents completed the interview. Of these, 208 completed all 16 items, whereas the remaining seven completed at least 12 items, allowing for the calculation of a global score for every participant. The mean CIA score was 11.57 (SD 5 10.92), and scores ranged from 0 to 43.Cronbach’s a calculated for respondents to both English and Fijian language questionnaires showed adequate to excellent internal consistency reliability; they were 0.91 for the English language version group, 0.94 for the Fijian language version group, and 0.93 for the entire study sample.Participants classified as symptomatic on 7 of 9 indicators of eating disorder psychopathology scored significantly higher on the CIA than participants not classified as symptomatic . The onlCIA scores were positively correlated with measures of eating disorder pathology as expected. Moreover, CIA scores were also correlated with measures of distress unrelated to disordered eating, as expected, with the exception of suicidal ideation, which exhibited only a trendlevel correlation with CIA, r 5 .13, p 5 .06. The observed correlations are summarized in Linear regression models of the relation between EDE-Q scores and the CIA are shown in Assessment of distress and impairment specific to eating disorders is critical to identifying appropriate clinical services and therapeutic strategies for individuals in need of treatment. Moreover, clarification of disease-specific burden of illness can inform health care policy decisions so that scarce mental health resource allocation most effectively meets local needs. Although eating disorders may be increasingly prevalent in populations under-going economic transition and have been designated as priority disorders by the WHO for child and adolescent mental health, little is known about the burden they impose in populations in developing areas of the world. Thus, measurement of distress and impairment specific to eating disorder symptoms can make an important contribution to the knowledge base for local mental health needs.Our results demonstrate that the CIA is feasible, internally consistent, and informative for this ethnic Fijian adolescent female study population. Notably, the CIA measured distress related to a measure of eating disorder symptom severity even when adjusted for generalized distress. The CIA also successfully discriminated between symptomatic and asymptomatic respondents for almost all of the categories examined. Reliability was comparable for the CIA in English and Fijian versions. This evidence supports the validity of the CIA as a measure of the distress and impairment associated with eating disorder pathology in this study population, even when adjusted for generalized distress and psychopathology. An illness-specific measure of distress and impairment can be a useful tool for health care providers in Fiji in assessing need for intervention in a setting in which mental health services are scarce and difficult to access. We believe that, in some cases, CIA data could also be potentially informative to educators in identifying and addressing antecedents to poor school performance. The demonstrated feasibility, reliability, and validity of the measure in a small-scale indigenous population without indigenous categories for eating disorders suggests that the CIA may also be useful across diverse populations, although further investigation is required before generalization can be made.Measurement of illness-related distress in this particular cultural context also raises concerns about whether distress and impairment for mental illness relate to a universal standard or rather can best be understood relative to their local cultural context. For example, several findings in our study were unexpected. Specifically, low weight and vomiting and laxative use were not significantly related to CIA scores whereas high weight and traditional herbal purgative use were. One possible explanation is that the CIA failed to identify distress related to low weight, vomiting, or laxative misuse. Another explanationone that we find more plausible, is that social norms and cultural experience moderate distress and thus attenuate the relation between these symptoms and expressed distress. For example, social advantages of low weight may offset distress in this social environment. Individuals may perceive that thinness enhances social opportunities or may elicit relatively greater social support.There are several limitations of our study. First, the CIA was administered here as an investigator-based interview rather than as a self-report. In some cases, it was desirable to offer phrasing in a related (standard) Fijian language. As such, phrasing may have varied across raters and interviews and may have been subject to bias if the respondent had difficulty with the local vernacular language. Although we believe these elements of our protocol would have enhanced the way respondents understood the questions, it is possible that social desirability biased results in an unknown direction . We did not assess either interrater or retest reliability of the CIA in this study population. Restricting our interviews to respondents present on the day of the site visit was a necessary compromise to logistical difficulties of travel in Fiji and could have also biased results. Also, only a small number of participants in our sample (n 5 2) met WHO criteria for under-weight, so the statistical power for detecting potential elevations in distress and impairment among low-weight participants was quite limited. Finally, our conclusions about construct validity rest on the assumption that the measures of related constructs were valid in this study population. For example, the items drawn from the GSHS as proxies for distress unrelated to disordered eating demonstrated acceptable but only modest reliability (kappas ranging from 0.41 to 0.69) . However, our data do support the reliability and validity of the EDE-Q in this study population.In sum, our study supports that the CIA is a valid measure of distress and impairment related to eating disorder symptoms in this study population. We believe that our findings suggest that some important core features of distress and impairment can be measured across diverse social environments. Our findings also underscore that sensitivity to local cultural meanings may be required to fully inform interpretation of measures of distress and impairment applied across diverse cultural settings.
Primary adenocarcinomas of the seminal vesicle (SVC) are very rare and poorly understood neoplasms with only somewhat more than 50 histologically confirmed cases reported in the literature. We demonstrate a case of SVC and discuss the problems related to diagnosis in this tumor. Primary adenocarcinomas of the seminal vesicle (SVC) are very rare and poorly understood neoplasms with only somewhat more than 50 histologically confirmed cases reported in the literature. We demonA 54-year-old man with dysuria and haematuria was first examined in January 2007. Laboratory parameters including serum prostate specific antigen (PSA) level were normal. Digital rectal examination detected a poorly defined hard tumoral mass on the site of the left seminal vesicle, under the intact rectal mucosa. This finding was confirmed by ultrasonography. Primary rectal tumor was ruled out by colonoscopy. Microscopic analysis of the colon biopsy showed infiltration of poorly differentiated adenocarcinoma restricted to submucosa with no evidence of tumor or dysplastic change in the mucosa.Contrast-enhanced magnetic resonance imaging was performed, which demonstrated a tumor mass in the area of left seminal vesicle, partly affecting the prostate and the anterior wall of the rectum . The patChest X ray and computed tomography (CT) of abdomen were also performed without showing any signs of malignancy. No previous diagnosis of malignant disease was registered in the patient's files. The clinical, radiological, histological, and immunohistological findings were consistent with primary SVC. The expert team of urological oncologists proposed radiotherapy before surgical intervention. However, the patient died of the disease 12 months after the histological diagnosis. Autopsy was not performed because relatives requested it.The tumor should be a microscopically verified carcinoma, localized exclusively or mainly to the seminal vesicle.The presence of other simultaneous primary carcinoma should be excluded.The tumor should preferably resemble the architecture of the non-neoplastic seminal vesicle.In 1956, Dalgaard and Giertson establisThese criteria can be readily applied to surgical resection specimens, but are less suitable in evaluating radiologically guided needle biopsies. Immunostains are helpful in these circumstances but vesicula-specific markers are not available.While primary SVCs are rare, secondary neoplastic involvement of the seminal vesicle is not so infrequent. Moreover, when clinical symptoms occur in a patient with SVC, they often reflect advanced disease with infiltration of adjacent organs obscuring the vesicular origin of the tumor. The most common symptoms are non-specific. These are hematuria and hematospermia, but dysuria; painful defecation and general pelvic pain have also been reported. CT scan,Histological examination is central in diagnosing SVC, despite the lack of organ-specific immunofenotype. Immunohistochemistry is rather helpful to exclude some metastatic tumors than to confirm the origin of the tumor in seminal vesicle . The com33According to the reported data in the literature, the immunohistohemical profile of SVC is not as consistent regarding other tumor markers as for PSA, CK7, and CK20.5 Carcino13In summary, the diagnosis of SVC is based on correlation of clinical, radiological, and histological findings. Immunohistochemistry may be helpful in ruling out some differential diagnostic options but cannot per se distinguish poorly differentiated SVC from metastatic spread of poorly differentiated carcinoma of prostate, certain adenocarcinomas of urinary bladder and from the rare carcinomas originating in müllerian duct cyst.
Abiotic stresses adversely affect plant growth and development. The hormone abscisic acid (ABA) plays a central role in the response and adaptation to environmental constraints. However, apart from the well established role of ABA in regulating gene expression programmes, little is known about its function in plant stress metabolism.Arabidopsis thaliana to ABA and high salt conditions. Our work shows that salt stress induces complex re-adjustment of carbohydrate metabolism and that ABA triggers the initial steps of carbon mobilisation.Using an integrative multiparallel approach of metabolome and transcriptome analyses, we studied the dynamic response of the model glyophyte These findings open new perspectives on how high salinity and ABA impact on central carbohydrate metabolism and highlight the power of iterative combinatorial approaches of non-targeted and hypothesis-driven experiments in stress biology. High soil salinity is a major environmental constraint for plant growth and development, and limits agronomic yield Genetic and biochemical studies indicate that distinct signal transduction pathways mediate different aspects of high salt stress, which ultimately lead to reprogramming of metabolism, physiology and morphology Salinity has a strong impact on gene expression Toxoplasma gondii and in human immune responses, thus indicating a conservation of ABA signalling across kingdoms ABA is an important regulator in many aspects of plant growth and development, and is pivotal for stress resistance Although global adjustment of metabolism is vital for adaptation to high salinity, our knowledge of the mechanisms involved in metabolic adaptation upon adverse environmental conditions is scarce. Early events largely determine whether plants can cope with stress Arabidopsis thaliana metabolome in response to high soil salinity on a large scale, we performed metabolic profiling on leaves of plants exposed to high salt stress. Four week old soil-grown plants were watered with 150 mM NaCl and analysed 6 h, 12 h, 1 d, 3 d and 5 d after the onset of stress. While prolonged stress exposure led to sodium accumulation in leaves, the concentration of soluble sodium did not increase during the early experimental timepoints for polar compounds. Additionally, we determined the levels of starch, the main carbohydrate store in plants, and of ascorbate and dehydroascorbate, as parameters of cellular redox-balance. Principal component analysis (PCA) of the metabolic data revealed a continuous change of the metabolome upon high salt stress. We observed minor overall changes in metabolite levels early after application of high salt conditions but a pronounced alteration after prolonged stress and S1 wLotus japonicus and Populus euphratica, indicating a conservation of this response to high salinity Analysis of individual metabolite levels showed that high salinity had a profound effect on central metabolism and induced complex changes in the metabolome in a time-dependent manner . TemporaRedox balance is crucial for cellular function. Application of high salt stress did not change the overall pool size of the antioxidant ascorbate. However, a rapid and sustained increase in oxidised dehydroascorbate (Dha) and a decrease in reduced ascorbate (Asc), which led to a marked shift in the Asc to Dha ratio, was observed . These dLotus japonicusHigh salt conditions induced a rapid alteration of carbohydrate levels. After irrigation of plants with NaCl, the levels of several soluble sugars and the glucose to fructose ratio changed significantly, indicating an impairment of central carbohydrate metabolism . NotablyThe dynamics of the overall changes in the metabolite composition induced by high soil salinity were confirmed in a second independent experiment .Arabidopsis transcriptome. We employed the MapMan gene ontology and image annotator To address the question of whether the observed changes in metabolite levels correlate with the transcriptional response of genes encoding the corresponding metabolic enzymes, we explored the AtGeneExpress microarray analyses were performed on young hydroponically-grown plants whereas metabolic profiles were obtained from soil-grown adult plants. To verify the transcriptional response in our experimental system and for direct comparison of the transcriptional and metabolic salt-induced patterns, we performed semi-quantitative RT-PCR on 48 selected genes from the same plant material which was used for metabolic profiling. 75% of the monitored genes displayed a similar expression pattern upon high salinity treatment in young hydroponically-grown and in adult soil-grown plants , emphasiBMY1 and BMY7), which cleaves maltose from starch-derived glucan molecules, was transcriptionally upregulated. Enhanced starch degradation by beta-amylase likely supplies energy and carbohydrate skeletons for adaptation processes (e.g. osmolyte production). Interestingly, genes involved in starch synthesis including an isoform of starch synthase and ADP-glucose pyrophosphorylase (AGPase1 and 2), which provides ADP-glucose, were also induced by high salinity. This observation might indicate that salt stress stimulates both starch mobilisation and starch synthesis. This interesting notion should be further addressed by metabolic flux analysis in future studies.Starch metabolism is very sensitive to the environment G6P DH), invertase (INV), hexokinase (HXK1 and 2) and UDP-glucose pyrophosphorylase (UGPase) were induced to different extents. In line with the accumulation of galactinol and raffinose, genes coding for galactinol synthase and raffinose synthase (RS) were strongly induced by high salt conditions.High salinity induced alterations in the levels of soluble sugars . SeveralP5CS1 and 2), phenyl ammonium lyase (PAL), nitrate reductase (Nry and NR) and proline oxidase (ERD5 and PrOX). Genes encoding different isoforms of glutamate decarboxylase (GAD) showed a diverse transcriptional response. GABA accumulated to high levels after 5 d in saline conditions. The expression of the glutamate decarboxylases GAD3 and GAD4 showed a biphasic induction whereas the expression of GAD2 decreased at later timepoints. The GABA transporters GABA trans1 and GABA trans2 (AtGAT1) were also induced transcriptionally. We observed a rapid change in the ascorbate to dehydroascorbate ratio by salt stress. However, with the exception of a glutathione reductase, genes of the ascorbate/glutathione cycle did not respond at the transcriptional level.Salt stress modified the expression of several genes encoding proteins involved in nitrogen metabolism. The majority of the monitored transcripts were strongly upregulated at later timepoints including pyrroline-5-carboxylate synthase , raffinose synthase (RS), pyrroline-5-carboxylate synthase (P5CS) and beta-amylase (BMY) which are strongly upregulated by high salinity. With respect to starch metabolism, the increase in maltose levels was positively correlated with the induction of beta-amylases (BMY7 and BMY8) at the transcriptional level but negatively correlated with the amount of starch. It is worth noting that starch showed the strongest negative correlation with raffinose. The negative correlated cluster also included phosphoric acid, shikimic acid and galactonic acid.To compare changes in metabolite and transcript levels comprehensively, we generated a correlation matrix. The variance of metabolite and transcript amounts was adjusted to equal levels by z-transformation and a pairwise correlation was subsequently undertaken. The resulting matrix revealed two prominent clusters with opposite behaviours . The firRAB18, ABI2 and ABA-induced PP2C were negatively correlated with starch contents but showed a strong positive correlation with the levels of the starch degradation product maltose, suggesting that this part of the high salinity-induced metabolic reaction might be triggered by ABA.In addition to the frequent association of metabolite levels with the expression levels of genes involved in the synthesis of these metabolites, a strong correlation between different metabolic pathways, e.g. starch mobilisation with raffinose and proline synthesis, was observed, suggesting a tight regulatory link between theses pathways in response to salt stress. Interestingly, the expression of ABA marker genes showed a strong co-regulation with numerous salt-induced metabolic changes. Transcript levels of To analyse the notion that ABA might be important for metabolic regulation and that ABA could be involved in carbon mobilisation, we studied the impact of ABA on metabolism. First, we examined the AtGeneExpress database for the transcriptional behaviour of salt-induced metabolic genes for their response to ABA. In agreement with the importance of ABA in the regulation of gene expression, the majority of the selected salinity-induced metabolic genes were also found to be upregulated by ABA .Arabidopsis metabolome to ABA in a time course experiment. Four week old soil-grown Arabidopsis plants were treated with 25 µM ABA for 2 h, 6 h, 24 h and 3 d. Subsequently, the metabolic profile was determined by GC-MS -ABA (Sigma) aqueous solution. For prolonged ABA treatment, plants were watered and sprayed every day. For both treatments, all plants were well-turgoured and stayed green during the entire experimental period.Soil grown Soluble sodium and potassium concentrations were measured by flame photometry (atom emission spectroscopy (AES)). For this, 50 mg of pulverised plant material was extracted with 20 ml of double distilled water and the supernatant was used for analysis.Arabidopsis plants were pooled and frozen in liquid nitrogen. 60 mg of ground plant material was extracted and analysed as described in Ten or fifteen rosettes of 4 week old Samples for metabolite profiling were prepared as described in 2O (0–5 min) to 19% MeOH, 81% H2O (5–12 min) and to 90% MeOH, 10% H2O (12–30 min hold time 5 min). The pH of the elution buffer was adjusted to 3.9 using 0.3 M acetic acid.Ascorbate levels and the redox-state of the ascorbate pool were determined by reversed phase HPLC. 100 mg of frozen and ground plant material was extracted with 0.1 M HCl under an argon safety atmosphere. Levels of free ascorbate were analysed directly. Total ascorbate levels were determined after a reduction step with 10 mM DTT. For this, the pH was adjusted with CHES buffer . Subsequently, extracts were acidified and analysed by HPLC. Separation of ascorbate was performed using an ODS column and a Dionex HPLC system with UV detection at 254 nm. For separation, a multi-step gradient was used starting from 5% MeOH, 95% Hhttp://www.uni-tuebingen.de/plantphys/AFGN/atgenex.htm). For high salinity stress, 18 day old Arabidopsis thaliana Col-0 plants were treated with 150 mM NaCl. A detailed protocol for the cultivation, stress exposure and sample harvesting was recently published Arabidopsis thaliana Col-0 seedlings were treated with 10 µM ABA. Samples were taken after 30 min, 1 h and 3 h http://affymetrix.arabidopsis.info/narrays/experimentbrowse.pl) and processed using ROBIN freeware (http://www-de.mpimp-golm.mpg.de/profil/services/index.html). Expression changes of more than 4-fold were counted as relevant. Additional genes involved in the pathways of interest were included in the analyses. Transcript levels of selected genes were analysed by semi-quantitative RT-PCR. Oligonucleotide primers were designed based on gene models from the MIPS database (www.arabidopsis.com). The resulting amplicon length of the PCR products was about 120 bp. Primer sequences are listed in Publicly available microarray data were generated in the frame of the AtGenExpress project (e.g. PCR was performed using MBI Fermentas Taq Polymerase according to the manufacturer's protocol. PCR products were separated on agarose gels stained with ethidium bromide.Transcript levels were determined by reading the pixel densities using Quantity-One 4.6.1 software (BIO RAD) and normalised to tubulin used as a constitutive control.http://metagenealyse.mpimp-golm.mpg.de/). The ICA algorithm applied is based on 5 components of a PCA and calculates the maximal independence of non-Gaussian distributed data. The directions of variance calculated by a PCA analysis in a multidimensional room are orthogonal to each other. The ICA calculates the directions of maximal independency of the pre-analysed data set. PCA and ICA algorithms are unable to operate with missing values, thus all substances measured at all timepoints were included in the calculations.The statistical screening for significant metabolic changes among the monitored metabolites was performed using Microsoft Excel. PCA and ICA analysis For comparison of gene expression with metabolite data, transcript and metabolite data were z-transformed prior to pairwise correlation (Pearson correlation coefficient) analysis.Figure S1Soluble sodium and potassium levels in response to high soil salinity. Soluble sodium and potassium ion concentrations were determined from the same plant material that was used for metabolic profiling. 50 mg of pulverised plant material was extracted with 20 ml of double distilled water. The supernatant was measured by flame photometry (atom emission spectroscopy).(0.01 MB PDF)Click here for additional data file.Figure S2Principal component analysis (PCA) of technical replicas of the salt stress time course experiment shown in (0.02 MB PDF)Click here for additional data file.Table S1Loadings of principal component analysis (PCA).(0.05 MB PDF)Click here for additional data file.Table S2Glucose to fructose ratio. The relative glucose and fructose contents of plants treated for 3 d with NaCl or ABA were calculated from the data presented in (0.01 MB PDF)Click here for additional data file.Table S3Dynamic changes in metabolite levels upon high soil salinity in an independent biological experiment. Four week old soil-grown Arabidopsis thaliana was watered with 150 mM NaCl. Metabolic profiles were determined from leaves of pools of 15 individual plants four times by GC-MS. Compounds were identified by the retention time (TAG RT), the quantitative mass tag (TAG MASS) and the reverse match value (REV MATCH) of the mass spectra comparison. Starch levels were quantified spectrophotometrically. Values are x-fold changes compared to the corresponding unstressed controls. Bold letters indicate significant changes in metabolite levels .(0.05 MB PDF)Click here for additional data file.Table S4Transcriptional response of metabolism-related genes to high salt and ABA treatment. Transcriptional response of genes encoding enzymes in selected metabolic pathways to NaCl and ABA treatment of young, hydroponically-grown seedlings (AtGeneExpress) and adult, soil-grown plants . ++, strong transcriptional induction ≥7-fold for microarray and ≥2-fold for RT-PCR; +, transcriptional induction ≥4-fold for microarray and ≥1.5-fold for RT-PCR; o, unchanged; -, transcriptional reduction ≤0.5-fold; –, strong transcriptional reduction ≤0.15-fold; −/+, transcriptional induction at late timepoints after transcriptional reduction at early timepoints; n.d., not determined.(0.06 MB PDF)Click here for additional data file.Table S5ABA-induced metabolic changes. Metabolite levels of 4 week old Arabidopsis thaliana after treatment with 25 µM ABA. Pools of ten plants were harvested after the indicated timepoints and the metabolite contents were analysed by GC-MS, HPLC or spectrophotometrically. Shown are the relative metabolite levels as an x-fold change normalised to untreated plants. Bold letters indicate significant changes of metabolites . TAG RT: retention time; TAG MASS: quantitative mass tag; REV MATCH: reverse match.(0.05 MB PDF)Click here for additional data file.Table S6Comparison of major NaCl- and ABA-induced metabolite changes.(0.12 MB PDF)Click here for additional data file.Table S7Table of independent component analysis (ICA) loadings.(0.05 MB PDF)Click here for additional data file.Table S8List of primers used for RT-PCR.(0.05 MB PDF)Click here for additional data file.
Lipomas are benign mesenchymal neoplasms composed of mature adipocytes, usually surrounded by a thin fibrous capsule. They are uncommon intra-oral tumors with 1% to 4% occurring in this region. The literature is scanty on lipomas occurring in the buccal soft tissue, especially in our environment.We present a case of a 35-year-old woman of the Tiv ethnic group of Nigeria who presented with a slow growing left cheek swelling that was treated by intra-oral local excision.The purpose of this report is to highlight the existence of this rare but not uncommon disease even in our environment and to emphasize that a high index of suspicion is needed in making a diagnosis. Surgical excision as treatment is associated with an excellent outcome. Lipomas are benign mesenchymal neoplasms composed of mature adipocytes, usually surrounded by a thin fibrous capsule . They arAdequate surgical excision in order to prevent recurrence is the treatment of choice ,5. We reAlthough an isolated case of buccal soft tissue fibrolipoma has been reported in our environment , this paA 35-year-old housewife of the Tiv ethnic group in Nigeria was referred to our Ear, Nose and Throat clinic by family physicians with a 6-year history of a slowly progressive, painless left cheek swelling not preceded by trauma and not associated with fever, weight loss or any other otorhinolaryngological symptoms. Examination revealed a 6 cm by 6 cm non-tender doughy mass in the left cheek with no overlying skin changes. Slipping sign was not demonstrable and there was no bruit over this mass. The intra-oral mucosa over the mass appeared normal. A provisional diagnosis of buccal soft tissue lipoma was made with epidermoid cyst as a differential diagnosis.Imaging using ultrasonography revealed a fairly well circumscribed echogenic mass in the left cheek measuring 1.67 cm by 1.23 cm with no evidence of neovascularization noted within. On this premise, the radiologist made an assessment of lipoma. A computerized tomographic scan was not done because the patient could not afford to pay for it. Other investigations performed included full blood count, serum urea and electrolyte, and urinalysis which were all within normal limits.She was prepared for and had excision under general anesthesia via naso-endotracheal intubation. During surgery, the mass was approached intra-orally by a transverse 5 cm linear incision made in the mucous lining over it Figure . The 4 cMicroscopic examination of the excised soft tissue mass revealed sheets of mature adipocytes containing large clear cytoplasms and eccentric nuclei with inconspicuous vascularity and no evidence of cellular atypia or metaplasia Figure . These fth postoperative day and has remained free of any symptoms for over 36 months of follow-up.Postoperatively, she was placed on ciprofloxacin, ibuprofen and vitamin C tablets with oral saline mouth wash after meals. She was discharged in good condition on the 5Lipomas are adipose mesenchymal neoplasms that rarely occur in the oral cavity with a 1% to 4% reported occurrence in this region ,4. The pIn the oral cavity, the most common sites are the cheek, tongue, palate, mandible and lip where lipomas occur as sessile or encapsulated masses . The etiThe classification for benign lipomas includes the following: classic lipoma; lipoma variants ; hamartomatous lesions; diffuse lipomatous proliferations and hibernoma .Oral lipomas are slow growing tumors and patients commonly present with a well circumscribed mass that has been growing for several years . Our patClinically, they present as soft and compressible masses with doughy consistency which are well defined clinically and radiologically using ultrasonography and computerized tomographic scan and moreUnlike oral lipomas, lymphoepithelial cysts are found in the floor of the mouth, soft palate and mucosa of the pharyngeal tonsil . AlthougAdequate surgical excision is the treatment for oral lipomas ,5. The sMicroscopically, it is difficult to differentiate between normal adipose tissue and lipomas, therefore, a clinician sending a surgical specimen to the pathologist for microscopic analysis must provide accurate clinical and surgical information in order to make a definitive diagnosis . The micBuccal soft tissue lipomas are rare tumors. A high index of suspicion is required in making a diagnosis. Surgical excision is the ideal treatment with excellent outcome. The importance of histological diagnosis cannot be overemphasized and the features of lipoma are usually straightforward and classical.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.AAA was the principal surgeon, performed the literature search and prepared the manuscript. TLN assisted in the surgery and postoperative management of the patient. ANM interpreted the slides and reviewed the manuscript. GOE prepared the slides and the photomicrographs of the specimen.
Executive committeeT Philip, Director of SOR, paediatricianB Fervers, Deputy director of SOR, medical oncologistP Bey, Representant from the FNCLCC Advisory Committee, radiotherapistD Maigne, General manager of the FNCLCCSOR specialist projectA Bataillard, Coordinator of the SOR specialist project, general practitionerG Gory-Delabaere, Methodologist, pharmacistM Haugh, Methodologist, biochemistF Farsi, Network referent, public health physicianE Luporsi, Associated methodologist, medical oncologistS Theobald, Associated methodologist, public health physicianL Bosquet, Assistant methodologistN Fabre, Assistant methodologistS Rousmans, Assistant methodologistSOR SAVOIR patient projectS Brusco, MethodologistJ Carretier, MethodologistV Delavigne, LinguistL Leichtnam-Dugarin, MethodologistScientific information serviceS Guillo, Information scientistAG Guy, Information technicianAdministration and editorial serviceS Debuiche, Head of serviceH Borges-Paninho, Document managerE Esteves, SecretaryD Ropé, AssistantScientific Advisory CommitteeI Durand-Zaleski (President), Hôpital Henri Mondor, FranceJ Clavier (Vice-President), Hôpital Morvan, FranceJP Boissel, Hôpital Neuro-Cardiologique, FranceC Chouaïd, Hôpital St Antoine, FranceF Cluzeau, St George's Hospital, UKP Durieux, Faculté de médecine–Hôtel Dieu, FranceC Esper, Centre National de l'Equipement Hospitalier, FranceR Gagnayre, UFR de Bobigny, FranceA Giraud, Hôpital Jean Verdier, FranceG Ollenschläger, Agency for Quality in Medicine, GermanyR Otter, Integraal Kankercentrum Noord Nederland, The NetherlandsR Pinkerton, Royal Marsden Hospital, UKD Serin, Clinique Ste Catherine, FranceG Viens, Chaire Essec Santé, FranceOne representative from each of the following institutions:B Besse), CNAMTS, DGS (MF Chedru), INSERM (C Moquin-Pattey), Ligue Nationale Contre le Cancer (H Pujol), OECI , UNHPC (N Albin), FHF , College of General Managers of University Hospitals (F Grateau), College of Presidents of University Hospital Medical Committees (J Lansac), FranceAERIO (S Delahaye), and for general practitioners (Y Zerbib)Also a representative for patients : 8–16Fervers B, Hardy J, Blanc-Vincent MP, Theobald S, Bataillard A, Farsi F, Gory G, Debuiche S, Guillo S, Renaud-Salis JL, Pinkerton R, Bey P, Philip T (2001) SOR: project methodology. Electronic J Oncol1: 1–12. Available: URL: http://www.elecjoncol.orgFervers B, Hardy J, Blanc-Vincent MP, Theobald S, Bataillard A, Farsi F, Gory-Delabaere G, Debuiche S, Guillo S, Renaud-Salis JL, Pinkerton R, Bey P, Philip T (2001) SOR: project methodology [online]. Recommandations pour la pratique clinique en cancérologie [CD-ROM] Fédération Nationale des Centres de Lutte Contre le Cancer (ed). 2nd edn. Paris: FNCLCC, John Libbey EUROTEXT. Standards, Options et Recommandations.Bey P, Blanc-Vincent MP, Bonichon F, Farsi F, Fervers B, Labrèze L, Lehmann M, Philip T, Renaud-Salis JL, Sarradet A, Theobald S (1998) Méthodologie d'élaboration des standards, options et recommandations diagnostiques et thérapeutiques en cancérologie de la Fédération Nationale des Centres de Lutte Contre le Cancer (ed). 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In Bull Cancer87: 557–591Pottecher B, Herbrecht R, Blanc-Vincent MP, Bussy Malgrange V, Escande MC, Fuhrmann C, Crokaert F, Gory-Delabaere G, Senet JM, Lesimple T, Raveneau J, Béal J, Biron P, Viot M (2000) Standards, Options et Recommandations (SOR) pour la surveillance et la prévention des infections nosocomiales en cancérologie. http://www.fnclcc.fr/-sci/sor/pdf/infections_nosocomiales_integrale_1199.pdfPottecher B, Herbrecht R, Blanc-Vincent MP (2000) In Standards, Options et Recommandations pour la surveillance et la prévention des infections nosocomiales en cancérologie [online], Fédération Nationale des Centres de Lutte Contre le Cancer (ed) November 2000. Available: URL: Infection et cancer, Fédération Nationale des Centres de Lutte Contre le Cancer (ed) Vol. 8, pp 129–179. Paris: John Libbey EUROTEXT. 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Standards, Options et Recommandations: marqueurs tumoraux sériques du cancer du sein [online], Fédération Nationale des Centres de Lutte Contre le Cancer (ed) January 2002. Available: URL: http://www.fnclcc.fr/-sci/sor/pdf/sein_oncobiologie_abregee_0400.pdfBasuyau JP, Blanc-Vincent MP, Bidart JM, Daver A, Deneux L, Eche N, Gory-Delabaere G, Pichon MF, Riedinger JM (2002) In Standards, options et recommandations: marqueurs tumoraux sériques dans les cancers du côlon [online], Fédération Nationale des Centres de Lutte Contre le Cancer (ed) January 2002. Available: URL: http://www.fnclcc.fr/-sci/sor/pdf/oncobiologie_colon_abregee_0601.pdfEche N, Pichon MF, Quillien V, Gory-Delabaere G, Riedinger JM, Basuyau JP, Daver A, Blanc-Vincent MP, Buecher B, Conroy T, Bidart JM, Deneux L (2002) In Bull Cancer88: 1177–1206.Eche N, Pichon MF, Quillien V, Gory-Delabaere G, Riedinger JM, Basuyau JP, Daver A, Blanc-Vincent MP, Buecher B, Conroy T, Bidart JM, Deneux L (2001) Standards, options et recommandations: marqueurs tumoraux sériques dans les cancers du côlon. http://www.fnclcc.fr/-sci/sor/pdf/oncobiologie_colon_integrale_0601.pdfEche N, Pichon MF, Quillien V, Gory-Delabaere G, Riedinger JM, Basuyau JP, Daver A, Buecher B, Conroy T, Dieu L, Deneux L (2002) Standards, options et recommandations: marqueurs tumoraux sériques dans les cancers du colon [online], Fédération Nationale des Centres de Lutte Contre le Cancer (ed) January 2002. 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Available: URL: Hospital cateringhttp://www.fnclcc.fr/pdf/dietetique_prestation_integrale_0900.pdfDayot F, Bataillard A, Keré C, Ducès F (2001) Standards, Options et Recommandations: Bonnes pratiques de diététiques en cancérologie: la prestation alimentaire [online], Fédération Nationale des Centres de Lutte Contre le Cancer (ed) October 2001. Available: URL: Bull Cancer88: 1007–1018Dayot F, Bataillard A, Keré C, Ducès F, Bachmann P, Blanc-Vincent MP, Besnard B, Bonneteau C, Champetier S, Claude M, Combret D, Cometto F, Duguet A, Duval N, Fink C, Freby-Lehner A, Garabige V, Lallemand Y, Massoud C, Meuric J, Montane C, Poirée B, Puel S, Rossignol G, Roux-Bournay P, Simon M, Tran M (2001) Standards, Options et Recommandations: Bonnes pratiques de diététiques en cancérologie: la prestation alimentaire. 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A generic feature in all intracellular biochemical processes is the time required to complete the whole sequence of reactions to yield any observable quantity-from gene expression to circadian rhythms. This widespread phenomenon points towards the importance of time delay in biological functions. Theoretically time delay is known to be the source of instability, and has been attributed to lead to oscillations or transient dynamics in several biological functions. Negative feedback loops, common in biochemical pathways, have been shown to provide stability and withstand considerable variations and random perturbations of biochemical parameters. The interaction of these two opposing factors-of instability and homeostasis-are features that are widespread in intracellular processes. To test the effect of these divergent forces in the dynamics of gene expression, we have designed and constructed simple negatively auto-regulated gene circuits consisting of a basic regulator and transcriptional repressor module, and compared it with one, which has delayed repression. We show, both theoretically and experimentally, that delayed repression induces transient increase and heterogeneity in gene expression before the gain of stability effected by the negative feedback. This design, therefore, seems to be suitable for conferring both stability and variability in cells required for adaptive response to a noisy environment. Networks of genetic and metabolic reactions, underlying intra-cellular processes, are interconnected multi-step chemical reactions having widely different time scales. The complex regulation of these metabolic and transcriptional networks is brought about by the interaction of simpler regulatory structures Much excitement has been generated in recent years to study how stochasticity/perturbation in the different reaction steps affects gene expression kinetics using both theory and experimental ‘forward engineering’ approach, based on the construction and analysis of artificial “gene circuits” for single genes, gene networks, and multi-step regulated pathways auto-regulation), or by any other factor E. coli being of the negatively auto-regulated type. From previous theoretical studies, negative auto-regulation has also been predicted to shift noise to higher frequencies, which is more easily filtered out by gene networks-a property conjectured to contribute to the prevalence of such auto-regulatory motifs in the regulation of 40% of E. coli genes Among the basic regulatory designs, feedback inhibition of gene expression is the most common motif in gene regulation, where the expression of a gene is down-regulated by either its protein product and slow reactions , is higher, and hence have been postulated to be a significant source of delay in the subsequent steps of the reaction Even though delay-induced instability is widely discussed in theoretical literature for negative feedback systems In the following sections we discuss the theoretical and experimental results and compare them for the non-delayed (Basic) and the delayed negative feedback (Delay) circuits (described in t in The time course of Green Fluorescent Protein (GFP) in the Basic and Delay circuits for the four variable deterministic model and stochastic model (average of 100 simulations) are shown in c . Thus, with increasing delay time, the system shows damped oscillations, and the steady state is reached with progressively larger excursion in the phase plane. Our theoretical analysis, therefore, predicts that our model gene circuits have the intrinsic property of transient “overshoot” dynamics with increasing delay.Delays can make an otherwise-stable system less stable, i.e., the rate of decay of perturbations can decrease with an increase in the values of delay parameters, and a system, which has been otherwise non-oscillatory, can show oscillatory tendencies before converging to the steady state The fact that both the deterministic and stochastic versions of the models show overshoot for the Delay circuit indicates that this feature is due to delay in repression and not solely due to intrinsic or extrinsic noise in the cell population. Hence from now onwards, we show results only from the stochastic model of the gene circuits.600), respectively. The Basic circuit shows an initial increase in GFP expression, which decays towards a steady level over 9 hours. The Delay circuit, on the other hand, shows a large increase (overshoot) in fluorescence, which also decays towards a steady level, although this level is not the same as that reached by the basic circuit. We have done flow cytometric analysis to show that this is due to increased size of cells in the Delay circuit during growth at later time points, which show high fluorescence. A size correction reduces the large increase to some extent in the Delay circuit compared to the Basic circuit.Both the Basic (TG) and the Delay (C2TG) circuits were indTo address the role of plasmid size/copy number variation and translational efficiency between the Basic and Delay circuits, we have constructed a Control Delay circuit with the Basic and Delay circuits having plasmid variation , we determined the distribution of the GFP expression levels in the cells at different time points. The frequency distributions of the GFP fluorescence in the model Basic and Delay circuits are shown in The GFP fluorescence in 10,000 cells in populations of Basic (TG) and Delay (C2TG) circuits were studied at different time points, after induction with 25 ng/ml Doxycycline, using Flow Cytometry ; (ii) InFor studying the bimodal distribution of fluorescence in the cell populations in It may be noted that there is no role of inducer concentration in the models, and hence all cells in both model circuits are induced equally, and their GFP increase with time in A broader distribution of GFP fluorescence is observed in the Delay circuit cell population compared to the Basic circuit. To make a comparative quantification of the intra-population cellular heterogeneity in gene expression in the Basic and Delay circuit at different time points, we plot in Considering the fact that the mean protein levels are higher in the Delay circuit populations , its higThe model Delay and Basic circuits do not predict any significant difference in their CV over time except at an early time point see . We showThe complex regulation of metabolic and transcriptional networks in the cell is brought about by the interaction of simpler regulatory structures. Study of these simpler structures using theoretical modelling and experimental gene circuit construction provide us with important clues regarding the function of larger, more complex networks. The advantage of this approach is the greater freedom available to dissect out and study specific properties of the regulatory structure of interest in isolation. It allows experimental testing of theoretical predictions, made using mathematical models of the regulation, more easily. The negative auto-regulatory motif has been well studied using this approach, since it is one of the simplest and ubiquitous control structure found in nature E. coli, obtained from public databases (Regulondb: http://regulondb.ccg.unam.mx/ and Biocyc: http://biocyc.org/) into “Delay” and “Basic” type, depending on the position of the repressor gene with respect to its promoter, as shown in Delay or Basic type is termed if the repressor is the first gene, or, is not the first gene in the operon. In The design adopted by us in the Basic and Delay circuits is a common motif in negative regulation, predominantly observed in biosynthetic pathways lac, trp or the gal operons have been extensively studied to estimate the time involved in basic biological processes such as transcription and translation The numerous sequences of biochemical reactions that underlie the complexities of expressing a single gene points towards the fact that the characteristics of such delay may be essential in understanding whole-genome regulation. Time delays involved in operons, such as, the is the cause of transient instability, which can be a simple mechanism for generating a pulse of protein through the overshoot mechanism that is observed even in the absence of forced population synchrony Delayed feedback strongly affects the dynamics of genetic networks-it can show transient behaviour, or transition to a new fixed point accompanied by oscillations. Theoretical analysis of three eukaryotic genetic regulatory networks showing oscillations have been attributed to a common design of a negative feedback loop with time delay underlying them The interesting observation in our work is the presence of larger heterogeneity in gene expression in the cells with the Delay circuit compared to the Basic negative auto-regulatory case. In the Basic circuit, we observe a more homogeneous distribution of GFP in the cell populations that gradually moves from a low fluorescence value to higher ones with increasing inducer concentration. In the Delay circuit, on the other hand, at intermediate inducer values, fluorescence distribution is spread wider, and some cells seem not to express GFP at all. A single population is seen only at high inducer concentration representing all induced cells. Similar bimodality has also been observed for gene expression without negative feedback at medium inducer concentration and intermediate repression values also contribute to population variability in gene expression. Our results add to the growing body of evidence regarding the importance of transient dynamics in bringing about variability that in some cases may aid in the choice of alternate cell fates Interestingly, our theoretical model circuits are a minimal representation of the real cells, and they have no consideration of the realistic details such as, cell size or, variation of cell size during growth. It also considers full induction of all cells from the beginning. Our simulation results clearly predict what the experimental circuits would do at high induction levels, viz, the variability in both circuits would reduce, indicating that the heterogeneity in gene expression in the cell population occurs primarily during the unregulated state at lower induction level, which finally returns to the same level as the basic circuit, albeit delayed, due to establishment of repression. This also points to the fact that all heterogeneity, that is attributed to noise alone, need not be so, as delayed negative auto-regulation can The generic origin of delay in biochemical pathways (transcription regulatory pathways or metabolic pathways) implies that there is a high likelihood that the two properties shown in our study-transient overshoot and generation of heterogeneity in gene expression in cell population-play an important role in gene regulation. This motif of regulation can do two things successfully even before the stabilising effect of repression sets in. The overshoot allows for gene products being available in large amount for multi-step pathways to function, and it can also act as a dominant source of large deterministic variability paving way to increase the phenotypic diversity of a population of cells before the negative regulation sets in. It has been documented that phenotypic diversity can be crucial for many processes E.coli DH5α as described in The different circuits were constructed and cloned in The design of our negative auto-regulatory transcriptional modules with and without delay is given in On induction by Doxycycline (inducer), gene expression ensues, and fluorescence increases. Depending on the promoter strength, the number of plasmid copies, the half-life of the repressor, and the concentration of inducer, a dynamic balance is achieved between the intracellular inducer molecules and the repressor, and repression is re-established. The detailed study of growth of the circuits TG and C2TG show similar behaviour (The fluorescence of a representative population (200 µl) was measured in live cells using a Fluorescence ELISA reader . The settings used were 491 nm absorbance, 535 nm emission, with 530 nm auto cut off to reduce background fluorescence. The instrument sensitivity was kept at maximum and an average of 6 measurements was taken. As controls for auto fluorescence, uninduced samples of each induced circuit were also measured at each time point. The auto fluorescence was subtracted from the induced fluorescence and normalised by the growth (measured as OD at 600 nm) of the circuit to give the final fluorescence value per unit time.Flow cytometry was carried out using a FACS Calibur or a FACS Vantage (Becton-Dickenson) with a 488 nm laser using fixed cells as described in Deterministic and Stochastic models of the gene circuits corresponding to the negatively auto-regulated pathways with and without delay in repression were constructed using coupled differential equations and the modified Gillespie Algorithm It is known that variation in plasmid copy number in populations of cells is an important source of variability Supporting Information S1Construction of circuits; Cell Culture; Fixing protocol; Growth and Induction kinetics of different circuits; Gating of the FACS data.(0.04 MB DOC)Click here for additional data file.Supporting Information S2Models, Methods and Parameters(0.05 MB DOC)Click here for additional data file.Figure S1Growth of the circuits with and without induction. Legend: Uninduced-TG: triangles with solid lines, C2TG: Black circles with dotted lines. Induced-TG: squares with solid lines, C2TG: diamonds with dotted lines.(0.04 MB TIF)Click here for additional data file.Figure S2Growth of the circuits at different inducer concentrations (a) 25 ng/ml, (b) 50 ng/ml and (c) 75 ng/ml of Doxycycline. .(0.05 MB TIF)Click here for additional data file.Figure S3Kinetics of GFP fluorescence of the Basic , Control Delay , and Delay circuits upon induction in four independent experiments at different inducer concentrations-(a) 25, (b) 50, and (c) 75 ng/ml of Doxycycline, at 1 hr interval.(0.35 MB TIF)Click here for additional data file.Figure S4SDS-PAGE results showing TetR kinetics at different time points after induction-(A) Basic (TG) and (B) Delay (C2TG) circuits. The arrow indicates the position of the TetR band. The lower panel in each figure indicates the control band.(0.10 MB TIF)Click here for additional data file.Figure S5Quantification of intensity of the bands .(0.03 MB TIF)Click here for additional data file.Figure S6Gating of cell populations according to size, showing the greater uniformity of cell size in the gated population as compared to ungated population. (A) and (C) for TG and C2TG populations at 0 hr before gating, and, (B) and (D) for TG and C2TG populations after gating.(0.14 MB TIF)Click here for additional data file.Figure S7The effect of gating on the fluorescence distribution of the cells. TG (A) ungated and (B) gated; C2TG (C) ungated and (D) gated. X- axis = different time points represented in serial numbers, Y-axis = percentage of cells .(0.70 MB TIF)Click here for additional data file.Figure S8Frequency distribution of the gated population at various time points after induction of the circuits (A) TG and (B) C2TG. The X- axis: fluorescence in arbitrary units; Y-axis Time in min; Z-axis: Frequency.(0.53 MB TIF)Click here for additional data file.Figure S9Kinetics of TetR in the Basic (solid circles with dashed lines) and the Delay (squares with solid lines) circuits for (A) deterministic model and (B) stochastic model (average of 100 simulations).(0.13 MB TIF)Click here for additional data file.Table S1Comparison of kc value with k values from Nyquist loci for increasing delay.(0.02 MB DOC)Click here for additional data file.Table S2Parameter values used for deterministic and stochastic simulations(0.02 MB DOC)Click here for additional data file.
Transthoracic echocardiography is a primary non-invasive modality for investigation of heart transplant recipients. It is a versatile tool which provides comprehensive information about cardiac structure and function. Echocardiographic examinations can be easily performed at the bedside and serially repeated without any patient's discomfort. This review highlights the usefulness of Doppler echocardiography in the assessment of left ventricular and right ventricular systolic and diastolic function, of left ventricular mass, valvular heart disease, pulmonary arterial hypertension and pericardial effusion in heart transplant recipients. The main experiences performed by either standard Doppler echocardiography and new high-tech ultrasound technologies are summarised, pointing out advantages and limitations of the described techniques in diagnosing acute allograft rejection and cardiac graft vasculopathy. Despite the sustained efforts of echocardiographic technique in predicting the biopsy state, endocardial myocardial biopsies are still regarded as the gold standard for detection of acute allograft rejection. Conversely, stress echocardiography is able to identify accurately cardiac graft vasculopathy and has a recognised prognostic in this clinical setting. A normal stress-echo justifies postponement of invasive studies. Another use of transthoracic echocardiography is the monitorisation and the visualisation of the catheter during the performance of endomyocardial biopsy. Bedside stress echocardiography is even useful to select appropriately heart donors with brain death. The ultrasound monitoring is simple and effective for monitoring a safe performance of biopsy procedures. Over the past decade heart transplantation (HT) has evolved from a rarely performed procedure to an accepted therapy for advanced heart failure. About 45% of the candidates to HT have ischemic cardiomyopathy while 55% have some form of dilated cardiomyopathy of various origin. The prognosis for HT patients following the orthotopic procedure has greatly improved over the past 20 years, and a recent report (August 2006) of Heart Transplants: Statistics of American Heart Association. informs that the 5 years survival rates is 66.9% in women and 71.2% in men. Although significant advances have been reached in surgical techniques, in donor and recipient selection criteria, and also in the management of transplant patients, allograft rejection remains the most important cause of morbidity and. the primary limitation for the survival of these patients.Acute allograft rejection (AAR) is frequent in the first months after HT. Because it is initially asymptomatic, regular rejection surveillance is obligatory by monitoring immunosuppressive treatment, clinical and laboratory data and, in particular, by performing endomyocardial biopsies (EMBs), which represent the gold standard for the detection of rejection. AAR is characterised histologically by inflammatory cell infiltrates, interstitial edema and myocite necrosis which ultimately translates into structural and functional abnormalities of the allograft. A first international grading system for cardiac allograft biopsies, adopted in 1990 by the International Society for Heart Transplantation .Long-term survival of allografted hearts is limited by a progressive fibro-proliferative disease, resulting in intimal thickening and occlusion of the grafted coronary vessels. This disease, variously defined as accelerated transplant coronary artery disease (CAD) or cardiac graft vasculopathy (CAV), is also known as chronic allograft rejection ). After Echocardiography is particularly useful for the assessment of HT recipients since it is easily performable and not associated with the risks of the invasive procedures. Its versatility allows it to be applied in a wide variety of situations in the post-transplant setting.biatrial surgical approach) an anastomosis is made between the residual recipient atrial tissue and the donor atria such that there is a characteristic unique echocardiographic atrial morphological appearance. Therefore, transplanted hearts have increased size in both atria, primarily caused by an increase in their long-axis dimension. Sometimes, an echo dense ridge is also visualisable at mid-atrial level , it being the site of anastomosis between the residual recipient atrial tissue and the donor atria ] .IMP is an index which combines LV systolic and diastolic parameters and gives information about LV global performance . The ratAfter HT, it is evident an increase in wall thickness and LV mass, which can be detected and followed during time by serial echocardiographic examinations. LV hypertrophy is due to several causes and its progression occurs mainly in relation to cyclosporine levels and blood pressure levels . It is wValvular regurgitation is a fairly common occurrence immediately post HT but structural abnormalities of the aortic and mitral valves are not observed frequently after HT. In a long-term follow-up study, only 13 of 65 patients had mitral valve regurgitation (no patients had severe regurgitation), three cases of aortic valve regurgitation were reported and no patient has significant aortic valve stenosis . PossiblIn contrast to the left-sided valves, tricuspid valve regurgitation is very common after HT, its etiology being multifactorial. When its appearance is early after HT, probably it is related to elevated pulmonary arterial pressures and vascular resistance in the recipient as well as to atrial structure and function . In the majority of the cases, the resolution of tricuspid regurgitation is possible within 1 month after HT, hand in hand with the normalisation of pulmonary arterial pressures . Tricuspm velocity of the lateral tricuspid annulus has demonstrated to be lower than in controls, showing a trend to a further reduction during time after HT (18) and specificity (= 74%) for diagnosis of AAR are limited The possIntegrated backscatter (IBS) appears able to identify AAR by the decrease of cyclic variation signal [strain rate imaging (SRI), a tool which quantifies myocardial wall deformation and distinguishes "true" active myocardial contraction from passive wall motion [n signal and the n signal . The offl motion , has beel motion . SRI migl motion and reduSeveral attempts have been performed during time to take advantage by M-mode and 2-D echocardiography to diagnose AAR. The main echocardiographic variables proposed for diagnosis of AAR include increased wall thickness and wall echogenity, pericardial effusion, LV diastolic dysfunction and regional/global LV systolic dysfunction . Table 2Dobutamine stress echocardiography (DSE) identifies patients at risk for events and facilitates monitoring of CAV. A normal DSE predicts an uneventful clinical course and justifies postponement of invasive studies [m velocity and negatively with E/Em ratio in HT recipients: this findings indicates a possible association of impaired coronary microcirculation with both myocardial systolic dysfunction and increase of LV filling pressures in this clinical setting [The development of progressive CAD in HT has been recognised increasingly as long-term recipient survival has improved. Because CAV remains the major cause of death during long-term follow-up, its diagnosis is very important. It is remained for years in the prevalent domain of invasive techniques. Serial coronary angiography permits the study of coronary arteries and also the visualisation of coronary arteries by intravascular ultrasound (IVUS), that represents the reference gold standard in this clinical setting. Rapidly progressive vasculopathy by IVUS, defined as an increase of ≥ 0.5 mm in intimal thickness within the first year after HT, is a powerful predictor of all-cause mortality, myocardial infarction and angiographic abnormalities . However studies ,54,56. I studies . DSE has studies . The use studies . Also di studies ,62. Alth studies . Adenosi studies . A short studies . Figure setting .Table Another use of transthoracic echocardiography is the monitorisation and the visualisation of the catheter during the performance of endomyocardial biopsy . The useHeart donor storage is a main problem since patients in HT waiting list have a 7.3% death rate and the average waiting time is 2 to 3 years. In addition, there is a large amount of ''marginal'' recipients, it being due to either advanced age (> 65 years) or co-morbidity. Of consequence, a gradual trend toward liberalizing donor selection criteria has been developed and an expansion of the cardiac donor pool has involved accepting hearts of older donors, tolerating longer organ ischemic times and accepting hearts with structural and/or functional abnormalities, such as mild LV hypertrophy and mild valvular abnormalities -72. In tTransthoracic Doppler echocardiography is a primary non-invasive modality for investigation of cardiac transplant recipients. It is a versatile tool which provides comprehensive information about cardiac structure and function. Echocardiographic examinations can be easily performed at the bedside and serially repeated without any patient's discomfort. Although sustained efforts to develop and echocardiographic technique able to predict the biopsy state have been performed, it has fair to recognise that EMBs are still regarded as the gold standard for detection of acute allograft rejection. Conversely, stress echocardiography is able to identify accurately CAV and has recognised prognostic value, which is comparable to that of IVUS or angiography. A normal stress-echo justifies postponement of invasive studies. Bedside stress echocardiography is even useful to select appropriately heart donors with brain death. Finally, echocardiographic monitoring is simple and effective for monitoring a safe performance of biopsy procedures.The author(s) declare that they have no competing interests.SM conceived the study, participated in its design and drafted the manuscript, MM and MG reviewed the manuscript and participated in the design of the study, All the authors read and approved the final manuscript.
The F atom is disordered over four positions, on the two ortho positions of each ring, with occupancies of 0.287:0.213 (5). In the crystal structure, molecules are linked by intermolecular N—H⋯O and C—H⋯O hydrogen bonds.In the crystal structure of the title compound, C Å b = 5.2435 (10) Å c = 23.913 (5) Å β = 100.89 (3)°V = 587.3 (2) Å3 Z = 2 Kα radiationMo −1 μ = 0.11 mmT = 90.0 (1) K 0.25 × 0.20 × 0.10 mm Oxford Diffraction Xcalibur diffractometerAbsorption correction: none3819 measured reflections1205 independent reflectionsI > 2σ(I)1060 reflections with R int = 0.044 R[F 2 > 2σ(F 2)] = 0.034 wR(F 2) = 0.094 S = 1.07 1205 reflections118 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.28 e Å−3 Δρmin = −0.28 e Å−3 Δρ CrysAlis CCD used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008SHELXTL (Sheldrick, 2008SHELXL97.Data collection: 10.1107/S1600536808014645/bq2073sup1.cif Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808014645/bq2073Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:
Angiogenesis is fundamental to the progression of many solid tumours including prostate cancer. Sodium selenate is a small, water-soluble, orally bioavailable activator of PP2A phosphatase with anti-angiogenic properties.This was a dose-escalation phase I study in men with asymptomatic, chemotherapy-naïve, castration-resistant prostate cancer. The primary objective was to determine the maximum tolerated dose (MTD). Secondary objectives included establishing the safety, tolerability and pharmacokinetic profile.A total of 19 patients were enrolled. The MTD was 60 mg per day. Dose-limiting toxicity (fatigue and diarrhoea) was observed at 90 mg per day. The most frequently reported treatment-related adverse events across all treatment cohorts were nausea, diarrhoea, fatigue, muscle spasms, alopecia and nail disorders. No grade 4 toxicities were observed and there were no deaths on study. Linear pharmacokinetics was observed. One patient had a PSA response >50%. Median time to PSA progression (for non-responders) was 14.2 weeks. Mean PSA doubling time increased during the main treatment phase from 2.18 months before trial to 3.85 months.Sodium selenate is well tolerated at a dose of 60 mg per day with modest single-agent efficacy similar to other anti-angiogenic agents. Further trials in combination with conventional cytotoxic regimens are warranted. Prostate carcinoma is a common cancer, which is lethal in up to 15% of patients , a histological correlate of angiogenic potential, in human prostate samples. The MVD of prostate cancer is consistently higher than normal tissue, and is positively associated with more advanced tumour stage, higher Gleason scores, and the presence of metastatic disease. In addition, MVD predicts biochemical recurrence after both radical prostatectomy and external beam radiotherapy . On the The primary aims of this study were to assess the safety, tolerability and pharmacokinetics of sodium selenate in men with CRPC.μg l–1 required at study entry. LHRH agonists were continued and allowed concurrently. In addition, patients were required to be asymptomatic or have only minor symptoms due to their prostate cancer, have a WHO performance status ⩽2, and an estimated life expectancy of at least 6 months. Patients were also required to have adequate renal (serum creatinine <1.5 ULRR), liver (serum bilirubin <1.25 and AST/ALT <2.5 ULRR) and haematological reserve (absolute neutrophil count >1 × 109 l–1 and platelets >100 × 109 l–1), and have no evidence of severe or uncontrolled systemic disease.Patients with CRPC defined by at least three successive rises in serum PSA at least 2 weeks apart, in the presence of castrate levels of serum testosterone, were eligible for enrolment in this study. Anti-androgen therapy must have been discontinued at least 4 weeks before entry into the trial, with evidence of continuing PSA rises after this time, with a PSA level ⩾5 Patients treated previously with cytotoxic chemotherapy or strontium therapy, or who had coexisting malignancies or malignancies diagnosed within the last 5 years (with the exception of non-melanomatous skin cancer), were ineligible for enrolment. Treatment with any investigational agent within 4 weeks of study entry was not allowed, and patients with unresolved chronic toxicity greater than Common Terminology Criteria grade 2 from previous anti-cancer therapy or incomplete healing from previous surgery were excluded. Although the previous use of commercially available selenium supplementation was not a specific exclusion criterion, concomitant use over the study period was not permitted. Similarly, the concomitant use of unapproved or herbal remedies for prostate cancer was prohibited during the study period.The study received institutional review board approval and all patients gave written informed consent.This was an open-label phase I dose-escalation study. After signing informed consent, patients underwent baseline testing to confirm eligibility. Patients who satisfied the inclusion and exclusion requirements were invited to enter the study according to the dose-escalation criteria. Baseline evaluations were conducted within 2 weeks of starting therapy. Sodium selenate was administered daily for 3 weeks (one cycle). After four cycles of therapy (12 weeks), patients with stable or responding disease, and who wished to continue on study, were offered ongoing treatment for a further 12 weeks. During the main treatment phase (weeks 0–12) patients underwent assessments for safety and tolerability at the end of each cycle. Patients opting to continue the drug beyond this time were similarly evaluated at six weekly intervals. All patients were assessed for safety 28 days after the last dose of study drug, and where possible, all patients were evaluated 3 months after their final treatment with sodium selenate.At study commencement, sodium selenate was administered as an oral capsule taken once a day on an outpatient basis. Patients were given a fixed dose and there was no intra-patient dose-escalation. After the initial pharmacokinetic analysis demonstrated a short drug half-life in serum, the same total daily dose was given in three divided doses in one 24-h period to generate steady-state plasma selenate levels (beginning with patient 5).For the initial dose levels, an accelerated titration design was used with one patient at each dose treated and evaluated at the end of 3 weeks. Toxicities were graded according to Common Terminology Criteria for Adverse Events version 3. Drug-related toxicity (DRT) was defined as any grade 2 non-haematological toxicity and/or any grade 3 haematological toxicity. In the absence of a DRT, commencement of a patient at the next dose level occurred. Three patients were subsequently enrolled to each fixed-dose cohort, with planned dose levels of 60, 90 and 120 mg per day. The protocol was later amended to include 45 mg per day to collect additional safety information. Dose-escalation continued until dose-limiting toxicity (DLT) was observed in one-third of patients during cycle 1. The DLT was defined as any grade 3/4 non-haematological toxicity and/or and grade 4 haematological toxicity. If DLT was observed in one of three patients in a cohort, a further three patients were recruited to that dose level. If one further patient experienced DLT, then this defined the DLT level. If no DLT was observed, patients were recruited to the next dose level. The maximum tolerated dose (MTD) was defined as the dose level below that in which ⩾33% (2 of 6) of patients were observed to have a DLT. The MTD was then expanded to six patients.If patients developed ⩾ grade 2 haematological and/or non-haematological toxicity during the main treatment phase, sodium selenate was not administered. If treatment was deferred because of toxicity, it could be re-initiated once toxicity was ⩽ grade 1. Treatment delays of up to 2 weeks were allowed, but no dose reductions were permitted. In the absence of treatment delays due to adverse events, treatment continued for up to four cycles or until the disease progressed, the occurrence of an inter-current illness that prevented further administration of treatment, or the patient decided to withdraw from the study. In the absence of toxicity, patients were allowed to remain on selenate beyond the main treatment phase if they chose to do so.Sodium selenate was blended with the inert pharmaceutical excipient lactose and filled into capsules using a Dott Bonapace semi-automatic capsule filler, under GMP conditions, using the appropriate size capsules. Three dosage strengths were prepared, containing 5, 20 or 50 mg of sodium selenate. Independent quality control testing was performed on the finished product, and stability at 25°C for the time course of the study confirmed.–1 confirmed on a second reading ⩾3 weeks later. For patients not experiencing a decline in baseline PSA, the date of progression is defined by the date of documentation of PSA ⩾25% with an absolute increase ⩾2 ng ml–1 above baseline, after 12 weeks on treatment. In addition, PSA doubling time was calculated using the log-slope method as previously described . All patients entering the trial had an additional safety visit at 4 and 12 weeks after completion of therapy to assess for residual toxicity. To assess for tumour response, serum PSA was measured at the completion of each treatment cycle. Although the trial was initiated before the publication of the Prostate Cancer Trials Working Group 2 criteria for PSA end points, PSA data are analysed according to their recommendations and its various metabolites using ion chromatography dynamic reaction cell inductively coupled plasma mass spectrometry , post-dose at 30 min, 1, 2, 4, 6, 24, 48 and 72 h. In addition, trough samples were taken at days 7, 14 and 21. Samples of 10 ml were collected into potassium–EDTA tubes from an indwelling arm vein catheter, and centrifuged at 2000 trometry .Cmax, Cmin, Tmax and AUClast. AUC was determined by the linear trapezoidal rule, where AUClast was the AUC from time zero until the last concentration point. The elimination rate constant, Kel (h−1) was determined by linear regression of a minimum of three points. Half-life (t1/2) was determined according to t1/2=0.693 Kel–1.Pharmacokinetic results were processed according to standard non-compartmental analytical procedures . The actual times of sample collections were used in the calculations. Parameters measured included All data presented are descriptive and no formal statistical evaluation was performed.μg l–1, with an average PSA doubling time of 2.2 months (1.1–8.9). In all, 7 of 19 patients (36.9%) had previously received treatment to the primary tumour, three with radical surgery and four with external beam radiotherapy. The remaining 12 patients had androgen deprivation as their only prostate cancer treatment. In all, 10 of 19 patients (52.6%) had metastatic bone disease at the time of enrolment.Between June 2006 and November 2008, a total of 19 patients were enrolled in the study and their clinical characteristics are summarised in Of the 19 patients enrolled into the study, 12 patients (63%) completed the 12-week treatment period . In all,Two events considered to be DLTs were reported by two patients in the 90-mg (30 mg t.d.s.) cohort. Thus, 60 mg (20 mg t.d.s.) was determined to be the MTD. The DRTs were seen in the 60-mg (20 mg t.d.s.) and 90-mg (30 mg t.d.s.) cohorts. Fatigue was the most commonly reported DRT.–1 (as compared with 90 mmol l–1 at screening) when scheduled biochemistry assessments were performed at the end of the first cycle of treatment. At this visit, the patient was also noted to have decreased bicarbonate levels (19 mmol l–1 compared with 30 mmol l–1 at screening), associated with diarrhoea and dehydration. The study drug was stopped immediately and the patient was admitted for investigation of acute renal impairment. Examination of the patient's clinical records revealed the patient had a history of proteinuria, macro-haematuria and underlying kidney disease before commencing sodium selenate. Despite investigation, no cause for the acute deterioration in renal function could be determined, and a contribution of sodium selenate to the existing underlying renal disease cannot be ruled out. Creatinine levels were further elevated to 290 mmol l–1 when re-tested 12 weeks after the last study dose, although bicarbonate levels had normalised. The four serious adverse events considered not to be associated with the study drug were episodes of pelvic pain, bowel obstruction, renal colic and lower respiratory tract infection.A total of five serious adverse events were reported by five patients, only one of which was considered to have a potential causal relationship to sodium selenate. This was a patient in the 90-mg (30 mg t.d.s.) cohort who presented with an elevated creatinine level of 260 mmol lApart from the patient described above, there were no clinically significant laboratory results considered to be potentially related to the study drug, and no patients were found to have vital sign assessments or ECG results that were abnormal and clinically significant.vs dose, across the dose ranges assessed in this study, with the caveat of small cohort size. The selenate t1/2 ranged from 1.2 to 2.9 h in the 5–30 mg single-dose cohorts, respectively. The short half-life necessitated a three times a day daily dosing regimen to maintain adequate blood levels of sodium selenate. Peak selenate plasma concentrations were achieved within 1–4 h across all dose levels. At the recommended phase II dose, that is, 20 mg t.d.s., the mean tmax was 2.5 h and the mean t1/2 was 2.9 h.The PK parameters are detailed in tmax ranging from a mean of 90 h for the 10 mg t.d.s. cohort to 294 h for the 30 mg t.d.s. cohort (data not shown). Plasma selenite levels reached steady-state levels by 3 weeks. The ratio of selenite AUC504vs selenate AUC504 ranged from 1.3 × at 10 mg t.d.s. to 7 × at the 30 mg t.d.s. dose level. Seleno-methionine, seleno-cyanate and methyl selenium species were other significantly minor metabolites identified in plasma. For the t.d.s. dosing cohorts, 16–37% of total daily dose of selenium was recovered in the 24-h urine. Selenate was the major selenium species identified in the 24-h urine accounting for 10–24% (based on the mean for each t.d.s. dosing cohort) of total daily selenium administered. Seleno-methionine and miscellaneous methyl selenium species were also major selenium compounds identified in 24-h urine; however, levels varied significantly across the patients in the t.d.s. dosing cohorts. Selenite was barely detectable in the urine, being <0.3% of total daily dose.The major selenium metabolite identified in plasma was selenite. Its plasma concentrations showed signs of accumulation, with n=12), median time to progression was 14.2 weeks (13.1–41.1)Although the study was not designed to determine clinical efficacy, PSA was monitored throughout the trial as a surrogate marker of tumour response. The percentage change in PSA from baseline for each individual patient is shown in Mean doubling time increased from 2.2 months (1.1–9.0) before trial to 3.9 months (1.1 to −2.0) during the main treatment phase .The management of metastatic prostate cancer has changed significantly in the past decade. Cytotoxic chemotherapy with docetaxel is a standard of care in castrate-resistant disease and new therapies, including hormone agents such as abiraterone, show promising activity. However, no current therapy is curative and novel therapies are still needed. Most recently, many clinical trials have focussed on anti-angiogenic agents targeting critical kinases.We have previously identified that the specific selenium-containing compound, sodium selenate, significantly boosts the activity of the protein phosphatase PP2A, inhibits VEGF-induced growth and survival signalling in endothelial cells, and impedes tumour neovascularisation.This ‘first-in-human’ study examined the use of sodium selenate in men with CRPC before they received cytotoxic chemotherapy. Sodium selenate was well tolerated at doses up to 45 mg per day t.d.s. and the MTD was attained at a dose of 60 mg per day t.d.s. DLT was fatigue and diarrhoea, while other frequently reported adverse events included, nail disorders, muscle spasms, alopecia and nausea. Some of the side effects observed at the higher doses, in particular the fatigue, diarrhoea, alopecia and vomiting are perhaps attributable to the accumulation of the inorganic metabolite selenite, a selenium compound with marked cytotoxic properties and a very different pharmacokinetic profile compared with selenate, as confirmed in this study , significant or sustained responses are rarely observed with single-agent therapy. This clinical difference may simply reflect the strength of angiogenic drive within different tumour types, as renal cancers, together with glioblastomas, have the highest rates of endothelial cell proliferation and levels of immature blood vessels of tumour types studied, certainly significantly higher than prostate cancer, and thus may be more intrinsically sensitive to angiogenic inhibition (http://www.roche.com/media/media_releases/med-cor-2010-03-12.htm). As only the VEGF signalling pathway was targeted, it is possible that combining anti-angiogenic agents with different mechanisms of action can overcome the inherent redundancy in angiogenic signalling in prostate cancer, and improve clinical response rates. This is supported by recently published phase II data exploring the combination of docetaxel, bevacizumab, thalidomide and prednisone, with 90% of patients experiencing a PSA decline of ⩾50% (Despite their lack of single-agent activity, anti-angiogenic agents have shown favourable effects in boosting the efficacy of standard chemotherapy regimens in different tumour types . InteresFurther exploration of sodium selenate as a single-agent in CRPC is not warranted; however, its combination with conventional cytotoxics in particular docetaxel, as well as with other anti-angiogenics with different modes of action is worthy of further exploration.
Alexandrium are the proximal source of neurotoxins associated with Paralytic Shellfish Poisoning. The production of these toxins, the toxin biosynthesis and, thus, the cellular toxicity can be influenced by abiotic and biotic factors. There is, however, a lack of substantial evidence concerning the toxins' ecological function such as grazing defense. Waterborne cues from copepods have been previously found to induce a species-specific increase in toxin content in Alexandrium minutum. However, it remains speculative in which context these species-specific responses evolved and if it occurs in other Alexandrium species as well. In this study we exposed Alexandrium tamarense to three copepod species and their corresponding cues. We show that the species-specific response towards copepod-cues is not restricted to one Alexandrium species and that co-evolutionary processes might be involved in these responses, thus giving additional evidence for the defensive role of phycotoxins. Through a functional genomic approach we gained insights into the underlying molecular processes which could trigger the different outcomes of these species-specific responses and consequently lead to increased toxin content in Alexandrium tamarense. We propose that the regulation of serine/threonine kinase signaling pathways has a major influence in directing the external stimuli i.e. copepod-cues, into different intracellular cascades and networks in A. tamarense. Our results show that A. tamarense can sense potential predating copepods and respond to the received information by increasing its toxin production. Furthermore, we demonstrate how a functional genomic approach can be used to investigate species interactions within the plankton community.Marine dinoflagellates of the genus Alexandrium possess a high ecological impact due to their ability to form Harmful Algal Blooms associated with Paralytic Shellfish Poisoning (PSP). PSP is a threat to marine aquaculture and shellfish consumers, occurring worldwide with increasing frequency and distribution Dinoflagellates of the genus Alexandrium with no adverse effects on the grazers as well as (2) enhanced mortality of the grazer Alexandrium strain investigated, as well as on the grazer species Microcystis spp. strains, for example, can differ significantly in their toxin content Daphnia spp.) shows different levels of impairment upon grazing, potentially due to differences in detoxification abilities or adaptations to the toxins following post-exposure Alexandrium populations are composed of different strains, producing different amounts of PST, and possessing different PST profiles Alexandrium blooms, are less affected by PST compared to copepods originating from regions devoid of AlexandriumAlexandrium over toxic cells by copepods AlexandriumAlexandrium minutum after exposure to waterborne cues from copepods, which correlated with a decreased copepod grazing. Further, Bergkvist et al. A. minutum only increased its PST production significantly when exposed to waterborne cues from two out of three different copepods, which indicates that toxin production might be target specific.Saxitoxin is one of ∼two dozen naturally occurring PST and was first discovered in 1975 Alexandrium tamarense when exposed to copepod grazing or the waterborne cues from copepods. By collecting the copepods from the same geographic regions as the origin of the A. tamarense strain used, we assumed that the copepods had a history of co-existence with A. tamarense. It has been suggested that co-existence is an important driver for the chemical cue specific responses A. tamarense cells to actively grazing copepods or only their waterborne cues should induce an increase in PST production by A. tamarense. The impact of PST on the copepods, post exposure to A. tamarense, was estimated by relating their internal PST concentration, ingestion rates and behavioral response to the toxins. The response of A. tamarense after exposure to copepods and potential waterborne cues was assessed via screening of gene expression patterns through microarray analyses for all treatments and related to the PST measurements. This functional genomic approach allowed us to trace cue perception to changes in gene expression, since potential waterborne cues recognized by A. tamarense may alter gene expression through receptor stimulation.This study aimed to investigate the effects on PST content in C. helgolandicus individuals and water borne cues caused the largest increase in A. tamarense PST content per cell compared to the control whereby both the test treatments differed significantly from the control treatment, and also from each other (Holm-Sidak method: p<0.05). In contrast, no significant change in total PST content of A. tamarense was observed when exposed to O. similis individuals or O. similis waterborne cues . Similarly after exposure to A. clausii, no significant changes in cellular PST concentrations of A. tamarense could be verified .Exposure to control . IncubatC. helgolandicus had two folds higher average ingestion rates of A. tamarense cells compared to the average ingestion rates of both O. similis and A. clausii, which had ingestion rates of ∼50 and ∼60 cells per female per hour, respectively (p>0.05). The lowest weight-specific PST content was found in C. helgolandicus, which had an average weight-specific PST content 3.7 and 2.5 times lower than the average weight-specific PST content for O. similis and A. clausii, respectively . After 48h incubation with Alexandrium cells, 57% of the C. helgolandicus individuals showed an escape response, interpreted as normal response behavior, while 29% showed no escape response and 14% of C. helgolandicus died during the incubation. In contrast, O. similis individuals revealed a significantly higher proportion of affected copepods : 86%. Here, the group of affected copepods is composed of 65% individuals with no escape response and of 21% dead individuals. No noticeable change in escape behavior and therefore no effect were observed for 14% of the Oithona individuals. Acartia individuals exposed to A. tamarense showed an equal mean distribution of not-affected and affected copepods (50% each). Due to the standard deviation no significance could be determined (A significantly higher proportion of unaffected copepods was only observed within the p>0.05) . Affectet-test against control treatment; p<0.05; C. helgolandicus individuals and C. helgolandicus waterborne cues whereas the lowest numbers were observed in the treatments containing O. similis individuals and O. similis waterborne cues. Numbers of up-regulated genes observed from the A. clausii treatments were intermediate. Regarding the numbers of down-regulated genes, numbers observed for the treatments with C. helgolandicus and A. clausii individuals and waterborne cues exceeded numbers achieved for the C. helgolandicus waterborne cue treatment and the O. similis treatments.A significant change in the gene expression compared to the control treatment was observed for every treatment . Hence, the species-specific alarm cues are not activated when non-threatening copepods are present, e.g. O. similis.Necessary for this adaptive response to different copepod species is that Alexandrium must have receptors transmitting the received signals into the cell where they are processed, and finally induce the expedient response to the received infochemicals. Such receptors comprise fast evolving proteins that are coupled to signal cascades and are capable of adapting to new or changing environmental cues in a relatively short period of time through slight mutations of an ancestor receptor Oxyrrhis marinaAlexandrium to integrate waterborne cues from the environment and to respond appropriately.On a molecular level this means that Alexandrium gene expression with observed overlapping responsive genes in the respective copepod/copepod-cue treatment for every species tested. Thus, the observed gene expression patterns indicate that the presence of all three tested copepod species and their cues was sensed by A. tamarense, yet elicited a different response into the class of mitogen-activated protein kinases (MAPKs) and respective downstream kinases (MAPKKs) and four are grouped into the class of calcium dependent protein kinases (CDPKs). In the A. clausii treatment, one kinase could be grouped into the class of CDPKs whereas no MAPK or CDPK was among the regulated genes in the O. similis treatment. MAPK cascades are known to be an important pathway downstream of receptors regulating cellular responses to several external stimuli and are evidenced to be involved in regulating defensive responses through jasmonic acid formation after wounding in tobacco species Nicotiana attenuate and Solanum nigrumN. attenuate is described in Wu et al. C. helgolandicus inducing the highest increase in internal PST levels of A. tamarense showed the strongest change in altering expression levels of MAPKs and CDPKs transcripts. A direct involvement of MAPKs and CDPKs in coordinating the response towards copepod grazers in A. tamarense should therefore be considered.Different expression rates of serine/threonine kinases observed in this study suggest that these serine/threonine kinases are involved in the different responses of cues see . Three o2+ signaling highlight their importance in responding to environmental changes A. tamarense.Besides CDPKs, further transcripts of proteins with a dependency of calcium ions as second messengers have been identified as differently expressed in this study. The regulation of intracellular calcium concentrations and oscillations is a well-described mechanism to transduce environmental changes into the cell . Also in marine phytoplankton, several studies concerning CaA. tamarense is inducible through the presence of copepod grazer in a species-specific manner and that these specific responses are detectable through functional genomic approaches. The role of PST in acting as defense compound should therefore be reconsidered with respect to genotypic variation and phenotypic plasticity of Alexandrium species and tested copepod grazers. Since the present study investigated the effect of one clonal strain of A. tamarense on the copepod species we cannot exclude different outcomes of the same study when using different strains. The treatments with copepods or copepod-cues resulting in altered gene expression patterns can be considered as an outcome of the ongoing dialog between Alexandrium and its environment and the result of integrating and processing the received information into the most appropriate response selected through evolutionary processes. Different abundances of MAPKs together with CDPKs and further Ca2+-dependent proteins potentially influence the overall cell response through directing the external stimuli into different intracellular cascades and networks.This study demonstrates that increased PST production in Alexandrium tamarense clonal strain was isolated in May 2004 from the North Sea coast of Scotland −1 prior to the experiment. The used clonal strain contains intermediate toxin content in comparison with 9 other A. tamarense strains at 15°C in darkness before each experiment. To avoid anoxia in the 20 L containers the seawater was gently bubbled with air (5–10 cm between each bubble). Females were chosen because they release metabolites that attract males and function as a chemical trail potential perceived by A. tamarense in this experiment Female copepods were collected with vertical WP2 net hauls (200 µm) from 50 m depth to the surface in the North Sea during 12–29 August 2007. A. tamarense we incubated the clonal strain in two types of K-medium prepared from filtered (0.2 µm) sea water collected in situ; a) K-medium with no further manipulation (control treatment) and b) K-medium in which copepods had been kept for 24 hours and potential released metabolites during the incubation (copepod-cue treatment). In each experiment A. tamarense was incubated in 1.15 L bottles at a concentration of approximately 4×106 cells L−1. This concentration was necessary to ensure enough material for downstream analysis . Nine bottles were incubated for each treatment, three bottles containing copepods, three bottles containing the K-medium with potential copepod-cues (treatments) and three bottles without copepods or any trait of them (control). For copepod containing bottles, we either used (per bottle) 10 C. helgolandicus females, 15 A. clausii females or 30 O. similis females. The K-medium was consequently pretreated with potential copepod cues from the corresponding number of copepod females. All copepod species used for this experiment were dominating species in the waters investigated. The feeding range of the selected copepod species is within the right range to enable them grazing on A. tamarense cells feeding on A. tamarense in the treatments (see To investigate if the presence of copepods has an effect on the toxicity of ents see . Incubat−1) for 45 s. Afterwards, cells were frozen in liquid nitrogen and stored at −80°C until further use.Culture bottles were taken off the plankton wheel after 48 h, careful turned over head 7 times to ensure equal mixing of the culture. A sterile pipette tip was used to take out 10 mL of the culture and preserved in acid Lugol's solution for cell counts. Afterwards the culture was poured through a 100 µm mesh followed by a 10 µm mesh to filter out the copepods and to sample the cells. The copepods were transferred into a container with sterile filtered sea water for further examination of their condition. The 10 µm mesh containing the cells was washed out into a 50 mL collection tube with sterile sea water and adjusted to a volume of 40 mL. From this concentrated cell culture 4 mL were used for toxin analysis and the remaining 36 mL where immediately centrifuged at 4°C for 5 min for RNA samples. Cell pellets were immediately mixed with 1 mL 60°C hot TriReagent , transferred to a 2 mL cyrovial containing acid washed glass beads. Cells were lysed using a Bio101 FastPrep instrument at maximum speed . The RNA-sample was further cleaned with the RNeasy Kit (Qiagen) according to manufactures protocol for RNA clean-up including on-column DNA-digestion. RNA quality check was performed using a NanoDrop ND-100 spectrometer for purity and the RNA Nano Chip Assay with the 2100 Bioanalyzer device was just to examine the integrity of the extracted RNA. Just high quality RNAs (OD 260/280>2 and OD260/230>1.8) as well as with intact ribosomal peaks were used for further experiments.A. tamarense total RNA samples derived from the copepod treatment and the waterborne cues treatment were hybridized against the respective control treatment. The microarray hybridization procedure was carried out using 500 ng total RNA from each sample and the Agilent two color low RNA Input Linear Amplification kit . Prior to the labeling, the Agilent RNA Spike-In Mix (Agilent) was added to the RNA. The RNA was reverse transcribed into cDNA, amplified towards cRNA and labeled following the ‘Agilent Low RNA Input Linear Amplification Kit’ protocol (Agilent). Dye incorporation rates (cyanine-3 and cyanine-5) and cRNA concentrations were measured using the NanoDrop ND-100 spectrometer (PeqLab). Hybridization was performed onto 4x44k microarray slides at 65°C for 17 h (Agilent). The microarrays contained 60mer oligonucleotide probes designed by Agilent's E-array online platform from three Alexandrium spp. EST libraries. For each hybrization, 825 ng of cyanine-3 and cyanine-5 labeled cRNA were used. After hybridization, microarrays were disassembled and washed according to manufacturer's instructions (Agilent). Microarrays were scanned using an Agilent G2565AA scanner. Raw data were processed with the Agilent Feature Extraction Software version 9.1.3.1 (FE). Array quality was monitored using the Agilent QC Tool (v1.0) with the metric set GE2_QCMT_Feb07. The array design, raw data and normalized data and the detailed experimental design are MIAME compliant and deposited in a MIAME compliant database . Testing for differential expressed genes was performed using the GeneSpring GX software platform version 11 (Agilent) with the implemented T-test against zero statistics combining biological replicates. Genes were considered to be differential expressed when test statistics reply p-Values were less than 0.05 and calculated fold changes between the control and the treatment exceeded 1.5.−1) for 45 s. and centrifuged afterwards for 15 min at 13.000 g and 4°C. From the supernatant, 400 mL were passed through a spin filter (pore size 0.45 µm) by centrifugation for 30 sec. at 3000 g. For the copepods the same procedure was applied with the following chances: after removing the remaining seawater from the copepods, some liquid nitrogen was filled in the tube and the copepods were crushed. After evaporation of the nitrogen the acetic acid was filled in the tube. The samples were further processed as described for the cell pellet samples. The filtrate was injected into the HPLC/FLD equipped with a fluorescence detector The LC-FD analysis was carried out as previously described in detail in Krock et al PSP toxins were extracted according to Krock et al. Table S1C. heloglandicus treatments with live copepods and respective cues.Full list of regulated ESTs with annonations from the (XLS)Click here for additional data file.Table S2O. similis treatments with live copepods and respective cues.Full list of regulated ESTs with annonations from the (XLS)Click here for additional data file.Table S3A.clausii treatments with live copepods and respective cues.Full list of regulated ESTs with annonations from the (XLS)Click here for additional data file.
Ineach treatment, the most rapidly-germinating seeds were selected, grown to maturity, andsubjected to molecular marker analysis. A selective genotyping approach detected between 6 and9 QTL affecting germination rate under each of the four conditions, with a total of 14 QTLidentified. Ten QTL affected germination rate under 2 or 3 conditions, which were consideredgermination-related common QTL. Four QTL affected germination rate only in one treatment,which were considered germination-related, condition-specific QTL . The results indicated thatmostly the same QTL affected seed germination under different stress and nonstress conditions,supporting a previous suggestion that similar physiological mechanisms contribute to rapid seedgermination under different conditions. Marker-assisted selection for the common QTL mayresult in progeny with rapid seed germinability under different conditions.The purpose of this study was to determine whether the rates of tomato seed germination underdifferent stress and nonstress conditions were under common genetic controls by examiningquantitative trait loci (QTL) affecting such traits. Seedsof BC Solanum lycopersicum L. Seed germination is particularly important if the target environment is lessthan optimal during germination. Unfavorable conditions may lead to decreasedrate and final percentage of seed germination, which may result in poor standestablishment and low crop yield. Under optimal germination conditions , most tomato seeds germinatewithin 2–5 days. Understress conditions, such as extreme temperatures, high soil salinity, and waterdeficit, however, germination is delayed or completely inhibited depending onthe intensity and duration of stress as well as genetic background of the seed.In some tomato-growing areas, the crop is established by sowing seeds directlyinto the field instead of using transplants. Presence of environmentalstresses, however, restricts establishment of direct-seeded crops. Mostcommercial cultivars of tomato are sensitive to environmental stresses duringseed germination and early seedling growth [Theability of the seed to germinate rapidly and uniformly under differentenvironmental conditions is a desirable characteristic for most crop plants,including tomato, g growth –3. Such Solanum species for rapid seedgermination under stress conditions [Genetic variation exists within tomato nditions , 4–6. Sunditions –5, 7, 8.nditions , suggestnditions . HoweverTomato seed germination under differentconditions exhibits continuous distributions, typical of quantitative traits , 7. DuriAn effective approach to identifying genetic linkage between markerloci and QTL is trait-basedmarker analysis , 12, alsS. pimpinellifoliumL. and F1 progeny wasproduced. NC84173 is a horticulturally superior advanced tomato breeding line(PVP) that is sensitive to cold, salt, and drought stress during seedgermination and LA722 is a self-compatible accession that germinates rapidlyunder most conditions, including nonstress and cold, salt, and drought stress.Original seed of NC84173 and LA722 were obtained from RG Gardner, NorthCarolina State University and CM Rick Tomato GeneticsResource Center, University of California , respectively. Asingle F1 plant was used as pollen parent to hybridize plants ofNC84173 to produce BC1 seed. The BC1 population was usedfor trait evaluation, genetic mapping, and identification QTL.The tomato breeding line NC84173 washybridized (as pistillate parent) with a fast germinating accession (LA722) oftomato wild species NaCl + 15 mM2CaCl and that for the drought treatment included 14% PEG.Germination media were prepared in 15-cm round Petri plates. The waterpotentials (ψ) of thetreatment media were −30, −30, −690, and −680 kPa for thecontrol, cold, salt, and drought treatments, respectively, as measured on aWescor-5100 vapor pressure osmometer . Seeds ofparental lines (NC84173 and LA722) and BC1 population weresurface-sterilized with 0.5% NaOCl solution for 10 minutes, rinsed withsterile, distilled water, and briefly blotted. For each of the control, cold,salt, and drought treatments 1000 seeds of BC1 generationand 192 seeds of each of the parental lines were sown on germination mediaunder aseptic conditions. Each Petri plate contained 64 seeds and wasconsidered as one replicate. Petri plates were placed in a completelyrandomized design (CRD) in incubators maintained in dark at either 20 ± 0.5°C or 11 ± 0.5°C (coldtreatment). Germination responses were scored visually as radicle protrusion at8-hour intervals for 37 consecutive days. To estimate mean germination time,germination distributions of the parental lines and BC1 populationin the four treatments were subjected to survival analysis, [Sterile germination media, containingeither 0.8% w/v agar or 0.3% w/v Phytagel (for drought-stress treatment), were prepared.For the drought treatment, Phytagel was used as agelling agent as agar does not gel with drought agent polyethyleneglycol (PEG).The germination medium for the salt treatment also included 150 mM 1 seeds (thefirst 3% germinated) were selected (hereafter referred to as “selectedclasses”). Selected seedlings from the different treatments were transplantedinto greenhouse seedling trays and subsequently into a field, where they weregrown to maturity and self-pollinated to produce BC1S1 progeny seed. The BC1S1 progeny were examined for rate ofseed germination under different conditions, as described elsewhere [In each of the control, cold, salt, anddrought-stress treatments the 30 most rapidly germinating BClsewhere .1 plants wascollected for DNA isolation and marker analysis. Nuclear DNA was extracted using standard protocols for tomato [DraI, EcoRI, EcoRV, HindIII, and XbaI according to the manufacturer's instructions and subjected to gelelectrophoresis. Genomic blots were prepared and hybridized with 119 DNAprobes, which previously were determined to detect polymorphism between the twoparents [32P-dCTP byprimer extension [Leaf tissue from each of the 120 selected BCparents , includixtension . Agarosextension .1 population(N = 119) of the same cross (NC84173 ×LA722) was previously genotyped with 151 RFLPmarkers, including the 115 markers used in the present study, and a geneticlinkage map was developed [1 population, which thenwere used to calculate differences in marker allele frequencies betweenselected and nonselected populations and identify QTL, as described below.A nonselected BCeveloped . In the 1 plantsfrom each of the four selection treatments were determined for the 119 RFLP markers. Using the genotypic numbers obtainedfor the 119 RFLP markers, marker allele frequencies were determined for each ofthe four selected classes. The variance of allele frequency for each marker wascalculated as a binomial variance were determined, where Sq is the frequency of the ith allele at the kth marker locus in each of the selected classes (N = 30) and NSq is the frequency of the ith allele at the kth marker locus in the nonselected population (N = 119). Allele frequency differencesbetween the selected classes and the nonselected population were consideredsignificant when S-qNSq ≥2σq, where σq = SqNS/2NS(pNSqNS/2NNS)1/2+p is the standarderror of the difference between marker allele frequencies, SN is the number of BC1 progeny in eachselected class, and NSN isthe number of individuals in the nonselected BC1 population. Thistest provides a confidence of more than 95% on the identified QTL [S-qNSq was smaller than σq2 butgreater than 1σq, the marker was judged to be associatedwith a QTL with minor effects.Marker allele frequency differencesbetween each of the selected control, cold, salt, and drought classes and thenonselected BCtion N = 9. Allelfied QTL , 23, 24.fied QTL , 17, 25.While selective genotyping is more powerful thanstandard marker-based analysis in detecting linkage betweenmarkers and QTL (primarily because of the use of large population), it is lessefficient in determining QTL effects. Individuals in the high class tend tohave a large number of positive QTL alleles and individuals in the low classtend to have a large number of negative QTL alleles, and there is a deficiencyof individuals with a mixture of positive and negative alleles in the subpopulationsbeing analyzed (this is particularly true for traits with high heritability).This deficiency hampers the ability to measure the effect of individual QTLusing traditional analysis of variance. However, the approximate effects of QTLcan be estimated using an equation that relates the change in marker allelefrequencies due to selection with the QTL effects (described below). Thisequation assumes no recombination between the marker locus and the QTL. Whenthis assumption is met, the larger the effects of the QTL, the greater would bethe difference in marker allele frequency in response to selection. However, ifrecombination occurs between the marker and the QTL, the effect of the QTLwould be underestimated.i, with the coefficient of selection, s, acting on an individual gene (or QTL) as follow:D = 2 d/σP is the standardized effect of the QTL and d is the phenotypic difference between the two homozygotes at the QTL. Withfurther substitution for s andassuming no recombination between the marker and the QTL, the standardizedeffect of a QTL, as a function of selection intensity and the difference inallele frequencies at a linked marker resulting from a one-step directionalselection in a BC1 population, can be estimated asδq is the difference in marker allelefrequencies between a selected class and the nonselected population , i is the selection intensity , and q is the allelefrequency at the QTL-linked marker locus in the nonselected population. Usingthis expression, the approximate standardized effects of the marker-linked QTLwas estimated. It should be noted that the calculated values are consideredminimum effects of QTL due to likely recombinations between markers and QTL.Falconer provided1 populationgerminated intermediate between the two parental lines, indicating theinheritance of rapid germination from LA722 to the progeny (Seed of the wild accession LA722 germinatedsignificantly more rapidly than seed of the breeding line NC84173 under nonstress (control) as well as cold, salt, anddrought stress conditions; the difference between the two parents, however, wasgreater under stress than nonstress conditions . This is progeny .1 population of the same cross from a previous study [Using a nonselected BCus study and the us study . The prous study , 27. Thius study .QTLfor germination under nonstress (control) conditions. Four major QTL and 3 minor QTL were identified for germinationunder nonstress conditions (see D) of the QTL ranged from 0.38 to 0.87 phenotypicstandard deviation.ions see . All QTLQTLfor germination under cold stress conditions. Three major QTL and 6 minor QTL were identified for germination under cold stress (see D) of the identified QTL ranged from0.31 to 0.69 phenotypic standard deviation.ress see . For allress see . The staQTLfor germination under salt stress conditions. Four major QTL and twominor QTL (on chromosomes 4 and 12) were identified for germination under saltstress (see D) of the identified QTL ranged from0.45 to 1.01 phenotypic standard deviation.ress see . All QTLress see . FurtherQTLfor germination under drought stress conditions. Two major QTL (on chromosomes 8 and 9)and seven minor QTL were identified for germination underdrought stress (see D)of the identified QTL ranged from 0.33 to 1.02 phenotypic standard deviation.ress see . For allress see . FurtherA total of 14 QTL were identified with significanteffects on germination rate under one or more conditions. Of these, 4 QTL (29%)affected only one trait and 10 QTL (71%) affected 2 or 3 traits. The QTLaffecting one trait included one on chromosome 1 affecting germination undercontrol (nonstress) condition, two on chromosome 8 affecting germination undercold or drought stress, and one on chromosome 12 affecting germination underdrought stress. Three QTL affected 2 traits, including one on chromosome 4affecting germination under drought and nonstress and one on each ofchromosomes 7 and 12 affecting germination under cold and salt stress. Seven QTL affected germination rate under three different conditions,including 3 on chromosomes 1 and 9 affecting germination under cold, drought,and control conditions, 2 on chromosomes 4 and 9 affecting germination undercold, salt, and drought conditions, one on chromosome 5 affecting germinationunder salt, drought, and control conditions, and one on chromosome 11 affectinggermination under cold, salt, and control conditions see . Tenof 1S1 progeny indicatedthat selection for rapid seed germination under any of the four conditions inthe BC1 generation resulted in progeny with improved germinationunder all four conditions of the trait, geneaction, the type of mapping population, the intensity of selection, the numberand individual effects of QTL, the extent of marker coverage, and the distancebetween marker loci and QTL affecting the trait [N = 1000), an intensive selection (p = 3%), and a relativelygood marker coverage provided sufficient power to detect many putative QTL. Foreach trait, between 2 and 4 major QTL and 2–7 minor QTL (1σq ≤ dq < 2σq) were detected. However, due to moderateheritabilities of these traits or backcross inbred lines(BILs) are used. Also, the calculated standardized effects of QTL and f QTL D; should b2 generation, in BC1 generation of a cross between two inbred lineslinkage disequilibrium is large and consequently loosely-linked flankingmarkers may also show association with QTL and it may not be possible todetermine the exact position of QTL [The intervals fora few QTL, including those identified on chromosomes 1 and 4, were rather largepopulations of the same cross as in this study, QTL were identified for rapidseed germination under cold stress and sal1 and 4; ). The prTenof the 14 identified QTL (71%) affected germination rate under 2 or 3 conditions,of which 7 affected germination rate under three differentconditions . This fiIn comparison, only 4 of the 14 QTL (29%) affected germinationonly in one treatment see . The ideExcessive salt in thegermination medium depresses water potential, making water less available tothe seed, which may reduce the rate or completely inhibit seed germination. Lowrate of germination under salt stress could be due to osmotic and/or ioniceffects of the saline medium. The available evidence, however, suggests thatlow water potential of the germination medium rather than its ion toxicityeffects is the major limiting factor to germination under salt stress indifferent crop species, including tomato , 34–36. r = 0.82, P < .01) betweengermination rate under salt and drought stress in tomato [Under drought stress, reducedwater potential of the germination medium is the cause of slow seed germination. This isUnder cold stress, the delay inseed germination could also be due to water stress as low temperature doesaffect water status of the cell . HoweverIn the present study, 7 majoror minor QTL were identified affecting germination rate under nonstress(control) conditions see . Of thesFormajority (71%) of the identified QTL, the positive alleles were contributedfrom the rapid germinating donor parent, LA722. This was not surprising becauseof the significant differences between the two parents in germination rate inall four treatments . HoweverThe present study identifiedbetween 6 and 9 QTL for each of the four germination traits. While four QTLwere identified, each affecting only one trait, the majority of the QTL (71%)were common across the treatments and affected rate of seed germination undertwo or three conditions. The identification of germination-related common QTLindicates that the rate of tomato seed germination under different conditionsis at least partially under the same genetic controls, confirming previousreports of presence of correlations among the rate of tomato seed germinationunder different conditions. It further suggests that similar physiologicalmechanism(s) may facilitate rapid seed germination under different conditions,congruent with the findings of previous physiological studies of tomato seedgermination. It is, therefore, expected that tomato seed germinability underdifferent conditions can be improved by marker-assisted selection for common QTL.In this regards, the seven QTL on chromosomes 1, 4, 9, and 11 which wereidentified with effects on germination rate under three conditions should b
Uncomplicate regurgitation in otherwise healthy infants is not a disease. It consists of milk flow from mouth during or after feeding. Common causes include overfeeding, air swallowed during feeding, crying or coughing; physical exam is normal and weight gain is adequate. History and physical exam are diagnostic, and conservative therapy is recommended. Pathologic gastroesophageal reflux or gastroesophageal reflux disease refers to infants with regurgitation and vomiting associated with poor weight gain, respiratory symptoms, esophagitis. Reflux episodes occur most often during transient relaxations of the lower esophageal sphincter unaccompanied by swallowing, which permit gastric content to flow into the esophagus. A minor proportion of reflux episodes occurs when the lower esophageal sphincter fails to increase pressure during a sudden increase in intraabdominal pressure or when lower esophageal sphincter resting pressure is chronically reduced. Alterations in several protective mechanisms allow physiologic reflux to become gastroesophageal reflux disease; diagnostic approach is both clinical and instrumental: radiological series are useful to exclude anatomic abnormalities; pH-testing evaluates the quantity, frequency and duration of the acid reflux episodes; endoscopy and biopsy are performed in the case of esophagitis. Therapy with H2 receptor antagonists and proton pump inhibitors are suggested. Regurgitation is defined as the passage of refluxed gastric content into the oral pharynx whilst vomiting is defined as expulsion of the refluxed gastric content from the mouth. The frequency of regurgitation may vary largely in relation to age and younger infants up to first month of age are more frequently affected by regurgitation. Gastroesophageal reflux (GER) is the backward flow of stomach contents up into the esophagus or the mouth. It happens to everyone. In babies, a small amount of GER is normal and almost always goes away by the time a child is 18 months old. The consensus statements that comprise the definition of gastroesophageal reflux disease (GERD) in the pediatric population were developed through a rigorous process .Diagnostic investigation of infants who regurgitate, but gain weight satisfactorily and do not exhibit other signs or symptoms is not indicated in clinical practice. The North American Society for Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) recommenIn children is important distinguishing between normal, physiologic reflux and pathological one. Most infants with physiologic regurgitation are happy and healthy even if they frequently spit up or vomit, and babies usually outgrow GER by their first birthday. These patients have no underlying predisposing factors or conditions, growth and development are normal, and pharmacologic treatment is typically not necessary. Patients with pathologic gastroesophageal reflux or GERD frequently experience complications noted above, requiring careful evaluation and treatment.Symptoms and signs associated with GER are non-specific. Regurgitation, irritability, and vomiting are common both in infants with physiologic GER or GERD and in iAs reported above, a diagnostic approach for the "happy spitter" infants, gaining weight satisfactorily and without other signs or symptoms, is not needed. However, diagnostic instrumental tests can be performed in infants frequently present complications, in which it is not easy to identify individuals who truly have GERD. The list of current available diagnostic tools in the management of GERD are reported in table Ambulatory 24-h esophageal pH monitoring is currently the best available test for quantifying esophageal acid exposure, particularly in patients presenting with atypical symptoms. Esophageal pH recording provides quantitative data on both esophageal acid exposure and on the correlation between patient symptoms and reflux events. Esophageal acid exposure is defined by the percentage of the 24-h recording time that the pH is < 4.0. Values > 3.5% are considered abnormal. However, pH monitoring is of limited use in preterm infants whose gastric pH is >4 for the 90% of the time making it almost impossible to detect GER by this technique ,14. WireAlthough important contributions have been made to assess the diagnostic value of the long-term pH monitoring in any age pediatric groups, only few reports in children have attempted to correlate the pH pattern of reflux with the clinical severity of gastro-oesophageal reflux disease and to determine the ability of the test to differentiate normal subjects from patients with various degrees of reflux disease ,17. The Development of esophagitis was associated with increased acid exposure of the esophagus. The number of reflux episodes lasting more than five minutes was the most significant variable that differentiated patients with esophagitis from those with simple gastro-esophageal reflux disease. The five minute value is currently regarded as the most accurate variable in predicting the occurrence of esophagitis because it reflects the mechanisms of esophageal acid clearing. However, symptoms may not correlate with acid exposure or the presence of esophagitis. This may be because symptoms may result from nonacidic as well as acidic refluxate . A surprGastroesophageal reflux can be acid, nonacid, pure liquid, or a mixture of gas and liquid. Esophageal pH and impedance were used to identify acid reflux (pH drop below 4.0), minor acid reflux (pH drop above 4.0), nonacid reflux (pH drop less than 1 unit + liquid reflux in impedance), and gas reflux . Non-aciAdditionally, there are many children that are continuously fed through gastrostomy tubes such that the pH of the stomach is neutral for the majority of the day. Other factors can explain a negative pH monitoring in subjects with gastro-esophageal reflux disease. First, episodes of alkaline gastroesophageal reflux might be overlooked using the standard routine pH measurement. Increased flow/volume of saliva can reduce the exposure acid time of the esophagus neutralising the acidity of the refluxed content . EsophagPrevious pediatric studies have shown that between 30-88% of reflux in children is non-acid . The litA recent work have attempted to characterize the proportion of acid and nonacid esophageal reflux events in young infants with suspected GER using combined pH-multichannel intraluminal impedance (pH-MII) monitoring. To determine the symptom index correlation with nonacid reflux and acid reflux events in children, aged 2 weeks to 1 year, 1890 reflux events were detected by pH-MII, and 588 reflux events were detected by pH probe alone. The percent of reflux that was acid was 47% versus 53% of nonacid reflux events. The proportion of nonacid reflux decreased with age and with increasing time elapsed from last meal. The most frequently reported symptom was fussiness/pain, which correlated with nonacid reflux events 24.6% and acid reflux 25.2%. The proportion of nonacid reflux to acid reflux events in infants was more similar to adults than previously reported. Combined pH-MII esophageal monitoring identifies more reflux events and improves clinical correlation with symptoms .The pH-MII catheter is a small tube that is inserted through the nose into the esophagus and is identical in size to the standard pH probe. The catheter remains in place for 24 hours during which it continuously measures the amount of both acid and non-acid reflux that is entering the esophagus from the stomach. Another significant advantage to pH-MII is the ability of the catheter to measure the height of the refluxed stomach contents; impedance sensors are positioned throughout the esophagus so reflux extends along the entire length of the esophagus, and even up into the mouth and potentially the airway, can be determined. Pediatric studies have suggested that the pH-MII catheter is as sensitive as the pH probe in the detection of reflux.This tool has been very useful in the evaluation of patients with atypical reflux symptoms and in patients who continue to have symptoms while taking acid blocking medicines. Studies in adults and children have shown that the addition of pH-MII monitoring significantly improves the physicians' ability to diagnose reflux-related disease. In studies of infants, the use of pH-MII has been particularly important in clarifying the relationship between respiratory diseases. While the association between apnea and reflux in infants has been debated, there is some evidence that non-acid reflux may be associated with breathing problems in these young patients. In a study of infants with primarily respiratory symptoms who underwent pH-MII testing, the standard pH probe failed to detect 88% of reflux episodes that were associated with breathing problems . There iBecause the understanding of the role of non-acid reflux is in its infancy, very few studies have addressed the treatment options for patients with pathologic non-acid reflux. Adult and pediatric studies suggest that proton pump inhibitors such as omeprazole and lansoprazole do not decrease the total amount of reflux in patients. Instead, they convert the reflux from acid to non-acid reflux which may explain why some patients continue to have symptoms despite therapy with proton pump inhibitors . Adult sEndoscopy associated with histology is a reliable and accurate method to demonstrate esophageal damage induced by GERD, such as inflammation and strictures. However, up today optimization and standardization of pediatric endoscopy procedure have not yet realized . The finAll patients with erosive esophagitis presented reflux esophagitis on histology. The esophageal biopsy plays an important role, as much in cases of normal examinations or mild abnormalities as in cases of erosive esophagitis. If the edema, erythema and friability commonly observed in children are non-specific, findings from histological examination and morphometric studies of the esophageal mucosa allow an etiologic diagnosis of eosinophilic esophagitis if characteristic alterations such as eosinophil infiltrates, increased total epithelial and basal cell thickness, and elongation of stromal papillae are seen . FurtherEosinophilic oesophagitis results in inflammation the esophagus, and in most cases are seen in people with allergies such as hay fever and asthma. There is some evidence that this may be an unusual form of food allergy. It is important to rule out it since eosinophilic esophagitis can progress to esophageal stenosis, and not responding well to anti-GER treatment, corticoid therapy being indicated instead. In such cases, the high eosinophil density (> 20 per high power field) and the presence of eosinophils in the proximal esophagus favor the hypothesis of eosinophilic esophagitis . To deteMotility disorders are postulated to potentially cause reflux since an association between diminished LES tone, transient LES relaxations, delayed gastric emptying and GER have been recognized. Esophageal manometry measures movement and pressure in the esophagus. In particular, it measures esophageal motility pattern and coordinated peristalsis, and the upper and lower esophageal sphincter pressures. There are two main types of manometric recording systems: perfused and solid state. Both have strengths and weaknesses, and the choice of any particular system depends on how these strengths and weaknesses are viewed. Esophageal motility develops during infancy and early childhood, and may be influenced by various factors, including maturation, dietary and postural habits, arousal state, ongoing illnesses, congenital anomalies, and effects of medical or surgical interventions. Esophageal motility is particularly important because it regulates the movement of a bolus during swallowing or during GER. Infantile reflux is different from adult reflux in that regurgitation or vomiting is quite common, even in normal infants . DespiteManometric study is useful in identifying transient relaxations of the LES as a pathophysiological mechanism of GERD and for Plain radiographic findings are not useful in evaluating patients for GERD, but they are helpful in evaluating pulmonary status and basic anatomy. Esophageal inflammatory and neoplastic diseases are better detected with double-contrast techniques . ConversEarly esophagitis is not well demonstrated and decreases the overall sensitivity of barium swallows . This isConventional ultrasonography have reported to be a reliable non invasive method to detect reflux events and as well to describe anatomical conditions such as hiatal hernia, length and position of the LES and the magnitude of the gastro-esophageal angle of His. Although conventional sonography is not a diagnostic tool for achalasia, it provides interesting sonographic information. It cannot reveal each layer of the wall of the lumen as endoscopic ultrasound does, but it may tentatively differentiate achalasia from malignancies and assists clinicians when endoscopic ultrasound is not available . Few impLast, dinamic ultrasound may be useful for the study of the gastric emptying time ,70. AntrIn young children suspected of GERD, the gastroesophageal junction was examined with ultrasonography directly after a feeding while these children were on overnight extended esophageal pH monitoring (EEpHM). The two tests showed 81% to 84% agreement in the detection of the presence or absence of GER, depending on whether the whole period of EEpHM or only the part of it covering the ultrasound observation period . The two99mTc sulfur colloid is placed in the stomach. Control subjects do not have peaks exceeding a value twice that of the baseline count levels. Reflux patients exceed this value, either spontaneously or after Valsalva maneuvers. This technique has a sensitivity which is greater than that of barium and equal to the sensitivity of a pH probe in patients with both moderate and severe reflux. Scintigraphic reflux was shown in 62% of moderate refluxes and 85% of those with severe reflux as defined clinically. This test can be performed rapidly with minimal radiation exposure and is noninvasive [Gastroesophageal reflux and clearance of the refluxed material can be measured by plotting a time-activity curve from an esophageal area of interest after 1 mCi ofinvasive . The seninvasive . Scintiginvasive . Besidesinvasive . Even itThe treatment of GER/GERD should be individually tailored according to the clinical manifestation and possible complications. Treatment options for regurgitation and GERD include conservative measures, dietary management, pharmacologic therapy and surgery. Table Because most cases are functional GER, reassurance is the only treatment needed . ConservAlthough some authors consider conservative therapy to be an efficient first choice for improving regurgitation even compared with thickened formula , the latAs a result, the last European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) guidelines suggested avoidance of formula thickened with locust bean in infants up to six months because the possible risk of enterocolitis . Thus, tThe leading symptoms of GERD are present in the case of cow milk allergy. This disorder should be considered in preterm infants with recurrent vomiting and irritability . ConfirmIn the case of pharmacologic intervention, "step-up" therapy involves progression from diet and lifestyle changes to H2RAs and to PPI . Both clH2RAs decrease acid secretion by inhibiting H2 receptors on gastric parietal cells . The faiPPIs inhibit acid secretion by blocking Na+, K+ ATP-ase, the final common pathway of parietal cell acid secretion, often called the proton pump. PPIs currently approved for use in children in North America are omeprazole, lansoprazole and esomeprazole. In Europe, only omeprazole is approved. No PPI has been approved for use in infants < 1 year of age. Most studies of PPIs in children have demonstrated the efficacy of PPIs in the controlling of symptoms and haeling of erosive esofagitis ,104. ChiWhen medical therapy has failed, or when complications of gastroesophageal reflux are present , the antThere are no controlled studies of fundoplication versus medical therapy and studies evaluating different surgical treatments. In fact, there is no randomization of children undergoing partial versus complete wraps, even if some studies suggest that the results of partial one was better than those of Nissen fundoplication ; there aStandard approaches to infants who regurgitate gastric contents (often the overflow from an overly generous feeding) differ from that recommended for children who reflux and have resultant disease manifestations (GERD). For infants with functional GER, a rational and conservative approach is to reassure the parents of the benign nature of the "spitting". Pathologic gastroesophageal reflux or gastroesophageal reflux disease refers to infants with regurgitation and vomiting associated with poor weight gain, respiratory symptoms, esophagitis. In such case clinical and instrumental diagnosis are needed. Among the latter upper radiology, pH-testing and MII testing are useful for diagnosis. Endoscopy and biopsy are performed in the case of esophagitis. The therapy with H2 receptor antagonist is currently suggested.AGA: American Gastroenterology Association; EE: Eosinophilic esophagitis; ESPGHAN: European Society for Pediatric Gastroenterology Hepatology and Nutrition; EEpHM: Extended esophageal pH monitoring; H2RA: Histamine2 receptor antagonist; LES: Lower esophageal sphincter; GABA: Gamma-aminobutyric acid; GER: Gastroesophageal reflux; GERD: Gastroesophageal reflux disease; NASPGHAN: North American Society for Pediatric Gastroenterology Hepatology and Nutrition; pH-MII: pH-Multichannel intraluminal impedance; PPI: Proton pump inhibitor; RI: Reflux index.The authors declare that they have no competing interests.FI: Conceived the study and helped draft the manuscript. GR: Drafted the manuscript. FR: Participated in the drafting and polishing the manuscript in the diagnostic approach section. LC: Participated in its design and coordination RF: Participated in its design and coordination. All authors read and approved the final manuscript.
Limited information is available in Iraq regarding the causes of under-five mortality. The vital registration system is deficient in its coverage, particularly from rural areas where access to health services is limited and most deaths occur at home, i.e. outside the health system, and hence the cause of death goes unreported. Knowledge of patterns and trends in causes of under-five mortality is essential for decision-makers in assessing programmatic needs, prioritizing interventions, and monitoring progress. The aim of this study was to identify causes of under-five children deaths using a simplified verbal autopsy questionnaire.The objective was to define the leading symptoms and cause of death among Iraqi children from all regions of Iraq during 1994–1999.To determine the cause structure of child deaths, a simplified verbal autopsy questionnaire was used in interviews conducted in the Iraqi Child & Maternal Mortality Survey (ICMMS) 1999 national sample. All the mothers/caregivers of the deceased children were asked open-ended questions about the symptoms within the two weeks preceding death; they could mention more than one symptom.The leading cause of death among under-five children was found to be childhood illnesses in 81.2%, followed by sudden death in 8.9% and accidents in 3.3%. Among under-five children dying of illnesses, cough and difficulty in breathing were the main symptoms preceding death in 34.0%, followed by diarrhea in 24.4%. Among neonates the leading cause was cough/and or difficulty in breathing in 42.3%, followed by sudden death in 11.9%, congenital abnormalities in 10.3% and prematurity in 10.2%. Diarrhea was the leading cause of death among infants in 49.8%, followed by cough and/or difficulty in breathing in 26.6%. Among children 12–59 months diarrhea was the leading cause of death in 43.4%, followed by accidents, injuries, and poisoning in 19.3%, then cough/difficulty in breathing in 14.8%.In Iraq Under-five child mortality is one of the highest in the Middle East region; deaths during the neonatal period accounted for more than half of under-five children deaths highlighting an urgent need to introduce health interventions to improve essential neonatal care. Priority needs to be given to the prevention, early and effective treatment of neonatal conditions, diarrheal diseases, acute respiratory infections, and accidents. This study points to the need for further standardized assessments of under-5 mortality in Iraq. Children are the promise and the future of every nation, being the core of its development. Investing in children's health and development means investing in the future of a nation. Children are also a vulnerable group whose needs and rights must be protected, including the right to health and development . Child mIraq has a young population; 45% are under the age of 15 years, and those below 5 years of age constitute 17% of the population (3.9 million); two-thirds of the population lives in urban areas . In thisTwo cross-sectional household surveys were conducted in the South/Center and Kurdistan regions in February-March and April-May 1999, respectively. Altogether 40,477 married women between 15 to 45 years were interviewed in 23,978 households in the South/Center and 16,499 in Kurdistan by trained interviewers using a structured questionnaire that was developed using the Demographic and Health Surveys (DHS) questionnaire . Iast Asia , but it ast Asia . The cauast Asia . We did ast Asia . HoweverThe high percentage of neonatal deaths could be explained by the deterioration of the health services and the socioeconomic conditions of the population at large during the nineties, especially that of women and children. All these factors had a deleterious effect on delivery and neonatal services. In 2001, an assessment of newborn care undertaken in 35 Maternal and child hospitals in Iraq showed that policies and guidelines on newborn care are non-existent. Only 46.7% of the doctors in these hospitals were trained on newborn resuscitation, with 51.6% receiving practical training for less than one hour on the procedure. Of the nurses working in the delivery rooms, 22.5% were trained on newborn resuscitation, and 36.7% of the medical and nursing staff still practiced holding the baby upside-down to stimulate breathing. Drying the baby with warm towels was practiced by only 13.5% of the staff working in delivery rooms .Among neonates cough and/or difficult breathing accounted for 42.3% of neonatal deaths, these causes include pneumonia and respiratory distress syndrome. Other causes include congenital malformation (10.3%), prematurity (10.2%), sudden death (11.9%) and disorders of pregnancy and perinatal conditions (3.6%). These figures are different from the global estimates which indicate that causes of neonatal deaths are preterm birth (28%), severe infections including sepsis/pneumonia (26%), tetanus (6.5%), diarrhea (2.8%), complications of asphyxia (23%), and congenital abnormalities (7%) .Preterm births as a cause for neonatal deaths was lower in our study than the global estimates , which cGlobally, injury mortality is about 4 to 5% annually in most countries, and constitutes 5–10% of deaths among children less than five years, although the relative importance in the one to four year period is much greater (in the order of 25 to 35%) . Deaths The causes in this category included deaths due to traffic accidents, drowning, burning, falling from heights, and poisoning by kerosene, other petroleum products, agricultural pesticides and household cleaners. It is important to recognize the burden of injury among young children in Iraq; most deaths following injury occur in the very hot summer because of poisoning with kerosene and petroleum products when thirsty toddlers drink petroleum products and detergents kept in drinking water cans in households. Fire is another important cause of death, especially in the cold winter months where unsafe kerosene heaters are used for warmth. Falls from unprotected roofs accessible for children is another important cause of death among children.Globally congenital malformations contribute to 8% of neonatal deaths , which iCongenital malformation rate in the middle Euphrates region is more than double that of the North region 9.2% versus 3.9% which could be explained by the widespread economic hardship and environmental factors which effected negatively the populations' health of this region, among which was the use of mustard gas during the 1991 gulf war . In a laThe higher undr-5 mortality rate observed in the South and Middle Euphrates region compared to the North region is mainly due to the South region being on the forefront of the 8 years Iran-Iraq war and the 1991 Gulf War, compounded by the economic hardships of the region.In the last 20 years of the twentieth century, the whole country experienced one of the most catastrophic economic declines in modern history; households' income witnessed a continuing decline; the GDP per capita has been declining since around 1980 due to a combination of wars, sanctions, and economic mismanagement. During the Iran-Iraq war real GDP per capita fell by an astonishing 57% between 1980 and 1988, and even further before the implementation of the UN's oil-for-food programme in 1996 .Broad economic sanctions were applied on Iraq during the nineties of the last century; many studies documented the adverse effects of sanctions on the health and survivor of children ,28. Per The dramatic changes in the child survival rates in southern central Iraq have been attributed to a variety of factors, including the declining economic well-being of the population, its effect on mother and child nutrition levels, as well as the declining access to healthcare and food security . UNICEF The Millennium Development Goals (MDGs) represent the widest commitment in history to addressing global poverty and ill health . The MilTo develop effective strategies for reducing neonatal and under-five child mortality in the 21st century, studies of the underlying factors that contribute to morbidity and mortality of children in Iraq should be conducted. These studies must understand not only the biologic factors but also the social, economic, psychological, and environmental factors that contribute to maternal and infant deaths. A thorough review of the quality of health care, especially that of neonates, and access to care for all women and infants is needed, to avoid preventable mortality and morbidity, and to develop public health programs that can eliminate disparities in health .Research and focused programs, especially for neonates, to enable mothers to identify acute lower respiratory infections, particularly pneumonia, and encourage timely and appropriate seeking of care, strengthening of acute lower respiratory infections case management at the primary care facilities being important priorities. Another priority is the systematic introduction of corticosteroid in premature birth, which can assist in the reduction of cases of Respiratory Distress Syndrome and hence deaths among neonates.Introducing preventive programs to decrease mortality because of accidents in the age group of 1–4 is important. Innovative interventions to reduce childhood mortality from road accidents, drowning, burning, falls from heights and poisoning, should be designed and tested. While promotion of oral rehydration for diarrhea and antibiotic treatment for dysentery should continue. Broader preventive interventions including provision of safe water and sanitation, and improvements in personal hygiene require more attention. Further intensification of immunization programs is essential to reduce child mortality.To be on the track for achieving the MDG-4, Iraq is facing very serious challenges, with the political instability, the ongoing – albeit reduced – violence which has hindered access to primary care in the south and middle region, and the insecurity which makes it difficult for people to travel to the services they need. Other constraints external to the health system include factors, such as the protracted failure of the electricity and the sanitation systems.The overall policy environment and the political instability, are both inconsequential to materializing the MDGs in Iraq. However Iraq would be capable of a "dramatic" improvement in child survival and achieving the MDG to reduce U 5 mortality in the country if peace and political will prevailed.Malnutrition was not included in the questionnaire design, as well as questions about breast feeding and immunization status of deceased children. All of these have great effect on mortality in children. Steps and criteria for assigning causes of childhood deaths were not incorporated in the study design. Training in Kurdistan was done separately and by different facilitatorsCause-of-death information and the epidemiogical facts are needed to provide evidence for health planning, and to prioritize the delivery of the necessary interventions to reduce child mortality. The information regarding causes of death in under-five children provided by this study can assist in guiding national plans and program interventions for reducing their mortality in Iraq; at the same time it will assist in assessing changes over time.Our findings illustrate the urgent need for more comprehensive improvement of prenatal and postnatal care in Iraq, as well as the need for robust programs for combating respiratory, diarrheal diseases and accidents as the main causes of death among children younger than five. Children living in the Southern and Middle Euphrates region are at a higher risk of dying before reaching the age five, there is the need to emphasize interventions to reduce mortality in both regions.The authors declare that they have no competing interests.NAA, KS and NJA were principal investigators, and were involved in writing the survey protocol, participated in its design and coordination, supervised the survey implementation and analysis of the data. NAA drafted the manuscript. MMA was the principal statisticians who supervised all steps of data management, data analysis and drafted the manuscript. FAM, MA, and NA assisted in the analysis of data, paper design and in revising the manuscript, as well as providing critical comments.The pre-publication history for this paper can be accessed here:
A bioluminescence technique involving single photon imaging was used to quantify the spatial distribution of the metabolites ATP, glucose and lactate in cryosections of various solid tumours and normal tissue. Each section was covered with an enzyme cocktail linking the metabolite in question to luciferase with light emission proportional to the metabolite concentration. The photons emitted are imaged directly through a microscope and an imaging photon counting system. In some cases, good agreement was observed between the distribution of relatively high concentrations of ATP and glucose in viable cell regions of the periphery, while the reverse was seen in more necrotic tumour centres with comparatively high lactate levels. In general, lactate was distributed more diffusely over the sections while ATP was more highly localised and glucose assumed an intermediate pattern. In contrast to the large degree of heterogeneity seen in tumours, distribution patterns of metabolites were much more homogeneous in normal tissue, such as heart muscle. Mean values for metabolite levels in cryosections using bioluminescence are in good agreement with those obtained from the same tumour by conventional methods.
In vitro and in vivo evidence suggests that these redox-active compounds can contribute to the functioning of oxidative folding. This review focuses on the participation of small molecular weight redox compounds in oxidative protein folding.Oxidative protein folding is mediated by a proteinaceous electron relay system, in which the concerted action of protein disulfide isomerase and Ero1 delivers the electrons from thiol groups to the final acceptor. Oxygen appears to be the final oxidant in aerobic living organisms, although the existence of alternative electron acceptors, e.g. fumarate or nitrate, cannot be excluded. Whilst the protein components of the system are well-known, less attention has been turned to the role of low molecular weight electron carriers in the process. The function of ascorbate, tocopherol and vitamin K has been raised recently. After synthesis, these proteins are transferred to the lumen of the ER, where their native conformation evolves by several co-/post-translational modifications, e.g. the disulfide bond formation . The oxiRecent studies have indicated the role of vitamin C (ascorbate) and vitamin E (tocopherol), the most abundant water-soluble and lipid-soluble antioxidants in the ER, in oxidative protein folding. Both antioxidants have two transmittable electrons, but are also able to donate only one electron. In this case free radicals - ascorbyl radical and vitamin E radical - are formed. Vitamin E radical is generated e.g. when a reactive oxygen species (ROS) oxidizes tocopherol. The redox connection between ascorbate and vitamin E is well-known; the tocopheryl radical can be re-reduced to vitamin E by ascorbate, while ascorbyl radical is produced . AscorbyAscorbate is synthesized in the hepatocytes of most animals . The syn2.In intact cells, the oxidative environment in the ER enables disulfide bond formation in newly synthesized proteins. It is, however, no longer the case in ER-derived microsomal vesicles, which suggests that the process needs a cytosolic factor or a membrane-permeable compound, which is lost during the preparation of microsomes. GSSG was long considered to have a key role in the process, but this assumption was conquered, since it was shown that the disulfide bond formation in GSH deficient yeast is intact , and theIn the presence of rat liver microsomes, ascorbate was continuously transformed to ascorbyl free radical and then dehydroascorbic acid. When microsomes were incubated in a cytosol like concentration of ascorbate at 37°C, concentration of ascorbate was decreasing with a constant rate during the incubation. Dehydroascorbic acid was detectable after a few minutes and its level remained constant as long as ascorbate was present in the medium. As it was detected by electron spin resonance spectroscopy, when ascorbate was incubated in an adequate buffer without microsomes, the typical ascorbyl free radical signal was present. In the presence of microsomes, a much higher ascorbyl radical signal was detected, which remained on the same level during the whole time of the experiment. As both ascorbyl free radical and dehydroascorbic acid are very instable, the maintenance of their constant level could be ensured only by a continuous ascorbate oxidation .The fact that the addition of microsomes to the incubation medium radically increased ascorbate oxidation implied that there is a microsomal ascorbate oxidase activity. Pretreatment of microsomes with heat or protease significantly decreased the ascorbyl radical production and almost completely eliminated the ascorbate oxidase activity, indicating that ascorbate oxidation was indeed a protein mediated process.Further information was gathered about the ascorbate oxidase enzyme by the addition of different inhibitors. Among several types of inhibitors, only various metal chelators reduced significantly the ascorbyl free radical production in the presence of microsomes. Among the chelators, the copper-specific neocuproine caused tIn line with the consumption of the added ascorbate to liver microsomes, the oxidation of protein thiols is also observable, while without ascorbate addition remains negligible. The two processes – ascorbate consumption and thiol oxidation - showed good correlation with each other. In the presence of neocuproine both the ascorbate oxidation and the thiol oxidation were decreased .Since the thiol oxidation was coupled with the ascorbate oxidation, the role of dehydroascorbic acid in the process had to be supposed. Upon addition of dehydroascorbic acid, protein thiols were indeed oxidized, but dehydroascorbic acid proved to be less effective than ascorbate itself in this respect. This had a particular importance knowing the fact that, among the ascorbate/dehydroascorbic acid redox couple, the oxidized dehydroascorbic acid is the transported form through the ER membrane , while tin vitro observations, that ascorbate might have a role in oxidative protein folding, experiments were designed to show its role also under in vivo conditions. In cells, the defective oxidative protein folding results in the accumulation of immature proteins, which in turn causes ER stress and unfolded protein response (UPR).Based on the Since one of the most important functions of the ER is the synthesis and posttranslational modification of secretory and membrane proteins, the lumen of the organelle is equipped with a powerful protein-folding machine composed of chaperones, foldases and also with sensors that detect the presence of misfolded or unfolded proteins. Physiological and pathological effects or experimental agents that disturb the normal folding process provoke the UPR, an intracellular signaling pathway that coordinates ER protein-folding demand with protein-folding capacity and is essential to adapt to homeostatic alterations (collectively named as ER stress) that cause protein misfolding. These include changes in intraluminal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in the redox status. The principal events of ER stress and the UPR has been summarized in numerous recent reviews (see e.g. ).in vivo [The complex defense mechanism of UPR includes the up-regulation of ER chaperones and foldases and the inhibition of protein synthesis. When these mechanisms are not able to rescue the cell from the stress situation, apoptosis is initiated to eliminate the diseased cell. Guinea pigs, which are unable to synthesize ascorbate, were used to model the effect of ascorbate deficiency on protein maturation in vivo . Experimin vivo .3.The observation that ascorbate addition to microsomes promoted the protein thiol oxidation more effectively than dehydroascorbic acid foreshadowed that the formation of dehydroascorbic acid from ascorbate contributes to disulfide bond generation also in an indirect way. This suggested the involvement of an ER-membrane located lipid-soluble molecule, which connects the two processes: ascorbate oxidation on the outer surface, and protein thiol oxidation in the lumen.in vitro conditions the formation of disulfide bonds was linked to the vitamin K-dependent γ-carboxylation of glutamate residues and the PDI/thioredoxin system might serve as the donor of the reducing equivalents in the vitamin K cycle. Later further evidences were lined up to show the link between the PDI dependent oxidative folding and the vitamin K reduction. Reduced protein substrates increased the rate of vitamin K reduction and γ-carboxylation of an artificial substrate, as it was shown in a recent study. Furthermore, a stable complex of PDI and VKORC1 has been also suggested [in vivo has to be examined further.Among the lipophilic electron carriers, the role of vitamin K was brought on earlier. The reduced form of vitamin K has a crucial role in the γ-carboxylation of proteins which require γ-carboxylated glutamate to evolve their activity. During γ-carboxylation, vitamin K is epoxidated, which then has to be reduced in order to regain its activity. The enzyme responsible for the reactivation is an integral ER membrane protein facing to the lumen, called vitamin K epoxide reductase (VKORC1) . VKORC1 uggested . These din vitro addition of vitamin E the microsomal tocopherol level could be almost normalized. It was shown that in vitamin E deficient microsomes the intra- and extraluminal redox processes were partly uncoupled: while the intraluminal thiol oxidation decreased, the extraluminal ascorbate oxidation itself increased. Both effects were avoidable by in vitro tocopherol addition. The higher ascorbate oxidation was accompanied by an increased lipid peroxidation, which was probably caused by ROS produced on the extraluminal surface during ascorbate oxidation and also to nonflavin molecules – among which two hem binding proteins and one protein with a copper-center (azurin) were examined successfully [In yeast and mammals, the last protein component of the system, Ero1p also uses oxygen as electron acceptor ; in vitrs formed . It is qnditions –30, whicessfully . Free fl5.Although recent studies have revealed that an ensemble of enzymes, chaperones, and small molecules are involved in the oxidative protein folding, still PDI remained at the center of the process. PDI, a 57 kDa molecule that resides in the ER of eukaryotic cells, is a member of the thioredoxin family of proteins –32. ThisOne is the reduction potential (E°′) of the disulfide bond which is formed between the Cys residues in the active site . This vaa) of the N-terminal thiol group in the active site. Sulfhydryl group of free cysteine has a relatively high pKa and as a consequence it is relatively inert for redox reactions in physiological conditions. In contrast, some structural folds in thiol-disulfide oxidoreductases provide appropriated environments for changing the pKa values of sulfhydryl groups. If this constant is close to the pH of the solution, a large part of the Cys can be deprotonated and stabilized in the anionic form called thiolate (RS−). The formed thiolate can initiate nucleophile attack to compose disulfide bonds in the substrate proteins. The pKa value of Cys in the active site depends on the proteinaceous environment; e.g. in PDI the pKa value of this Cys is 6.7 [The other factor that governs the efficiency of thiol-disulfide oxidoreductases is the acid dissociation constant t of PDI .trans-1,2-bis(mercaptoacetamido)-cyclohexane (BMC), which has an E°’ and first-thiol pKa value close to that of PDI [in vitro from its scrambled analogue with random distribution of disulfide bonds. The monothiol analogue of BMC – however in less extent – also shows unscrambling activity. BMC is an effective folding catalyst in vivo as well – addition to the growth medium of S. cerevisiae increases the yield of the secretion of native, disulfide containing Schizosaccharomyces pombe acid phosphatase.Other small molecule folding catalysts were designed by using the advantageous properties of PDI without modeling the active site of the enzyme. Although one thiol group is enough for catalysis, the presence of two Cys groups enhances the overall effectiveness of the oxidative folding. The most prevalent dithiol used as oxidative folding catalyst is (±)-t of PDI . Indeed,a values respect to their aliphatic counterparts, which ensures a high reactivity for thiol-disulfide interchange reactions at physiological pH. The thiol pKa can be tuned by changing the substituent on the aromatic ring. A typical representative of the group is the monothiol 4-mercaptobenzeneacetate, whose pKa value (6.6) is similar to that of PDI, is 5–6 times more effective then the commonly used glutathione redox buffer. Among the dithiol components of the group even more active molecules can be found.Aromatic thiols are also efficient foldases . They haa values then thiols, which extends their advantageous kinetic properties to acidic pH. They have enhanced reactivity in both oxidized and reduced forms, although the diselenide bond is more stabile than the disulfide bond. Another organic selenide, selenocystamine is also a commercially available folding catalyst; however exhibits lower activity than GSeSeG, which become even more pronounced under acidic conditions. Nevertheless, besides their advantageous kinetic properties in protein folding, it is notable that selenide derivatives often exhibit high toxicity [The diselenide analogue of oxidized glutathione - GSeSeG – has also been shown to act as an effective oxidant during protein folding . GSeSeG toxicity .ero1-1 mutant from its temperature sensitivity and the growth defect under anaerobic conditions.The thiol-specific oxidant, dipyridyl disulfide (DPS) was lately shown to affect directly the Ero1-PDI pathway . DPS wasin vitro protein refolding with nonspecific manners, e.g. by stabilizing native-like folding intermediates, by increasing the kinetics of oligomeric assembly, or by inducing the conformational-order in the fully reduced polypeptide [Besides the above enumerated standard redox-active chemicals, non-redox-active small molecules were also shown to assist in disulfide bond formation. This class of compounds - called chemical chaperones - was shown to reverse the intracellular retention of several different misfolded proteins thus were used efficiently in conformational diseases. Among these molecules the most known are glycerol and other polyols, DMSO, trimethylamine-N-oxide and trifluoroethanol. All of them were shown to increase the rate of ypeptide –47.6.in vitro systems, and some in vivo findings support its involvement in the oxidative folding. The effects of these vitamins on oxidative folding can contribute to their in vivo action. Further work is needed for the mapping of the redox network composed by the small electron carrier compounds in the ER lumen.Oxidative protein folding is mediated by an electron relay system chiefly composed by luminal proteins of the ER. However, low molecular weight electron acceptors can also contribute to the process. Their participation has been evidenced in bacteria and is suggested also in eukaryotes. Several compounds have been designed, synthesized and used effectively as folding catalysts. Recent observations show that endogenous molecules – principally water- and lipid-soluble antioxidant vitamins – can fulfill the same role. The thiol oxidant effect of dehydroascorbic acid has been demonstrated in
Loss of hand use is considered by many spinal cord injury survivors to be the most devastating consequence of their injury. Functional electrical stimulation (FES) of forearm and hand muscles has been used to provide basic, voluntary hand grasp to hundreds of human patients. Current approaches typically grade pre-programmed patterns of muscle activation using simple control signals, such as those derived from residual movement or muscle activity. However, the use of such fixed stimulation patterns limits hand function to the few tasks programmed into the controller. In contrast, we are developing a system that uses neural signals recorded from a multi-electrode array implanted in the motor cortex; this system has the potential to provide independent control of multiple muscles over a broad range of functional tasks. Two monkeys were able to use this cortically controlled FES system to control the contraction of four forearm muscles despite temporary limb paralysis. The amount of wrist force the monkeys were able to produce in a one-dimensional force tracking task was significantly increased. Furthermore, the monkeys were able to control the magnitude and time course of the force with sufficient accuracy to track visually displayed force targets at speeds reduced by only one-third to one-half of normal. Although these results were achieved by controlling only four muscles, there is no fundamental reason why the same methods could not be scaled up to control a larger number of muscles. We believe these results provide an important proof of concept that brain-controlled FES prostheses could ultimately be of great benefit to paralyzed patients with injuries in the mid-cervical spinal cord. Many spinal cord injury survivors report that recovery of hand use would be the most desirable function to regain By now, a number of groups have shown that multi-electrode recordings from the primary motor cortex (M1) can be used to predict kinematic features of desired movement rhesus macaque monkeys (monkeys A and T). We performed a series of experiments with two We used recordings from a multi-electrode array chronically implanted in the primary motor cortex (M1) to generate real-time predictions of intended muscle activation. These predictions were used to control the intensity of stimulation to four forearm flexor muscles, thus providing a brain interface by which the monkey could voluntarily control its paralyzed muscles. We quantified the effectiveness of this control in terms of: 1) the increase in voluntary force generating capacity, 2) the similarity in the time course of the force under normal and FES conditions, and 3) the precision with which the force was controlled.The nerve block dramatically decreased the amount of wrist flexion force that the monkeys could generate voluntarily. It seemed apparent by watching the monkeys that some of the remaining force in the blocked state resulted from the action of unblocked, proximal muscles that was inappropriately registered by the wrist force transducer. Unfortunately, quantifying the magnitude of this effect is difficult. The magnitude of EMG from the blocked muscles was very near the noise level; its reduction from the normal level was greater than the corresponding reduction in force . For monkey T, the average ratio of the EMG between the normal and blocked MVC tests in nine sessions was only 1.5% for the major wrist and digit flexors. The results for monkey A . This logic leads us to conclude that the remaining force in the blocked state was primarily due to unblocked muscles, and that the estimated MVC force in the blocked state was probably overestimated . Unless otherwise noted, all subsequent force-related results are expressed relative to the Blocked MVC.Beyond simply generating larger forces, the brain-controlled FES system allowed both monkeys to grade the amount of force they produced as would be necessary for a useful clinical application. In several experiments with monkey T, the FES system was intentionally turned off for 20% of randomly selected “catch” trials is the average reaction time (RT). The right edge of the target rectangles indicates the end of the average hold time for successful trials.Overall, the time to success across all targets during FES was 60% greater than normal for monkey T and twice normal for monkey A . This difference was potentially the result of several factors, including the monkeys' reaction time, the time to initial target entry, and the stabilization time within the target. In the example in Finally, the monkey's ability to enter the target without subsequently over- or under-shooting was also an important determinant of the time required to achieve a successful trial. Beyond a comparison of the monkeys' normal behavior with that under FES, we sought to compare the real-time stimulator commands to the normal pattern of EMG to determine how well the brain interface replicated natural control . As in fAlthough the nerve block did not affect extensor muscles, targets requiring extension force were occasionally included in FES sessions . During normal, unblocked conditions, there was a low level of cocontraction of flexor and extensor muscles at the beginning of extension trials. The black curve in It is important to note that the electrical stimulation of muscles activates only a subset of fibers in each muscle, that the recruitment order of these fibers is approximately reversed from normal, and that there is no rate modulation component at all. Given these differences between normal and FES activation of muscles, we consider the overall similarity of the command signal to normal flexor EMG to be remarkable.We have demonstrated neurally activated FES that provided two monkeys with the ability to exert continuous voluntary, control over the wrist flexor musculature despite temporary paralysis. This was accomplished by using the activity of an ensemble of motor cortex neurons to control the simultaneous, stimulation-driven contraction of four paralyzed muscles. There is no obvious reason why these essential results cannot be scaled up to significantly larger numbers of muscles to allow control of more complex, dexterous movements. These experiments serve as a proof of concept that a similar neuroprosthesis might restore the voluntary control of basic hand movements to a spinal cord injured human patient. Some of the preliminary results for the first monkey (monkey A), dealing with the development of the nerve block and FES methods, have been previously reported In general, monkey T achieved greater gains in both strength and precision of control through FES than did monkey A. This was probably due in large part to our use of percutaneous muscle electrodes in monkey A, which were less stable and typically less effective in generating force than were the chronically implanted intramuscular electrodes used for monkey T. In addition, the need to rely on percutaneous lidocaine injections rather than the implanted cannula system for peripheral nerve blocks resulted in fewer, shorter duration experiments with monkey A. Not only did this affect our ability to refine our methods, it also limited the amount of time that monkey A was able to adapt to the system. Finally, we were typically able to record about 20% more neurons from monkey T than monkey A, which may also have had some effect. Despite these differences, we were pleased with how quickly both monkeys learned to control the FES system, typically making the transition from the blocked state to FES control in a matter of minutes. This was presumably the consequence of our having mapped the neural discharge onto muscle activity sufficiently closely to that of the natural pattern such that extensive learning was not required to perform the basic task.Other studies have explored the possibility of brain controlled FES. Limited wrist force generation was recently achieved by a system that used the discharge of 1 or 2 neurons to directly control the activity of 1 or 2 wrist muscles Several groups have used neural activity recorded from electrodes implanted in the cortex to predict the position of a monkey's limb during normal movement We are working to increase the force that can be produced by stimulation, as well as the number of controlled muscles. We anticipate that it will be possible to produce voluntarily controlled grasp movements using these brain-controlled FES methods. It is worth noting that the multi-electrode array used here to record neural activity from the monkeys' brains has been implanted in a small number of human patients All animal care, surgical, and research procedures are consistent with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Northwestern University. Nonhuman primates are an important experimental model in the investigation of motor control. The motor areas of the central nervous system as well as the musculoskeletal system are very similar to those of humans. Macaque monkeys are not endangered, and are in common use in many different laboratories studying motor control, which allows the efficient comparison of related experiments. There is currently no alternative method to record the activity of single cortical neurons during behavior. We take great care that these animals are comfortable and remain in good health, both because of the potential humane considerations, and because an animal in ill health is unlikely to cooperate as well as one that is healthy.Both monkeys had a 100-electrode array (Blackrock Microsystems) chronically implanted in the hand area of motor cortex. The surgical details have been previously described Peripheral nerve blocks were achieved with percutaneous injection of Lidocaine or Bupivacaine in combination with epinephrine directly to the median and ulnar nerves (monkey A) or via chronically implanted nerve cuffs and injection cannulae (monkey T). By blocking the median and ulnar nerves proximal to the elbow, the flexor muscles of the wrist and fingers and the intrinsic hand muscles were all paralyzed. Nerve blocks were checked periodically for an absence of EMG and sensation, and by the evaluation of dexterity and measurement of maximum wrist strength. Essentially normal muscle strength returned within 3–4 hours of the initial injection of Lidocaine.The monkeys viewed a cursor that was controlled by isometric wrist force and displayed on a video monitor. During six experimental sessions, Monkey T was given randomly intermixed force targets that included a single extension target and 1–3 flexion targets. In two experimental FES sessions, Monkey A was given blocks of trials, each consisting of a single flexion target, occasionally with an additional extension target. The monkeys had 2–3 seconds (4–5 seconds under FES conditions) to move the cursor to a target and to hold it there for 500 ms, for a liquid reward.Estimates of maximum voluntary contraction (MVC) were made under normal conditions prior to nerve blocks , after the nerve blocks were fully in effect, and then again under blocked conditions when the monkeys were using brain controlled muscle stimulation. In each condition, MVC was estimated by averaging peak force as the monkeys were encouraged to match increasingly high targets for several minutes. The five highest force peaks were averaged to determine the MVC. Both monkeys learned to generate some force with unblocked proximal muscles that tended to increase the Blocked MVC, such that the strength increase provided by FES was probably somewhat underestimated delivered monopolar, charge-balanced stimuli using a single common return electrode placed on the skin over the elbow. During each experiment, a single electrode was stimulated in each of four muscles: palmaris longus, flexor digitorum sublimis, flexor carpi ulnaris and either flexor digitorum profundus or flexor carpi radialis. In keeping with standard practice in FES applications, variation in stimulation pulse width was used to grade muscle contraction Methods S1This file contains a more extensive description of the methods.(0.04 MB DOC)Click here for additional data file.Brain Controlled FES S1This is a video taken of the screen, showing the required force target, and the cursor that is being controlled by the monkey through the brain-controlled FES system. “Interface off” indicates a catch trial during which the input to the stimulators has been turned off, in order to test the monkey's ability to do the task without FES assistance.(1.55 MB MOV)Click here for additional data file.
Saccharomyces cerevisiae. These approaches allow us to make novel qualitative and quantitative observations about the switching behavior of the galactose network, and provide a framework that might be useful to extract information needed for the development of quantitative network models.High throughput measurement of gene expression at single-cell resolution, combined with systematic perturbation of environmental or cellular variables, provides information that can be used to generate novel insight into the properties of gene regulatory networks by linking cellular responses to external parameters. In dynamical systems theory, this information is the subject of bifurcation analysis, which establishes how system-level behaviour changes as a function of parameter values within a given deterministic mathematical model. Since cellular networks are inherently noisy, we generalize the traditional bifurcation diagram of deterministic systems theory to stochastic dynamical systems. We demonstrate how statistical methods for density estimation, in particular, mixture density and conditional mixture density estimators, can be employed to establish empirical bifurcation diagrams describing the bistable genetic switch network controlling galactose utilization in yeast Decades ago, Waddington, and later Kauffman, likened the dynamics of a differentiating cell to a marble rolling downhill on bumpy terrain—the epigenetic landscape. In this metaphor, the valleys of the landscape represent the paths that cells can follow towards a stable cell type, and the fate of the cell is determined by the constant modulation of the epigenetic landscape by internal and external signals. With new technologies for measuring single-cell gene expression, it is increasingly feasible to map out these valleys and how external variables influence cellular responses. Moreover, it is possible to quantify population level effects, such as what fraction of a population of cells arrives at one valley or another, and variability at the cellular level, such as how individual cells bounce around within, and possibly between, valleys due to the stochasticity of cellular biochemistry. In this paper, we discuss which characteristics of the epigenetic landscape can readily be extracted from single-cell gene expression data, and describe computational methods for doing so. One of the primary goals of systems biology is to uncover the dynamics of cellular networks. Sometimes, this has meant collecting time-series data and applying tools for time-series analysis such as Fourier methods to identify periodically expressed genes Here, we are concerned with the experimental and computational quantification of bifurcation-like behavior in stochastic genetic switches. There is considerable evidence that signalling networks in a population of genetically-identical cells exhibit large cell-to-cell variability in their output, despite operating in a homogeneous external environment or environmental condition(s), in order to study how subpopulations change under varying conditions. This may mean changing the concentration of ligands or nutrients in the cellular environment or artificially manipulating the activity of regulatory factors inside individual cells. For example, Ozbudak et al. The organization of this paper is as follows. First, we discuss traditional bifurcation analysis in greater detail, introducing in particular saddle-node bifurcations, a type of bifurcation widely associated with the dynamics of gene regulatory switches. We then describe the necessity of generalizing the notion of bifurcation behavior to account for the inherent noise (stochasticity) in cellular networks. Next, we present the data that motivated our study—single-cell flow cytometry data measuring activity in the yeast galactose utilization network over a range of extracellular galactose concentrations. We then report on two broad approaches to analyzing this data and extracting estimates of bifurcation structure, namely, mixture density modeling and conditional mixture density modeling. We evaluate the relative strengths of these approaches, and describe a number of novel qualitative and quantitative observations about switching in the galactose network.Bifurcation analysis is a branch of dynamical systems theory concerned with steady-state or asymptotic behaviors of a dynamical system hes e.g. we descrbifurcation points and correspond in a deterministic system to the critical values of In contrast with deterministic models, real cellular networks can be significantly noisy, with system variables fluctuating over time for a variety of reasons, including, for example, fluctuations in biochemical reaction rates, random partitioning of cellular content at cell division, and variation in cell size and cell age .Our thoughts on stochastic bifurcation structure and methods to estimate it were motivated, indeed necessitated, by data we collected on activity in the galactose utilization network in gene see . Gal10 iA natural approach to modeling multi-modal data is to employ mixture distributions. We model data from each biological replicate separately, in order to avoid conflating replicate-to-replicate variation with cell-to-cell variation within a replicate. Consider a replicate, For a given replicate, fitting mixture distributions to each galactose concentration The only downside of this approach is that it does not explicitly model the dependence of these features of stochastic bifurcation structure on the external controllable bifurcation parameter—the galactose concentration For a given replicate We used two different approaches to fit mixture models and one approach to fit a conditional mixture model to the data. In all approaches, the mixtures contained one or more Gaussian components as well as a single uniform component. The uniform component was given a fixed mixture coefficient of In While the three fitting methods produce qualitatively similar results in many respects, a question arises as to whether any of the methods is better than the others in a quantitative sense. The first way we examined this question was to compare the log likelihood of the data under different models and replicates. In We have defined a notion of stochastic bifurcation structure suitable for studying the behavior of stochastic genetic switches, and we have generated an extensive map of the response of the canonical bistable yeast galactose utilization network to variation in external galactose concentrations. While the data broadly conforms to our expectations for stochastic switching between low and high expression states within the network, several additional properties are noteworthy. The establishment of a “high” expressing subpopulation occurs rather abruptly and fairly consistently at a concentration of approximately 0.003% galactose, although this state is initially overlapping the low expressing subpopulation. By contrast, the low subpopulation fades away more gradually at higher concentrations, while maintaining clear separation from the high subpopulation. Activity within the high subpopulation, in terms of fluorescent intensity, increases substantially as a function of galactose concentration—by approximately 300% over the range of concentrations tested. Activity within the low subpopulation is fairly constant, and is, in most cases, indistinguishable from that of cells not expressing the reporter gene (data not shown), though there may be a mild increase in expression as the galactose concentration increases. Hence, the response of the network to varying conditions appears to combine a boolean-type “binary” switch between “on” and “off” expression states with a continuous “graded” modulation of activity within the “on” state.From a methodological point of view, we proposed that mixture density estimation and conditional mixture density estimation are ideally suited to extracting stochastic bifurcation structure from real, noisy data. Our tests of two different mixture fitting methods and one conditional mixture fitting method suggested that, in most respects, the methods are equally accurate in fitting the data. It is possible that the conditional mixture model was less accurate. Visually, it appears to overestimate the location of the high subpopulation at smaller galactose concentrations, and underestimate it at higher concentrations see or 4A,B.Conditional mixture models have several additional advantages compared to fitting the data at each galactose level separately: they use fewer total parameters, and are thus less likely to overfit the data, and they explicitly represent and make predictions for the bifurcation structure at all values of the bifurcation parameter—not only the values tested experimentally. This approach worked well on our data. The drawback of this approach is that it requires choosing functional forms to represent the dependence of mixture probabilities and mixture component parameters on the bifurcation parameter. In this case, a proper means of representing mixture probabilities only became clear after doing the individual fits. In early conditional mixture model fits, we assumed the mixture probabilities were independent of galactose concentration. This had the unfortunate side affect that the high component would start to “capture” cells from the low subpopulation at low galactose levels, dragging down the whole mean curve for the high subpopulation until it intersected and overlapped with the low subpopulation. The form we chose for the mixture probabilities avoids this problem by definitively assigning cells to the low component at all galactose levels below some threshold. This illustrates that the strength of using few parameters and explicitly generalizing across bifurcation parameter values also implies a danger of poor performance if an inappropriate representation is chosen. While this is a truism in the statistics and machine learning communities, it is all the more important to keep in mind in systems biology where there is a greater focus on interpreting models, as opposed to, say, being concerned only about prediction accuracy.Despite our focus on mixture modeling, one can imagine other approaches for estimating stochastic bifurcation structure. For example, clustering methods such as K-means or self-organizing maps could readily be applied in much the same way as we applied mixture density estimation. Nonparametric density estimation techniques might also be applied, although it would take extra effort to extract subpopulations from a nonparametric density estimate. Investigating such alternative approaches is an important topic for future research.Part of our contribution is in specifying four types of information that should be included in a stochastic bifurcation analysis: the number of distinct subpopulations, the fraction of cells they contain, the level of expression and the variance within each subpopulation. Our notion of stochastic bifurcation structure is considerably different from ideas employed in stochastic bifurcation theory, which addresses the behavior of explicitly stochastic dynamical models, such as stochastic differential equations Stochastic bifurcation structure may provide useful information for the development of quantitative regulatory network models, however this remains to be investigated. The exact relationship between stochastic observables and model features is not yet clearly established. For example, models of gene regulatory networks are usually derived from molecular interactions within individual cells and rarely consider effects due to population dynamics. The gradual fading of the low-expressing subpopulation observed in our experiments could be due the stochastic dynamics of the regulatory network itself, or it could be due to a reduced growth rate of the low-expressing cells. Additionally, while we took steps to present the cells in each culture with homogenous extracellular conditions see , it is let al. (W303) is much less sensitive to galactose and displays an almost 10-fold shift of the bimodal region (to concentrations between approximately 0.02% and 0.3%) compared to our strain from the native promoter of the GAL10 gene agar plates without leucine and histidine supplemented with 2% w/vol glucose and 1% w/vol adenine. Individual colonies were used to inoculate 3 mL rich media (YPR) containing 20 g/L Yeast Bacto-Peptone, 10 g/L yeast extract, 1% w/vol adenine and 2% w/vol raffinose (Wisent) or YPR media supplemented with 2% w/vol galactose . Following growth for 24 hours at Reporter gene expression was quantified in individual cells using a Beckman-Coulter FC500 flow cytometer. A total of 60,000 events were collected for each condition and filtered using custom-written software script using a fixed elliptical forward/side-scatter autogate capturing approximately 50% of the events in each sample. The fluorescence intensity associated with these events was used to generate representative expression distributions for each sample condition. A total of four replicates were obtained, for each final galactose concentration and both pre-growth conditions.Mixture density estimation using EM used 100 runs in an effort to avoid problems with stopping at solutions that were only locally optimal. Each of the 100 runs began from different random initial parameters. The means of each Gaussian component were chosen uniformly between the lowest and highest data point. Standard deviations were initialized to 50—roughly the level observed at single-subpopulation galactose concentrations—and initial mixture probabilities for the Gaussians were set to The EM fitting employed cross-validation to determine the proper number of Gaussian components to have in the mixture for each replicate and at each galactose level. After fitting a model with Mode estimation for the mode-estimation-plus-EM approach began by smoothing the data by taking a running average over a window of size 71 channels. Call this For fitting the conditional mixture density models, we used only a single run of EM, as further runs did not improve accuracy. Updates are standard, as given in Bishop http://www.perkinslab.caAll code is written in MATLAB. Code and raw data are available upon request, as well as on TJP's website:
Ammophila, whose decline under decreasing sand accretion is argued to be caused by either biotic or abiotic soil properties.Plants are affected by several aspects of the soil, which have the potential to exert cascading effects on the performance of herbivorous insects. The effects of biotic and abiotic soil characteristics have however mostly been investigated in isolation, leaving their relative importance largely unexplored. Such is the case for the dune grass Ammophila arenaria seedlings and Schizaphis rufula aphid populations. Root mass fraction and total dry biomass of plants were affected by soil biota, although the latter effect was not consistent across regions. None of the measured plant properties were significantly affected by the abiotic soil component. Aphid population characteristics all differed between regions, irrespective of whether soil biota were present or absent. Hence these effects were due to differences in abiotic soil properties between regions. Although several chemical properties of the soil mixtures were measured, none of these were consistent with results for plant or aphid traits.By manipulating dune soils from three different regions, we decoupled the contributions of region, the abiotic and biotic soil component to the variation in characteristics of Plants were affected more strongly by soil biota than by abiotic soil properties, whereas the opposite was true for aphids. Our results thus demonstrate that the relative importance of the abiotic and biotic component of soils can differ for plants and their herbivores. The fact that not all effects of soil properties could be detected across regions moreover emphasizes the need for spatial replication in order to make sound conclusions about the generality of aboveground-belowground interactions. Plants are heavily affected by both the abiotic and biotic properties of the soil in which they are rooted. Abiotic properties include the availability of nutrients and water, which are necessary for plant growth. Soil biota comprise mutualists as well as antagonists, exerting positive or negative effects on plant growth respectively.These effects of soil properties on the plant can further affect leaf herbivores. Firstly, herbivores are generally limited by the nutrients they can obtain from plants, some of which these plants extract from soil Jacobaea vulgaris. As these compounds are toxic to generalist herbivores, but preferred by specialists, it is concluded that both the abiotic and biotic soil component have the potential to affect herbivores of J. vulgaris. To our knowledge only a few studies have explicitly tested the combined effect of abiotic and biotic soil components on aboveground herbivores. Haase et al. Folsomia candida) and aphids on the grass Poa annua under different levels of nutrient availability. They demonstrated that collembolans strongly increased aphid numbers at low and moderate nutrient availability, while this effect was much weaker at high nutrient availability. It has furthermore been demonstrated that the effect of mycorrhizal fungi on the performance of an insect can depend on the amount of nutrients in the soil Although both biotic and abiotic soil components clearly have the potential to affect the performance of plants and their herbivores, their relative contribution to these effects has rarely been addressed. In a cross-inoculation experiment of soil biota, Joosten et al. Ammophila species in the early succession of coastal dune vegetation. Both the North American A. breviligulata and the European A. arenaria exhibit strongly suppressed growth as sand accretion ceases, making way for later successional plant species. Therefore, vigorous stands of Ammophila are found in foredunes and large, dynamic inland dunes with sufficient sand-drift, while stands in stabilised dunes, often at the inner dune edge, occur as degenerate relics. This phenomenon of loss of vigour under conditions of stabilisation has been coined “the Ammophila problem” by Marshall A. arenaria revealed that the accumulation of biotic soil factors in stabilised soils was responsible for reduced growth A. arenaria in lime- and iron-poor dunes to the limitation by N as a result of the relatively higher availability of P when it is not sequestered into iron and aluminium phosphates.One of the best studied and most debated cases of negative plant-soil feedback is that of A. arenaria was capable of reducing the population growth of the specialist aphid Schizaphis rufula under laboratory conditions. Yet no correlation between nematode and aphid abundances could be detected in a field survey conducted at six spatially separated sites. Both nematode and aphid abundances could however be explained by several plant characteristics In a previous study, we demonstrated that the natural root-feeding nematode community of A. arenaria to investigate plant-soil interactions, we chose this species as a model system. Based on the evidence that soil-borne organisms are involved in the Ammophila-problem, we hypothesise that plants should grow better on sterile soils compared to soils with naturally occurring biota. On the other hand, if this phenomenon is to some extent caused by an increased availability of nutrients in dynamic dune soils, plants should perform better on dynamic than on stabilised dune soils, irrespective of their biotic state. We inoculated sterile soils from dynamic and stabilised dunes with biota from either location and used a fully sterile soil as control. On these soils we grew seedlings of A. arenaria, on half of which we let a population of S. rufula develop. This setup was replicated with soils from three distinct regions along the Belgian coast, to assess the generality of potential results across large spatial scales Given this multitude of studies using A. arenaria grows very vigorously and one situated at the inner dune edge, where conditions are more stabilised and the plant only occurs as degenerate relics. All these dune areas are spatially separated (distances between sites from different regions ranging from 2.1 to 12.6 km). At each site, a composed sample of soil was taken, collected from underneath different stands of A. arenaria, comprising a mixture of upper root zone soil and freshly deposited soil from above the root zone. In the laboratory each sample of soil was divided into two parts, one of which was sterilised by autoclaving for 1 hour at 120°C and 1 atm. For each site three types of soil were prepared: fully sterile soil, sterile soil with an inoculum of unsterile soil from the same site and sterile soil with an inoculum of unsterile soil from the other site within the same region. So for each region the combinations were: D, D+s , D+d, S, S+d, S+s, with D = sterile dynamic dune soil, S = sterile stabilised dune soil, d = unsterilised dynamic dune soil inoculum, s = unsterilised stabilised dune soil inoculum. The inoculum comprised 21 volume percent of the soil mixture, which did not affect any of the purely abiotic properties of the soil mixture (see Soil was collected from three different regions at the coast on 5 November 2008: nature reserve Westhoek at De Panne (Belgium), nature reserve Ter Yde at Oostduinkerke (Belgium) and Le Perroquet at Bray-Dunes (France). In each region soil was collected from two sites; one situated in dynamic dunes with sand-drifts where ture see . This waA. arenaria were collected from the nature reserve Westhoek from a single stand. Seeds were surface sterilised by submersing in 4% household bleach solution, rinsing 10 times with demineralised water, submersing in 10% ethanol and rinsing another 10 times with demineralised water. This sterilisation method effectively eliminates endophytic fungi that otherwise could colonise the young seedling. Seeds were subsequently germinated at a light regime of 9/15 hours dark/light in plastic 1 L pots filled with 190 cm3 of commercial white sand that was autoclaved for 1 hour at 120°C and 1 atm. The sand was saturated with demineralised water. Plastic foil that covered the pots was perforated to allow of enough ventilation. Moisture level was reset to near saturation 3 times a week.Seeds of −1 tap water).For each unique soil type, twenty replicate 1062 mL glass jars were each filled with 300 mL of the treatment soil. Each jar received one six week old seedling, and from then on water was added twice a week, alternately with and without fertiliser , NH4-N (mg kg−1 of dry soil), plant available P , pH-KCl, percentage organic matter per dry soil and percentage CaCO3 , NOThe effects of the soil treatments on plant performance were tested within the control group of plants that did not receive aphids, because the effect of aphids on plants can potentially interact with the effects of the soil treatments. Tested plant variables were total dry weight, the root proportion of total dry weight and relative water content of the shoot, which was highly correlated with the relative water content of the entire plant. These variables were chosen because they represent a measure of total biomass produced, the relative allocation of biomass to roots and shoots, and the vitality of the plant tissue respectively.0.ekt served as a measure of population growth speed.Several aphid population parameters were tested in function of the soil treatments. The number of days between introduction of the first instar and the appearance of the first offspring was used as an approximation of generation time. The number of aphids at the population peak equates the maximum population size a plant can sustain. For each population, an exponential growth curve was fitted through the aphid abundances from day one until te day of population peak. The growth constant k of the curve N = NP>0.05) was performed to obtain robust P-values in the final model. If the final model happened to retain only one predictor variable, the test was repeated with unrestricted permutation of raw data, which provides an exact test for the one-way case P-values.To determine which treatments or interactions between treatments significantly affected plant and aphid characteristics, a permutational 3×2×3 ANOVA was performed for each dependent variable. The treatment “region” was composed of levels “Westhoek” (WE), “Ter Yde” (TY) and “Perroquet” (PE). The treatment “soil” refers to the sterile part of each treatment soil with levels “dynamic dune” (D) and “stabilised dune” (S). The treatment “inoculum” refers to the unsterile soil inoculum with levels “none” (/), “dynamic dune” (d) and “stabilised dune” (s). Tests were based on type III sums of squares and 99.999 permutations of the residuals under a reduced model. A backward stepwise pooling of non significant terms were replaced, but since this replacement affected the measured characteristics, only the initial plants were retained in all analyses. There were no significant differences in seedling mortality between regions, soil types, or inocula .To determine whether the soil properties of our treatment soils significantly differed according to region, soil, inoculum, or an interaction, a permutational 3×2×3 ANOVA with a backward selection procedure was performed for each soil parameter in a similar way as described above.4,93: 3.7674, P: 0.0068, 2,93: 3.4922, P: 0.0336) and the main effect of region being marginally significant . The type of sterile soil did not affect plant dry weight. Pairwise comparisons revealed that on soils from Westhoek and Le Perroquet there was no effect of soil biota on total dry weight, regardless of their origin. On Ter Yde soils, however, dry weight clearly increased when no soil biota were inoculated (Plant dry weight differed significantly according to an interaction between region and inoculum (pseudo-Foculated .P: 0.0269), the F-test of ANOVA is very robust against unequal variances, and a significance level of 0.01 has been suggested for homogeneity tests prior to ANOVA 4,93: 3.2103, P: 0.0165).Although for plant dry weight the Levene's test was significant (P: 0.001), and according to the non-parametric test, none of the treatments had a significant effect.Homogeneity of variances could not be confirmed for the relative water content of the shoot , as a larger proportion of the total dry weight was allocated to roots in plants grown on soils inoculated with biota from dynamic dunes (P: 0.3768). Summarising these results, it can be concluded that A. arenaria seedlings were most affected by soil biota.The root fraction of dry weight was only affected by soil inoculum (pseudo-Fic dunes . This efThe plant feature that correlated best with aphid maximum density was the fresh weight of the shoot (Pearson's r: 0.74221). Total fresh weight correlated best with generation time (Pearson's r: 0.49349), while the exponential growth constant correlated best with relative water content of the shoot (Pearson's r: 0.32240).2,161: 7.174, P: 0.001, 1,160: 194.88, P: 0.00001) still resulted in a significant effect of region . This indicates that regional soil effects do not only operate through changes in plant shoot weight, but also through additional mechanisms. Again only region was retained as a significant factor affecting aphid generation time, both in the models without and with total fresh weight as a covariate. No significant pairwise differences could be detected, but the differences between Le Perroquet and Westhoek (P: 0.0554) and between Westhoek and Ter Yde (P: 0.0601) were almost significant . A similar result was obtained when the relative water content of the shoot was modelled as a covariate . Aphid populations on plants from Ter Yde were characterised by stronger exponential growth, short generation times and larger maximum population sizes (P>0.05).Results of the ANOVA demonstrate a significant effect on maximum aphid density only of region Click here for additional data file.Table S1Results of the final permutational ANOVA models of the different soil parameters, obtained after a stepwise backward selection procedure. R: region where soil was collected: Westhoek, Ter Yde or Le Perroquet. S: sterile, abiotic component of the soil: dynamic dunes or stabilised dunes. I: unsterile, biotic soil inoculum: none, dynamic dunes or stabilised dunes. No significant effects were detected for the percentage moisture and NH4-N (mg/kg) of the soils.(0.05 MB DOC)Click here for additional data file.Table S2Results of pairwise comparisons among levels of factors that significantly affected the soil parameters in the final permutational ANOVA models. Comparisons were made by means of permutational t-tests. When the number of unique permutations was lower than 100, Monte Carlo sampling was used to obtain reliable P-values. Region - PE: Le Perroquet, WE: Westhoek, TY: Ter Yde. Soil - D: sterile soil component of dynamic dune, S: sterile soil component of stabilised dune. Inoculum - /: no inoculum, d: dynamic dune biota, s: stabilised dune biota.(0.28 MB DOC)Click here for additional data file.Table S3Results of pairwise comparisons among levels of factors that significantly affected plant and aphid characteristics in the final permutational ANOVA models. Comparisons were made by means of permutational t-tests. When the number of unique permutations was lower than 100, Monte Carlo sampling was used to obtain reliable P-values. Region - PE: Le Perroquet, WE: Westhoek, TY: Ter Yde. Inoculum - /: no inoculum, d: dynamic dune biota, s: stabilised dune biota.(0.09 MB DOC)Click here for additional data file.Figure S1Differences in NO3-N content of treatment soils with different unsterile inocula (mean + SE). Significant pairwise differences are indicated by different letters above the bars (P < 0.05). Inoculum - /: no inoculum, d: dynamic dune biota, s: stabilised dune biota.(0.05 MB TIF)Click here for additional data file.Figure S2Differences in soil parameters of treatment soils according to region and abiotic soil component (mean + SE). A) Percentage CaCO3. B) pH-KCl. C) Percentage organic matter per dry matter. Significant pairwise differences are indicated by different letters above the bars (P < 0.05). Region - PE: Le Perroquet, WE: Westhoek, TY: Ter Yde. Soil - D: sterile soil component of dynamic dune, S: sterile soil component of stabilised dune.(0.12 MB TIF)Click here for additional data file.Figure S3Differences in plant available P of treatment soils according to region, abiotic soil component and unsterile soil inoculum (mean + SE). Significant pairwise differences are indicated by different letters above the bars (P < 0.05). Region - PE: Le Perroquet, WE: Westhoek, TY: Ter Yde. Soil - D: sterile soil component of dynamic dune, S: sterile soil component of stabilised dune. Inoculum - /: no inoculum, d: dynamic dune biota, s: stabilised dune biota.(0.17 MB TIF)Click here for additional data file.
Both aliphatic and aromatic carboxylic acids participated in trifluoroacetic anhydride/phosphoric acid mediated C-C bond forming reactions under solvent-free conditions affording acyl benzothiophenes in good overall yields.A simple and single-step synthesis of 2- and 3-acyl substituted benzothiophenes has been described Additionally, formation of unidentified side products is very common in this reaction. Therefore, the use of this method especially in large scale preparation might present major drawbacks. To overcome these difficulties we, therefore, focused on the alternative methods available in the literature [3 and 4) that involves the first use of benzothiophene in the aromatic acylation process mediated by the mixed anhydrides of trifluoroacetic acid (While a number of methods are known for the synthesis of acyl benzothiophenes –20 many terature –30 that terature –30 the utic acid . The res3*Et2O or ii) acetyl chloride in the presence of AlCl3. In a typical procedure, to a mixture of commercially available acid 1 in TFAA was added acetic acid dropwise followed by 85% H3PO4 with vigorous stirring at 0°C. The mixture was then warmed to 25–30°C, stirred for 4 h and poured into ice-cold water (25 mL) with vigorous stirring. The solid separated was filtered, washed with petroleum ether (2 × 5 mL) and dried to give the mixture of product 3a and 4a in 70 % overall yield. Based on HPLC data, [see 3a and 4a. After separating these compounds using column chromatography, the structures of 3a and 4a was confirmed by spectral data [see 3a and 4a successfully we then tested the reactivity of other carboxylic acids under these reaction conditions and subsequently, a variety of acylated benzothiophenes were prepared [see 1 (1 equiv), acid 2 (1 equiv), 85% H3PO4 (1 equiv) and TFAA (4 equiv) at 25–30°C for 4–5 h and the % yields shown in 1H NMR, IR and mass spectra. As shown in 1H NMR data. The C-3 proton of compound 3 appeared in the region of 7.90–7.95 δ and 7.97–8.0 δ (when R = aryl) whereas C-4 proton of compound 4 appeared in 8.27–8.29 δ and 8.40–8.60 δ (when R = aryl).Initially, we conducted the acylation reaction of benzothiophene with acetic acid in the presence of 85% phosphoric acid and excess trifluoroacetic anhydride (TFAA) at room temperature (25–30°C) for 30 min where no significant product formation was observed. Then the reaction was continued for a longer time and the progress of the reaction was monitored by TLC over time. This exercise resulted in isolation of acylated products and the best results were observed when the reaction was carried out for 4 h. However, a mixture of 2- and 3-acylated benzothiophene was always isolated in all these cases. An attempt to prepare a single regioisomer by varying all the reaction parameters failed perhaps due to the high reactivity of the benzothiophene ring under the conditions studied. A similar observation was noted during acetylation,23 of be3 as a dehydrating agent. However, P2O5 is more convenient in small scale preparation of TFAA.We have developed a very simple and single-step process for the synthesis of acyl benzothiophenes. All the starting materials are commercially available and can be used directly. The process does not require the use of additional organic solvent but involves use of excess TFAA. While the excess of TFAA and TFA (trifluoroacetic acid produced during the reaction) was removed by treating the reaction mixture with water, their removal by distillation is more appropriate for large scale preparations. This alsin situ.[3 and 4. Phosphoric acid as a source of proton helps in activation. The isolation of compound 4 as the major product can be explained by the higher reactivity of C-3 over C-2 of the benzothiophene ring.A probable mechanism for the acylation of benzothiophene is shown in in situ. The acylIn conclusion, we have described a general synthesis of a variety of acylated benzothiophenes via acylation of benzothiophene rings with in situ generated arylacetyl trifluoroacetates. The reaction proceeds at room temperature and does not involve the use of any expensive reagents or catalysts. Other advantages of the present protocol include i) ready availability of the starting materials and mild reaction conditions, ii) environmentally safe as the protocol is free from the use of inorganic Lewis acids as well as chlorinated hydrocarbons as solvent, iii) simple operational procedure. However, formation of a mixture of regioisomers is the major drawback of this protocol. Nevertheless, the present process is certainly superior to the classical Friedel-Crafts acylation technique and other multi step synthesis. Further studies on establishing the regeioselectivity and application of this methodology in organic synthesis are under investigation.File 13a and 4a, spectral data for other selected compounds .Transition-metal/lewis acid free synthesis supporting info. HPLC conditions and spectral data for compounds
For successful photodynamic diagnosis (PDD) and effective photodynamic therapy (PDT) with the clinically used 'photosensitiser' 5-aminolaevulinic acid (ALA), knowledge of the maximal fluorescence intensity and of the maximal tumour-host tissue fluorescence ratio following systemic or local application is required. Therefore, time course and type of porphyrin accumulation were investigated in neoplastic and surrounding host tissue by measuring the kinetics and spectra of ALA-induced fluorescence in vivo. Experiments were performed in the amelanotic melanoma A-Mel-3 grown in the dorsal skinfold chamber preparation of Syrian golden hamsters. The kinetics of fluorescent porphyrins was quantified up to 24 h after i.v. injection of 100 mg kg-1, 500 mg kg-1 or 1,000 mg kg-1 body weight ALA by intravital fluorescence microscopy and digital image analysis (n = 18). In separate experiments fluorescence spectra were obtained for each dose by a simultaneous optical multichannel analysing device (n = 3). A three-compartment model was developed to simulate fluorescence kinetics in tumours. Maximal fluorescence intensity in the tumour arose 150 min post injection (p.i.) and 120 min p.i. . The fluorescence in the surrounding host tissue was far less and reached its maximum at 240 min and 360 min p.i. and . Maximal tumour-host tissue ratio (90:1) was encountered at 90 min after injection of 500 mg kg-1. The spectra of tissue fluorescence showed maxima at 637 nm and 704 nm respectively. After 300 min (host tissue) and 360 min (tumour tissue) additional emission bands at 618 nm and 678 nm were detected. These bands indicate the presence of protoporphyrin IX (PPIX) and of another porphyrin species in the tumour not identified yet. Tumour selectivity of ALA-induced PPIX accumulation occurs only during a distinct interval depending on the administered dose. Based on the presented data the optimal time for PDD and PDT in this model following intravenous administration of 500 mg kg-1 ALA would be around 90 min and 150 min respectively. The transient selectivity is probably caused by an earlier and higher uptake of ALA in the neoplastic tissue most likely as a result of increased vascular permeability of tumours as supported by the mathematical model.
The relationship between oxygen delivery and consumption in sepsis is impaired, suggesting a microcirculatory perfusion defect. Recombinant human erythropoietin (rHuEPO) regulates erythropoiesis and also exerts complex actions promoting the maintenance of homeostasis of the organism under stress. The objective of this study was to test the hypothesis that rHuEPO could improve skeletal muscle capillary perfusion and tissue oxygenation in sepsis.Septic mice in three experiments received rHu-EPO 400 U/kg subcutaneously 18 hours after cecal ligation and perforation (CLP). The first experiment measured the acute effects of rHuEPO on hemodynamics, blood counts, and arterial lactate level. The next two sets of experiments used intravital microscopy to observe capillary perfusion and nicotinamide adenine dinucleotide (NADH) fluorescence post-CLP after treatment with rHuEPO every 10 minutes for 40 minutes and at 6 hours. Perfused capillary density during a three-minute observation period and NADH fluorescence were measured.p < 0.001). Treatment of CLP mice with rHuEPO resulted in an immediate and significant increase in perfused capillaries in the CLP group at all time points compared to baseline from 28.5 to 33.6 capillaries per millimeter at 40 minutes; p < 0.001. A significant increase in baseline NADH, suggesting tissue hypoxia, was noted in the CLP mice compared to the sham group and improved with rHuEPO from 48.3 to 44.4 FU at 40 minutes (p = 0.02). Six hours after treatment with rHuEPO, CLP mice demonstrated a higher mean perfused capillary density and a lower mean NADH fluorescence as compared to CLP+normal saline mice .rHuEPO did not have any effects on blood pressure, lactate level, or blood cell numbers. CLP mice demonstrated a 22% decrease in perfused capillary density compared to the sham group (28.5 versus 36.6 capillaries per millimeter; rHuEPO produced an immediate increase in capillary perfusion and decrease in NADH fluorescence in skeletal muscle. Thus, it appears that rHuEPO improves tissue bioenergetics, which is sustained for at least six hours in this murine sepsis model. On a ma and use . Whether persist . Persistrsist [2 . These orsist [2 -8. Even rsist [2 ,9-14.Studies in critical care support a reduction in the red blood cell (RBC) transfusion threshold and the rHuEPO exerts multiple protective actions on the circulatory system, including the microcirculation, which is known to be dysregulated during sepsis. In addition, the blunted endogenous EPO response in critically ill patients with sepsis may contribute further to microcirculatory dysfunction and tissue dysoxia . Based oad libitum. All surgeries were performed with a clean technique.The University of Western Ontario Council on Animal Care approved the study protocol. Animals were managed according to guidelines set forth by the institutional Council on Animal Care. Mice were acclimatized to the laboratory for one week and had access to mice chow and water The C57BL/6 mice supplied by Charles River Laboratories, Inc. received general anaesthesia with ketamine/xylazine 80:10 mg/kg via intraperitoneal injection. Sepsis was induced by cecal ligation and perforation (CLP). An incision was made along the linea alba. The cecum was mobilized and gently exteriorized using swabs moistened with warm saline (37°C). After ligation, just distal to the ileal cecal valve with 1-0 silk, the cecum was punctured twice with an 18-gauge needle along the anti-mesenteric aspect and gently squeezed to ensure patency of the holes. The cecum was returned to the abdominal cavity and the incision was closed in two layers. In the sham mice group, a similar procedure was performed but without the ligation and puncture. The sham and the CLP animals were allowed to recover with free access to water and mice chow for 18 hours post-surgery prior to treatment with rHuEPO in the CLP group. All animals received buprenorphine 0.1 mg/kg in 1 ml of normal saline (NS) subcutaneously injected after surgery and every eight hours for analgesia and fluid resuscitation. The mice were monitored for signs of discomfort throughout the recovery period.The sham and the CLP mice were re-anaesthetized and placed on a heating pad, and core temperature was monitored using a thermocouple rectal probe and maintained between 36°C and 37°C. The extensor digitorum longus (EDL) muscle was exposed by gentle dissection. A suture was tied around the distal tendon, which was then separated, and the muscle was reflected over the microscope objective of a Nikon Diaphot 300 inverted microscope with the proximal neurovascular bundle intact. The preparation then was allowed 45 minutes to equilibrate . To visuNicotinamide adenine dinucleotide (NADH) fluorescence from the same area was measured by switching the microscope to an epi-fluorescence configuration using a 100-W mercury arc lamp source, a 365BP25nm excitation filter, a 450BP65 emission filter, and a 400CLP02 dichroic mirror . An additional 550-nm low-pass filter was installed within the C-mount of the microscope to prevent interference of emission light above 550 nm with the NADH fluorescence image. An ICCD (intensified charge coupled device) camera captured the images .Capillary density was assessed by observing capillaries of the EDL and counting the number of perfused and stopped capillaries crossing three equidistant lines drawn perpendicular to the direction of the muscle fibers on the observation screen. A capillary was counted as perfused if RBC flow was noted at any time during a three-minute observation period. If there was no flow for the entire three-minute period, the capillary was counted as stopped. Intermittent perfusion was not assessed and plasma-filled (no RBCs visible) capillaries were not detectable with this method. The width of the image field measured was 320 μm per objective field. Capillary density represents the number of capillaries visible across a distance of 1 mm as calculated from the magnification used during the study. NADH fluorescence intensity was measured using Sigma Scan and was expressed in arbitrary fluorescence units (FU). To account for fluctuation in daily intensity readings, all data were normalized using a standard NADH solution (41 μmol/l).n = 6; Sham+EPO, n = 6; CLP+NS, n = 7; CLP+rHuEPO, n = 7). The 400 U/kg rHuEPO dose was chosen after performing dose-response experiments using single sc doses of 200, 400, 800, and 1,000 U/kg, in which the 400 U/kg dose produced the optimal capillary perfusion during sepsis (data not shown). We measured mean arterial pressure (MAP) by cannulating the carotid artery with Intramedic polyethylene tubing (PE10) connected to a transducer and monitor . The heart rate (HR) was determined from a recording of the arterial pressure trace at time 0 and at 40 minutes post-sc injection. Blood samples were also drawn at 40 minutes post-sc injection of saline or rHuEPO for measurement of hemoglobin (Hb), white blood cell (WBC) count, platelets (PLTS), and arterial serum lactate. The complete blood count was measured on an LH750 Series Beckman Coulter hematology analyzer , and the arterial lactate was measured using a VSI 2300 Stat Plus glucose and lactate analyzer .Three sets of experiments were performed. In the first set of experiments, we assessed the acute effects of rHuEPO on hemodynamics, blood cell count, and arterial serum lactate level. In sham and CLP mice at 18 hours after surgery, the mice were re-anesthetized and were given either a 0.2 ml subcutaneous (sc) injection of NS or 400 U/kg rHuEPO and CLP mice (n = 8) 18 hours after surgery. The CLP animals then received a 400 U/kg bolus of rHuEPO by sc injection. Images of the capillaries and NADH fluorescence were recorded every 10 minutes for 40 minutes for both groups.In the second set of experiments, we wished to determined the acute effects of rHuEPO on the microcirculation and NADH levels in the EDL of CLP mice and using the mice as their own baseline control observation at time 0. We performed baseline intravital microscopy in untreated sham mice (n = 10) or rHuEPO 400 U/kg . After an additional six hours, the mice were re-anesthetized and intravital microscopy was performed as described above.In the third set of experiments, we wished to determine whether the effects of rHuEPO observed in the second experiment persisted in treated versus untreated CLP mice. Mice underwent CLP and 18 hours later received 0.2 ml sc injections of saline , we cannot rule out a change in total pool size contributing to the decrease in NADH fluorescence. Activation of poly (ADP-ribose) polymerase by oxidative stress in sepsis has been proposed as a pathway that could lead to NAD+ depletion, but there is evidence against this occurring in the CLP model [In the CLP animals, the initial NADH levels at 18 hours were higher than in non-septic controls and normalized following treatment with rHuEPO. The changes in NADH fluorescence were relatively small; however, this observation and the association with a change in microcirculatory perfusion are new observations. At present, we do not have any data that allow us to determine the relation between changes in NADH fluorescence of this magnitude and the prevention of cellular dysfunction or death. Because we did not measure oxidized NADH (NADLP model .Although we found changes in blood pressure, lactate, WBC count, and PLTS which are consistent with severe sepsis, there was no mortality in this study prior to euthanasia at the completion of each experiment. However, in preliminary studies with this model, we observed a 23% to 25% mortality between 18 to 24 hours post-CLP. This is in contrast to the findings of Hollenberg and coworkers , who repWe also recognize that the behavior of the microcirculation in the EDL skeletal muscle during sepsis may not be representative of the microcirculation of other tissues. We chose to use EDL skeletal muscle in this study because it is well characterized in our laboratory and in the literature . Similar2 via increased perfused capillary density. Further studies are warranted to determine the potential mechanisms for these observations and to determine whether this effect is sufficient to improve organ function and reduce morbidity and mortality in sepsis.rHuEPO treatment in a murine model of severe sepsis induces a rapid normalization in the perfused capillary density with a concomitant decrease in NADH fluorescence in skeletal muscle. Thus, rHuEPO appears to improve mitochondria oxidative phosphorylation and pyruvate metabolism in this septic mouse model in part by improving DO• Erythropoietin improves perfused capillary density in the skeletal muscle of septic mice.• Erythropoietin treatment can also decrease mitiochondrial NADH levels, suggesting improved oxidative phosphorylation and pyruvate metabolism.• Erythropoietin has a potential clinical application in the improvement of tissue bioenergetics during sepsis.2 = oxygen delivery; EDL = extensor digitorum longus; ETC = electron transport chain; FU = fluorescence units; Hb = hemoglobin; HR = heart rate; MAP = mean arterial pressure; NAD+ = oxidized nicotinamide adenine dinucleotide; NADH = nicotinamide adenine dinucleotide; NS = normal saline; PLTS = platelets; RBC = red blood cell; rHuEPO = recombinant human erythropoietin; sc = subcutaneous; WBC = white blood cell.CLP = cecal ligation and perforation; DOAX has been an invited speaker and medical monitor for Ortho Biotech and the recipient of unrestricted educational grant from Ortho Biotech. The other authors declare that they have no competing interests.RK conceived of and designed the study, performed data analysis, drafted the manuscript, and approved the final version of the manuscript. CMM conceived of and designed the study, revised the manuscript for critically important intellectual content, and approved the final version of the manuscript. AX conceived of the study, drafted the manuscript for critically important intellectual content, and approved the final version of the manuscript. TR revised the manuscript for critically important intellectual content and approved the final version of the manuscript. PY, WH, and JR carried out the animal experiments and acquisition of data. RK and AX contributed equally in drafting this manuscript.
Acremonium cellulolyticus and Trichoderma reesei are known to be potential cellulase producers. In this study, we aimed to reveal the advantages and disadvantages of the cellulase enzymes derived from these fungi.Bioethanol isolated from lignocellulosic biomass represents one of the most promising renewable and carbon neutral alternative liquid fuel sources. Enzymatic saccharification using cellulase has proven to be a useful method in the production of bioethanol. The filamentous fungi A. cellulolyticus and T. reesei cellulase activity against the three lignocellulosic materials: eucalyptus, Douglas fir and rice straw. Saccharification analysis using the supernatant from each culture demonstrated that the enzyme mixture derived from A. cellulolyticus exhibited 2-fold and 16-fold increases in Filter Paper enzyme and β-glucosidase specific activities, respectively, compared with that derived from T. reesei. In addition, culture supernatant from A. cellulolyticus produced glucose more rapidly from the lignocellulosic materials. Meanwhile, culture supernatant derived from T. reesei exhibited a 2-fold higher xylan-hydrolyzing activity and produced more xylose from eucalyptus (72% yield) and rice straw (43% yield). Although the commercial enzymes Acremonium cellulase demonstrated a slightly lower cellulase specific activity than Accellerase 1000 , the glucose yield (over 65%) from lignocellulosic materials by Acremonium cellulase was higher than that of Accellerase 1000 (less than 60%). In addition, the mannan-hydrolyzing activity of Acremonium cellulase was 16-fold higher than that of Accellerase 1000, and the conversion of mannan to mannobiose and mannose by Acremonium cellulase was more efficient.We compared A. cellulolyticus was superior to that from T. reesei, while the xylan-hydrolyzing activity was superior for the cellulase from T. reesei. Moreover, Acremonium cellulase exhibited a greater glucan and mannan-hydrolyzing activity than Accellerase 1000.We investigated the hydrolysis of lignocellulosic materials by cellulase derived from two types of filamentous fungi. We found that glucan-hydrolyzing activity of the culture supernatant from Lignocellulosic biomass represents a promising starting material for the production of bioethanol fuel, as it contains a large quantity of sugars in the form of cellulose and hemicellulose. Ethanol fuel production from lignocellulosic biomass is advantageous as it does not lead to competition for food resources . For ethTrichoderma reesei represent the best characterized, and are often used for enzymatic saccharification of lignocellulosic materials [T. reesei QM6a has been sequenced and the sequence information is readily available [T. reesei demonstrates a relatively weak β-glucosidase activity, and the reaction from cellobiose to glucose has been shown to be slow [et al. isolated enzymes from the filamentous fungus strain Acremonium cellulolyticus Y-94, which produces high levels of cellulase [T. reesei. However, the cellulase and hemicellulase produced by A. cellulolyticus have not been as well characterized as those produced by T. reesei. A number of cellulase hyperproducing mutants have also been obtained from A. cellulolyticus Y-94 and T. reesei QM6a following treatment with mutagens including ultraviolet (UV) and various chemical compounds [A. cellulolyticus and T. reesei may prove essential for the improvement of their hydrolyzing performance during bioethanol production processes.Filamentous fungal strains, also sometimes termed wood-degrading organisms, secrete a large quantity of cellulase and hemicellulase -10. Cellaterials . The genvailable . The cel be slow . Yamanobellulase . These eompounds ,16-18. UA. cellulolyticus and T. reesei by analyzing the hydrolysis of lignocellulosic biomass. We determined the specific activity of cellulase and hemicellulase, and performed enzymatic saccharification of three lignocellulosic materials.The aim of this study was to further understand cellulases derived from A. cellulolyticus CF-2612 [T. reesei CDU-11 [A. cellulolyticus CF-2612 was cultured in production medium in Erlenmeyer flasks [T. reesei was kindly supplied by Kyowa Hakko Kougyo Co. . The commercial enzymes used in this study were Acremonium cellulase and Accellerase 1000 .The cellulase hyperproducing strains used in this study were CF-2612 and T. ri CDU-11 . A. cellr flasks , and theet al. [et al. [p-nitrophenyl-β-D-glucopyranoside were added to 100 mM citrate buffer, and the enzyme reaction mixture was incubated at 45°C for 10 min. Absorbance at 420 nm was then measured. β-Xylosidase and β-mannosidase activities were measured under similar conditions, with the exception that p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-β-D-mannopyranoside were used as the substrates, respectively. For these experiments, one unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute.The soluble protein concentration was determined using the method of Lowry et al. . Filter-et al. , a metho [et al. . Briefly [et al. . Xylanaset al. [et al. [Eucalyptus and Douglas fir wood chips were kindly supplied by a pulp factory located close to our research center . Rice straw was kindly supplied by Dr Tokuyasu . Ball-milling pretreatment of cellulosic materials was based on the methods described by Inoue et al. . The iniet al. . The ini [et al. .et al. [Enzymatic hydrolysis was performed using an enzyme constituting 22.5 or 90.0 mg protein per gram of dry substrate. The soluble protein concentration was determined using the method of Lowry et al. . The dilSubstrate hydrolysates were analyzed using a high-performance liquid chromatography (HPLC) system equipped with a RI-2031 Plus detector . Glucose, xylose, mannose and mannobiose were analyzed using an Aminex HPX-87P column fitted with a Carbo-P micro-guard cartridge. The mobile phase used was doubly deionized water, and the flow rate was 1.0 ml/min at a column temperature of 80°C.A. cellulolyticus and T. reesei, we first measured FPase, Avicelase, CMCase and β-glucosidase specific activity to determine cellulase activity, and xylanase, β-xylosidase, mannanase and β-mannosidase specific activity to determine hemicellulase activity (Table A. cellulolyticus CF-2612 (SCF-2612) and T. reesei CDU-11 (SCDU-11). We found that SCF-2612 contained higher FPase, Avicelase, CMCase and β-glucosidase specific activity than SCDU-11. In particular, β-glucosidase activity in SCF-2612 demonstrated a greater than 16-fold increase in activity compared with that in SCDU-11. In contrast, xylanase, β-xylosidase and β-mannosidase specific activities for SCDU-11 were higher than those observed for SCF-2612. Mannanase specific activity remained similar for the two enzymes.In order to investigate the features of cellulase and hemicellulase derived from ty Table . The enz-1 substrate) glucose yield at 3 h for eucalyptus, Douglas fir and rice straw was 47%, 38% and 51%, respectively, while the SCDU-11 glucose yield was 32%, 27% and 32%, respectively , suggesting that the genes related to xylan-hydrolyzing performance of A. cellulolyticus CF-2612 may have been mutated, thus resulting in a reduction in xylan-hydrolyzing activity. The reduced xylan-hydrolyzing activity of A. cellulolyticus CF-2612 may also prove problematic for the saccharification of lignocellulosic materials and may require improvement. The maximum mannose yield produced from Douglas fir was less than 50% in all experiments in this study. This finding indicates that the mannan-hydrolyzing activity of A. cellulolyticus and T. reesei strains used was insufficient. We are currently focusing on producing an A. cellulolyticus strain that exhibits a higher mannan-hydrolyzing activity.It is well established that cellulose-degrading fungi produce cellulase and hemicellulase complexes -9,11. Inompounds ,17. A. cIn this study, AC generated glucose from lignocellulosic materials more efficiently than SCF-2612, while the glucose production curves were similar for both Accellerase 1000 and SCF-2612 (Figures A. cellulolyticus and T. reesei, and examined their performance during the saccharification of three lignocellulosic materials. The culture supernatant derived from A. cellulolyticus demonstrated a higher cellulase specific activity and glucose yield from lignocellulosic materials than the T. reesei supernatant. In contrast, the enzymes derived from T. reesei demonstrated a superior xylan-hydrolyzing activity than those derived from A. cellulolyticus. AC produced a greater amount of glucose from lignocellulosic materials and a higher mannan-hydrolyzing activity than Accellerase 1000. Further studies will aid in the development of cellulases and hemicellulases that hydrolyze lignocellulosic materials more efficiently during the bioethanol production process, for advancing the generation of alternative fuel sources.In this study, we investigated the cellulase and hemicellulase specific activities of both culture supernatants and commercial enzymes derived from The authors declare that they have no competing interests.TF carried out the measurement of cellulase and hemicellulase activities in SCF-2612 and SCDU-11, the enzymatic hydrolysis and drafted the manuscript. XF and HI carried out the measurement of cellulase and hemicellulase activities of the commercial enzymes. XF, HI, KM and SS designed and coordinated the study and helped in the drafting of the manuscript. All authors read and approved the final manuscript.
This study examined the prevalence and correlates of depressive symptoms in North Korean defectors who have been living in South Korea for more than one year.We used questionnaires developed by the authors to collect sociodemographic data in addition to the Center for Epidemiologic Studies Depression Scale (CES-D), the Psychosocial Well-being Index to measure stress, and a social support scale. A total of 367 subjects were included in this study.The results showed that 30.5% of the men and 34.7% of the women reported depressive symptoms, and 33.1% of the men and 36.1% of the women exhibited signs of severe distress. Correlates of depressive symptoms were lack of occupation , having escaped without family , and a poor subjective sense of health status .Continuing vocational training and career management, psychological support programs, and intensive physical health services are needed to improve the mental health of this population. The number of North Korean defectors has been skyrocketing recently due to national economic and diplomatic crises, food shortages, weakened political power, diminishing loyalty to the Communist Party, and personal reasons.2In response to the steady growth in the number of North Korean defectors settling in South Korea, government funding for settlement reached about one million dollars for 583 defectors in 2007, subsidies to employers for hiring reached about 2 million dollars in 2007, and other funds for supporting housing, education, and social security services have also increased continuously. Family violence, marital discord, divorce, runaways, adolescent defiance, and so on represent potentially major problems affecting the adaptation period.5Psychological stressors related to this adaptation period include alienation from South Koreans, feelings of inferiority, loneliness, uncertainty about the future, concern about government retaliation against family members left in North Korea,8Countries other than South Korea have reported that long and unstable settlement processes11These difficulties with the process of adaptation are clearly related to mental health.12Post-traumatic stress disorder (PTSD), anxiety disorders, and depressive disorders often affect recent escapees from North Korea.However, the aforementioned research was primarily conducted immediately after escape or during the initial resettlement period. Research about the psychopathology of defectors who actually settle in South Korea remains lacking, and the literature is devoid of follow-up studies.Although foreign studies have reported inconsistent results, agreement exists that the prevalence and severity of psychopathology tends to decrease with time.Long-term follow-up studies of North Korean defectors are lacking, but HongUntil now, no studies about the correlates of depressive symptoms among North Korean defectors, especially among those who have settled South Korea, have been conducted. Thus, this study was conducted to investigate the prevalence and correlates of severe distress and depressive symptoms among people who have resided for at least one year in South Korea in order to provide baseline data with regard to North Korean defectors.The study was conducted from July 2006 to March 2007. The study population consisted of North Korean defectors over the age of 20 who had been living in Jeju-do, Busan metropolitan city, and Daegu metropolitan city in South Korea for more than one year. A total of 367 subjects were selected through centers supporting North Korean defectors. The sample consisted of 151 men and 216 women. Written informed consent was obtained from all participants.Sociodemographic data on age, sex, marital status (in North Korea and South Korea), residential area (in North Korea and South Korea), education, occupation (in North Korea and South Korea), income (in North Korea and South Korea), religion (in North Korea and South Korea), party enrollment (in North Korea), military service (in North Korea), subjective sense of socioeconomic status (in North Korea and South Korea), time since escape from North Korea to South Korea, and family members participating in the escape were gathered via questionnaires developed by the authors.The Korean version of the Center for Epidemiologic Studies Depression Scale (CES-D)We used the Psychosocial Well-being Index-Short Form (PWI)The perceived level of social support was measured by the social support and social conflict items introduced by Abbey et al.;We estimated the prevalence of depressive symptoms among North Korean defectors according to sociodemographic characteristics, health status, family relationships, health habits, and degree of obesity. To identify the factors associated with depression, we first conducted logistic regression analysis for each potential factor after adjusting for age and sex. We then performed multiple logistic regression by including all the statistically significant factors in order to investigate their relationships with depressive symptoms. Odds ratios (OR) and corresponding 95% confidence intervals (CI) were used to measure the associations between depressive symptoms and the factors. Statistical Package for Social Science (SPSS) ver. 12.0 , was used for all analyses; the level of significance was set at 0.05.The total sample of 367 subjects consisted of 151 men and 216 women. The mean age of male subjects was 40.3 years (SD=14.0), that of female subjects was 40.6 years (SD=14.5), and that of the total sample was 40.4 years (SD=14.3). More than a majority of the sample (53.4%) resided in the City/district area of North Korea, 42.5% had been unmarried in North Korea, 24.4% had more than a college education, 63.6% had attended middle or high school, and 40.4% of male and 7.5% of female respondents had experienced military service, reflecting a statistically significant difference between the sexes in military experience. In terms of party enrollment, 34.7% of men and 8.5% of women had been members of the Communist Party, also reflecting a significant difference between the sexes. Nearly 60% (59.4%) of the subjects reported that their socioeconomic status in North Korea was low, and 55.6% of male and 56.9% of female participants had escaped from North Korea with family members.In South Korea, 55.3% of the men and 58.8% of the women in the sample lived with spouses. Family incomes below 1.5 million won were reported by 8.8% of the sample, 9.4% of the male and 8.4% of the female respondents. Family incomes below 1 million won were reported by 57.1% of the male and 76.3% of the female participants. Of the total sample, 70.3% reported having occupations in South Korea; 71.5% of the men and 69.4% the women had occupations. Of the total sample, 75.6% reported identification with religion .In terms of consumption of alcoholic beverages, 40.7% of all subjects, 66.2% of the men and 22.8% of the women, report current drinking. Among this group, 26.1% of the total sample, 39.6% of the men and 15.6% of the women, obtained CAGE questionnaire scores greater than 2, indicating the possibility of alcohol dependence. More than one-third (34.9%) of the total sample, 33.1% of the men and 36.1% of the women, met criteria for severe distress. Nearly one-third (32.9%) of the total sample, 30.5% of the men and 34.7% of the women, reported depressive symptoms (CESD scores greater than 21). No significant differences between men and women emerged with regard to severe distress and depressive symptoms .After controlling for sex and age, we calculated the ORs for depressive symptoms according to the values of the demographic variables. The prevalence of depressive symptoms according to residence before escape, marital status, education, military service experience, party enrollment, and socioeconomic status did differ significantly; however, those who identified with religion before escape were 3.579 times more likely than were those without religion in North Korea to report depressive symptoms. Nearly 40% (39.4%) of persons who did not escape with family reported depressive symptoms, and this group was 1.680 times more likely to have depressive symptoms than were those who escaped with family. The prevalence of depressive symptoms did not statistically differ according to marital status, place of residence in South Korea, time living in South Korea, and identification with religion. Those with family incomes below 1 million won were 6.092 times more likely to report depressive symptoms than were those with family incomes greater than 1.5 million won. People without occupations were 2.289 times more likely to report depressive symptoms than were those with occupations .Those with a subjective sense that their health was poor were 3.460 times more likely to experience depressive symptoms than were those with a subjective sense that they were in good health. Current smokers were 2.405 times more likely than were nonsmokers to report depressive symptoms. People with CAGE scores greater that 2 were 2.454 times more likely and those in the severe distress group were 9.355 times (95% CI 5.620-15.573) more likely to report depressive symptoms than were others .Multiple logistic regression analysis was conducted using the statistically significant variables in the simple regression analyses as well as those variables well known as risk factors for depressive symptoms as the independent variables; depressive symptoms were the dependent variable. Multiple logistic regression analysis showed that having no occupation , having escaped without family , and having a subjective sense that one's health was poor were correlated with depressive symptoms .Subjects in this study were North Korean defectors who had been living in South Korea for more than one year after leaving Hanawon, a government-sponsored educational facility for settling North Korean refugees during their initial phase in South Korea. Male participants had lived in South Korea for a mean of 4.3 years (SD=1.5), female participants had lived in South Korea for a mean of 3.8 years (SD=1.7), and all participants had lived in South Korea for a mean of 4.0 years (SD=1.6). Of the 59.0% of the subjects living in South Korea for more than 4 years, 66.9% were male and 53.5% were female. The longest period of residence in South Korea was 13 years; the shortest period was one year.Among North Korean defectors, depressive symptoms (CES-D scores greater than 21) were found in 30.5% of the men, 34.7% of the women, and 33.0% of the total sample, which is higher than previously reported results.Although most studies report higher rates of depressive symptoms in women than in men, this study showed no significant differences in this regard. This result is consistent with previous researchIn this study, having no occupation, escaping without family, and having a subjective sense of one's health as poor were correlated with depressive symptoms.Achievement of the personal goal of escaping from North Korea must constitute a main contributor to successful adaptation in South Korea. This study did not question subjects about their motives for escaping from North Korea. However, participants tended to be characterized by low socioeconomic status, urban residence, unmarried status, high school educations, and no history of army service; 7% of individuals leaving North Korea identified with religion and 43% left without family members. After settling in South Korea, 42.6% of respondents remained unmarried, 88.0% lived in urban areas, 68.7% had low monthly incomes (less than a million won per month), 72.5% were in a low socioeconomic group, and 29.7% were unemployed. The data pertaining to before and after escaping might not have shown subjective or objective improvements in socioeconomic status, but we could not confirm this possibility with statistical analyses. In other words, the socioeconomic status of defectors did not improve. The multiple logistic regression did not use family income as an independent variable, despite the high OR in the simple regression analysis, because 64.8% of the subjects earned less than 1 million won, and this variable was closely correlated with occupation. Continuous engagement in an occupation was thought to constitute the more important indicator of long-term income as well as a more central contributor to the emotional stability of the subjects, as compared to monthly income.2Previous studies have shown that traumatic experiencesEntry into South Korean society without family members represents another contributor to depressive symptoms. This finding is consistent with the results of a study focusing on foreign immigrants or refugees, but differs from the results of Han's research.In addition, guilt about distance from families might lead to compensatory pressures for success,In general, a psychologically and physically strong person is considered to be highly likely to succeed in escaping from North Korea.The principal limitation of our study is its cross-sectional design, which made it impossible to identify causal relationships between individual risk factors and depression. In addition, because this research did not use a nationwide sample, we cannot generalize our results to all North Korean defectors. The study was also limited by not including traumatic experiences as independent variables. However, this limitation is mitigated by previous researchIn summary, mental health programs could provide early interventions for North Korean defectors experiencing such psychological problems as depressive symptoms that could otherwise detract from their quality of life and their adaptation to South Korean society.A stable support system that provides vocational and technical education, not only in Hanawon but also as part of long-term follow-up to help defectors maintain jobs as they settle into South Korean society, is necessary to decrease the prevalence of depressive symptoms. Mental health programs are needed to address emotion problems related to guilt and depression for those who escape without families. In addition, programs to support physical health will improve not only physical health per se but also related symptoms of depression.
Alpinia katsumadai (AK) extracts and fractions were tested for in vitro antiviral activities against influenza virus type A, specially human A/PR/8/34 (H1N1) and avian A/Chicken/Korea/MS96/96 (H9N2), by means of time-of-addition experiments; pre-treatment, simultaneous treatment, and post treatment.50) of these one AK extracts and five AK fractions with exception of the AK-9 were from 0.8 ± 1.4 to 16.4 ± 4.5 μg/mL against A/PR/8/34 (H1N1). The two AK extracts and three AK fractions had EC50 values ranging from <0.39 ± 0.4 to 2.3 ± 3.6 μg/mL against A/Chicken/Korea/MS96/96 (H9N2). By the hemagglutination inhibition (HI) assay, the two AK extracts and five AK fractions completely inhibited viral adsorption onto chicken RBCs at less than 100 μg/mL against both A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2). Interestingly, only AK-3 was found with inhibition for both viral attachment and viral replication after showing extended antiviral activity during the post treatment assay and quantitative real-time PCR.In pre-treatment assay, the AK extracts and AK fractions did not show significant antiviral activity. During the simultaneous treatment assay, one AK extract and five AK fractions designated as AK-1 to AK-3, AK-5, AK-10, and AK-11 showed complete inhibition of virus infectivity against A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2). The 50% effective inhibitory concentrations (ECThese results suggest that AK extracts and fractions had strong anti-influenza virus activity that can inhibit viral attachment and/or viral replication, and may be used as viral prophylaxis. Orthomyxoviridae, including influenza viruses A, B, and C, and two other genera . To obtain polysaccharide fraction, we reexamined another procedure. The dried and pulverized seeds of AK (600 g) were mixed with 1.5 L of water and shaken at 80°C for 12 h. The water extract was filtered through a filter paper to remove debris, and then the solution was precipitated by the addition of ethanol in 1:4 ratio (v/v) at room temperature. After overnight precipitation, the precipitate was collected by centrifugation and washed with acetone and freeze-dried. This fractionation procedure was repeated three times. The corresponding fraction was light brown powder (polysaccharide fraction). The remained supernatant was concentrated in a rotary evaporator under reduced pressure, yielding a supernatant fraction .μg/mL streptomycin. The influenza strains A/PR/8/34 (H1N1) (ATCC VR-1469) and A/Chicken/Korea/MS96/96 (H9N2) were propagated in MDCK cells in the presence of 10 μg/mL trypsin .Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection and grown in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 5 cells/well for 24 h. The media in plates were replaced with media containing serially diluted extracts and incubated for 72 h. The solution was replaced with only media and 5 μL MTT solution was added to each well and incubated at 37°C for 4 h. The supernatant was removed, and 100 μL 0.04 M HCl-isopropanol was added to dissolve formazan crystals. Absorbance was measured at 540 nm with subtraction of the background measurement at 655 nm in a microplate reader. The 50% cytotoxic concentration (CC50) was calculated by regression analysis.MDCK cells were grown in 96 well plates at 1 × 105 cells/well for 24 h. Before virus inoculation, non cytotoxic concentration (≤ CC50) of AK extracts were added to the cells and incubated for 12 h. Then two AK extracts and five AK fractions were removed and the MDCK cells were washed 2 times with PBS. Influenza virus at 100 TCID50 (tissue culture infectious dose) were inoculated onto the MDCK cells for 1 h with occasional rocking. The virus was removed and the cells replaced with EMEM containing 10 μg/mL trypsin. The cultures were incubated for 72 h at 35°C under 5% CO2 atmosphere until the cells in the infected, untreated control well showed complete viral cytopathic effect (CPE) as observed by light microscopy. Each concentration of two AK extracts and five AK fractions was assayed in triplicate.Pre-treatment assay Figure : MDCK ce5 cells/well) for 1 h with occasional rocking. The solution was removed and the media was replaced with EMEM containing 10 μg/mL trypsin. The cultures were incubated for 72 h at 35°C under 5% CO2 atmosphere until the cells in the infected, untreated control well showed complete CPE as observed by light microscopy. Each concentration of two AK extracts and five AK fractions was assayed in triplicate.Simultaneous treatment assay Figure : Various50 were inoculated onto near confluent MDCK cell monolayers (1 × 105 cells/well) for 1 h with occasional rocking. The media was removed and replaced by EMEM containing 10 μg/mL trypsin and several two AK extracts and five AK fractions at different concentrations. The cultures were incubated for 72 h at 35°C under 5% CO2 atmosphere until the cells in the infected, untreated control well showed complete viral CPE as observed by light microscopy. The two AK extracts and five AK fractions were assayed for virus inhibition in triplicate.Post treatment assay Figure : Influen2O, and 50% ethanol) was added to each well. The plates were incubated in the dark for 15 min at room temperature. Absorbance was read at 540 nm using a microplate reader.After 72 h incubation in all antiviral assays, 0.034% neutral red was added to each well and incubated for 2 h at 35°C in the dark. The neutral red solution was removed and the cells were washed with PBS (pH 7.4). Destaining solution was mixed with an equal volume of AK extracts (25 μL) in a two-fold serial dilution in PBS (pH 7.4) for 1 h at 4°C. Fifty μL of the solution was mixed with an equal volume of a 1% cRBC suspension and incubated for 1 h at room temperature.The hemagglutination inhibition assay was performed to evaluate the effects of two AK extracts and five AK fractions on viral adsorption to target cells. Standardized chicken red blood cell (cRBC) solutions were prepared according to the WHO manual 2002 . The influenza virus solution (4 HAU/25 μg/mL) or tamiflu (10 μM). Medium was removed after 3 h and 18 h. Cells were scraped off, washed twice with PBS, and collected by centrifugation (500 g for 3 min). In order to determine the expression level of Matrix (M) gene mRNA of influenza virus, total RNA was isolated using Qiagen RNeasy mini kit (QIAGEN) according to manufacturer's instruction. The primer sequences used for quantitative real-time PCR of viral RNA were 5'-CTTCTAACCGAGGTCGAAACGTA-3' (sense) and 5'- GGTGACAGGATTGGTCTTGTCTTTA-3' (antisense) ). NA inhibition activities were determined by Enzyme-Linked Immunosorbent Assay (ELISA). All samples were dissolved in MeOH at 5 mM and diluted. Fifty C is the fluorescence of the control after 20 min of incubation, C0 is the fluorescence of the control at zero time, S is the fluorescence of the tested samples after incubation, and S0 is the fluorescence of the tested samples at zero time. To allow for the quenching effect of the samples, the sample solution was added to the reaction mixture C, and any reductions in fluorescence were assessed.Where 50). Confluent MDCK cells were incubated with EMEM media in the absence or presence of two-fold diluted AK extracts (0.39-200 μg/mL) for 72 h, and the MTT reagents were treated onto the cells. CC50 values of AK-1, AK-2, and AK-3 showed 8.9-27.1 μg/mL. CC50 values of AK-5, AK-9, AK-10, and AK-11 showed 92.3-over 200 μg/mL and A/Chicken/Korea/MS96/96 (H9N2) were less than 50% inhibition in pre-treatment assay. , AK-5 (40% methanol fraction), AK-10 (polysaccharide fraction), and AK-11 (supernatant fraction) exhibited inhibitory activities against A/PR/8/34 (H1N1) with EC50 values ranging from 0.8 ± 1.4 to 16.4 ± 4.5 μg/mL > AK-5 (1.3 ± 0.3 μg/mL) > AK-1 (3.6 ± 1.4 μg/mL) > AK-10 (16.7 ± 4.2 μg/mL) > AK-11 (37.5 ± 12.5 μg/mL) > AK-9 (66.7 ± 16.7 μg/mL) > AK-2 (100 ± 0 μg/mL) against A/PR/8/34 (H1N1) and AK-3 (1.6 ± 0.3 μg/mL) > AK-5 (3.1 ± 0.5 μg/mL) > AK-1 (9.4 ± 3.1 μg/mL) > AK-10 (37.5 ± 12.5 μg/mL) > AK-11 (50 ± 0 μg/mL) > AK-2 (75 ± 25 μg/mL) > AK-9 (100 ± 0 μg/mL) against A/Chicken/Korea/MS96/96 (H9N2). Among them, AK-1, AK-3, and AK-5 particularly showed strong inhibition of HA with 1.04 ± 0.3 to 3.64 ± 1.4 μg/mL against A/PR/8/34 (H1N1) and 1.56 ± 0.3 to 9.37 ± 3.1 μg/mL against A/Chicken/Korea/MS96/96 (H9N2). Results demonstrated strong interaction of AK extracts and AK fractions with hemagglutinin on the outer-layer proteins of influenza virus causing the blockage of viral attachment.The simultaneous treatment assay results indicated that treatment with AK extracts and AK fractions on virus entry completely abrogated virus infectivity. Hence, we evaluated whether AK extracts inhibit hemagglutination by influenza virus. The two AK extracts and five AK fractions completely inhibited viral attachment onto cRBCs in both A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2) at less than 100 μg/mL and A/Chicken/Korea/MS96/96 (H9N2) in MDCK cells. Two AK extracts and three AK fractions except AK-3 were showed no inhibitory effects against influenza viruses in the post treatment assay. However, AK-3 demonstrated a dose dependant antiviral activity against A/PR/8/34 (H1N1), and an effective antiviral activity against A/Chicken/Korea/MS96/96 (H9N2) at concentration below 12.5 μg/mL) treated cells comparison with the non-treated cells (05.% DMSO) in both A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2) treated cells showed a stronger in late stage than in early stage. These results indicate that AK-3 exerts antiviral effects by two mechanisms, blockage of viral attachment and virus replication.As influenza viral RNA synthesized in early and late-stage, their syntheses were compared between drug-treated (AK-3) and untreated infected cells. RNA extraction was performed at 3 h and 18 h after influenza virus infection and the levels of intracellular influenza RNA were measured. Quantitative real-time PCR showed a reduction of influenza RNA from the AK-3 . We found that the ICThe influenza virus replication cycle can be divided into 5 steps: 1) binding of viral HA to sialic acid (SA) receptor on host cell surface (adsorption step), 2) internalization of virus by receptor-mediated endocytosis and fusion of viral HA2 with endosomal membranes triggered by influx of protons through M2 channel (endocytosis and fusion step), 3) release of viral genes into the cytoplasm (uncoating step), 4) packaging of viral proteins with viral genes after viral RNA replication, transcription and translation, and budding of new viruses (packaging and budding step), and 5) release of new viruses by sialidase cleaving SA receptors (release step) . Recentl2O extract) showed antiviral effect against A/Chicken/Korea/MS96/96 (H9N2) only. These data suggest that two AK extracts and five AK fractions may directly interfere with viral envelope protein and not with the SA receptor at the cell surface. Therefore, we used HI assays to determine whether the two AK extracts and five AK fractions interacted with HA of influenza virus. Two AK extracts and five AK fractions exhibited complete inhibition of viral HA in both A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2), which agrees with the simultaneous treatment assay results. Overall, we strongly suggest that AK extracts and AK fractions could develop potent antiviral drug candidate via inhibition of viral HA protein.The time-of-addition assays during the pre-treatment and simultaneous treatment were used to identify which extracts block the viral adsorption to cells. The pre-treatment assay did not show significant antiviral activity Figure . AK-3 anin vitro effect of these extracts and fractions on viral replication. Grienke et al.[n-butanol inhibited NA protein. Therefore, we evaluated NA inhibition assay and confirmed that two AK extracts and four AK fractions except AK-11 inhibited NA protein of rvH1N1. We found that only AK-3 inhibited influenza virus infection suggesting two possible ways of viral inhibitions, 1) blockage of viral attachment by inhibition of viral HA protein, 2) blockage of viral replication and/or release by inhibition of NA. Interestingly, AK-3 showed greater inhibition of viral attachment than of replication, against both A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2). However, the other two AK extracts and four AK fractions showed only blockage of viral attachment of influenza virus to the cell.To evaluate the anti-influenza activity after virus infection, we employed the post treatment assay and quantitative real-time RCR to test the ke et al. reportedThis study has shown that AK extracts and AK fractions can inhibit both A/PR/8/34 (H1N1) and A/Chicken/Korea/MS96/96 (H9N2) influenza viruses by inhibiting viral HA binding to the SA receptors in the host cell. AK-3 offers a potential antiviral drug candidate by inhibiting viral attachment step, replication and release step. These results lead to further investigation about characterization of active compounds and their specific mechanism against influenza virus.The authors declare that they have no competing interests.HJK and HHK carried out most of the experiments, analyzed the data and participated in writing. SYY and JSC cellular studies and drafted the manuscript. YBR prepared extractions and fractions. KOC and MCR contributed to the data analysis. SJP and WSL conceived the study, participated in its design and coordination and accounted for the manuscript writing. All authors read and approved the final manuscript.
The north-east Indian states of Manipur and Nagaland are two of the six high HIV prevalence states in the country, and the main route of HIV transmission is injecting drug use. Understanding the pathways to injecting drug use can facilitate early intervention with HIV prevention programs. While several studies of initiation into injecting drug use have been conducted in developed countries, little is known about the situation in developing country settings. The aim of this study was to increase understanding of the contextual factors associated with initiation into injecting drug use in north-east India, and the influence of these factors on subsequent initiation of others.In mid 2006 a cross-sectional survey among 200 injecting drug users (IDUs) was undertaken in partnership with local NGOs that provide HIV prevention and care services and advocacy for IDUs in Imphal, Manipur and Dimapur, Nagaland. The questionnaire elicited detailed information about the circumstances of the first injection and the contexts of participants' lives. Demographic information, self-reported HIV status, and details about initiation of others were also recorded.Initiation into injecting drug use occurred at 20 years of age. The drugs most commonly injected were Spasmo-proxyvon (65.5%) and heroin (30.5%). In 53.5% cases, a needle belonging to someone else was used. Two-thirds (66.7%) had used the drug previously, and 91.0% had known other IDUs prior to initiation (mean = 7.5 others). The first injection was usually administered by another person (94.5%), mostly a friend (84.1%). Initiation is a social event; 98% had others present (mean = 2.7 others). Almost 70% of participants had initiated at least one other (mean = 5 others). Initiation of others was independently associated with being male and unemployed; having IDU friends and using alcohol around the time of initiation; and having been taught to inject and not paid for the drug at the time of initiation.Targeting harm reduction messages to (non-injecting) drug users and capitalising on existing IDU social networks to promote safe injecting and deter initiation of others are possible strategies for reducing the impact of injecting drug use and the HIV epidemic in north-east India. The north-east Indian states of Manipur and Nagaland, which lie along the border with Myanmar, are characterised by ethnic conflict, armed civil insurgency, a heavy military presence and high unemployment . ClassifAlthough Manipur and Nagaland are neighbouring north-east Indian states, they are different from each other in a number of important ways including ethnicity, culture, religion, insurgent movements, patterns of drug use and HIV, and the extent to which harm reduction approaches are accepted and integrated into the local response. Both the HIV epidemic and the public health response to it are more mature in Manipur. Sentinel surveillance data estimate that HIV prevalence among injecting drug users (IDUs) in Manipur was 24% in 2005, but only 4.5% in Nagaland .Approximately 2% of the population in Manipur and Nagaland engage in injecting drug use . The vasCurrent harm reduction programs in Manipur and Nagaland aim to reduce the risk of HIV infection among IDUs by offering a range of services including needle and syringe distribution and condom promotion. However, these programs are constrained in what they are able to achieve as both states are characterised by deeply felt social conservatism. There is an absence of interventions to address upstream factors that contribute to young people's decision to inject drugs. Transitioning from non-injecting drug use to injecting is not inevitable, and a range of individual and social network factors have been shown to influence this in developed country settings -16. FactTwo Australian studies of initiation into injecting drug use investigated factors associated with the initiation of others ,11. TheyIn contrast, little is known about initiation into injecting drug use in developing country settings where the legal, political, socio-cultural and economic contexts are very different, as are the epidemiology of HIV and the patterning of injecting drug use. A cross-sectional study in Thailand among 2231 drug users found that being ≥ 20 years, being single, having received education, living in an urban area, having a history of smoking or incarceration, having multiple sexual partners, having experienced sexual abuse, using heroin (rather than amphetamines), and younger age of drug initiation were significantly associated with transition to injecting .The aim of this study was to increase understanding of the contextual factors associated with initiation into injecting drug use in Nagaland and Manipur, and the influence of these factors on subsequent initiation of others into injecting. Understanding the pathways to injecting drug use will assist with identification of groups at risk of initiation into injecting, as well as mechanisms of initiation, so that prevention interventions can be better designed and targeted. Importantly, the findings from this study will also highlight opportunities for providing harm reduction interventions earlier in the 'career' of IDUs, and can be used to inform and enhance social and political advocacy for HIV prevention.This cross-sectional survey was conducted among IDUs from Imphal, Manipur and Dimapur, Nagaland between May and July 2006. The survey was conducted in collaboration with three Indian non-governmental organisations (NGOs): Social Awareness Service Organisation (Manipur); Bethesda Youth Welfare Centre ; and Community Awareness Development Foundation . These NGOs provide a range of services for IDUs including needle and syringe programs, peer education, primary health care, counselling and rehabilitation. Qualitative data were also collected via in-depth interviews, and findings from that component of the study will be reported elsewhere.A total of 200 IDUs aged ≥ 18 years were surveyed. An IDU was defined as a person who has injected illicit drugs within the last three years. The sampling approach used a combination of convenience and snowball sampling and was stratified by state and sex . In an Australian study of initiation into injecting drug use, 47% of IDUs had initiated at least one other person into injecting [The questionnaire was based on one used previously to describe initiation into injecting in Melbourne, Australia , but wasEleven bi-lingual peer outreach workers (ORWs) were trained to collect the data. The ORWs were supervised and supported by a locally appointed research officer. Individual IDU clients were approached by the ORWs in the course of their work. Although not formally recorded, feedback from the ORWs indicated that the number of IDUs refusing to participate was very small. Participants were paid Rs100 (~USD 2.3) for their time. Interviews took place in a range of settings including drop-in-centres, clients' homes, hotels and tea-shops.The data were entered into Microsoft Excel 2003 and analysed using Statistical Package for the Social Sciences (SPSS) Version 14.0. The statistical tests used to assess the strength of associations between variables included Pearson's chi-square and t-test for independent samples. Nagaland and Manipur have different histories, ethnicities and religion, so all variables were analysed for state differences. Variables associated with being an IDU who initiates others were investigated using logistic regression, and odds ratios and 95% confidence intervals (CIs) were calculated. A stepwise technique was used, and variables were selected for inclusion in the model on the basis of their p value. A model of best fit was chosen with variables included on the basis of a change in the log likelihood at p < 0.1.The study was funded by the United Kingdom's Department for International Development (DFID) through the Research and Learning Fund. It was approved by the Human Research Ethics Committee at the University of Melbourne, Australia, and the Institutional Review Board of the Emmanuel Hospital Association, New Delhi, India. All participants gave verbal informed consent and confidentiality was assured. No participant names were recorded at any stage of the study.The mean age of participants was 24.5 years . The majority identified ethnically as Naga (45.5%) or Meitei (39.5%). More than half were Christian (57.0%) and 40.0% were Hindu (Table Only 17.9% of participants were employed. The mean monthly income was Rs3662 (~USD 84) . Participants were asked the source of their income other than through employment and most received money from their family (83.8%). State differences in all of these variables were observed (Table The majority of participants (70.5%) lived in their family home and 72.9% were sharing their living space with their parents. Only 14.6% lived with a partner/wife/husband, 12.6% with other relatives, 6.5% with friends, 4% with others such as parents-in-law, and 1.5% lived alone . One quarter (25.1%) had children of their own. The average number of children was 1.5 , and 78.6% of those with children were currently living with them. In terms of mobility, all participants were born in north-east India, but 38% were currently residing in a district that was not their birthplace.The mean age of the first injection of illicit drugs was 20.1 years . There was no difference in age of first injection by state or by drug injected. For their first injection, 65.5% of participants injected SP and 30.5% injected heroin. Two-thirds (66.7%) had used the drug previously. In 53.5% of cases the needle used for the first injection belonged to someone else . For the majority (79.0%), the first injection was remembered as happening spontaneously. Almost half of the participants paid for the drug themselves (48.5%) or shared the cost with another (13.1%). The drug was most often obtained by someone other than the participant (63.3%). The most common places for the first injection were a friend's house (43.5%) or the participant's own house (17.5%). Only 2.5% were initiated at the drug dealer's place. More than half of the participants (53.3%) had other drugs in their system at the time of the first injection (Table Initiation into injecting drug use was a companionable event. The mean number of other people present (not counting the participant) was 2.7 , and most of those present also injected. Only 2.0% of participants were alone when they injected for the first time and 95.0% had up to five other people present. More than four-fifths (85.5%) had at least one friend present, 14% had at least one relative, 13.0% had at least one acquaintance/stranger, 3.5% had a drug dealer (known as 'peddlers' in north-east India), and 1.5% had a partner/wife/husband present the first time they injected .Most participants (94.5%) were injected by another person the first time, most commonly a friend (84.1%), followed by a relative (9%), an acquaintance/stranger (4.7%), a partner/wife/husband (1.6%) or a drug dealer (0.5%). They had known this person for an average of 7.0 years . A very small proportion (2.9%) had known the person who initiated them for one month or less; 14.9% had known the person for between one month and one year; 18.3% had known them for between one and three years; and close to two-thirds (64.1%) had known the person who initiated them for more than three years. In 87.3% of cases the participant asked the other person to inject them. Another person prepared the drug 93.3% of the time, taught the participant how to inject 69.8% of the time, and told the participant about the need to use new injecting equipment 36.5% of the time.All eleven participants who injected themselves the first time were shown how to inject by either friends or relatives. The participants had known this person for an average of 6.4 years (range 6 months – 20 years). In six cases the participant asked the other person to show them how to inject, in eight cases the participant prepared the drug, having been taught by another person in six of those cases, and in three cases the other person mentioned the need to use new injecting equipment.A number of state differences in the circumstances of first injection were observed Table . Those fParticipants were asked an open-ended question about their reasons for injecting at that point in time. The reasons were coded as: curiosity about the high/pleasure seeking (46.7%); the influence of others (34.7%); economic reasons (10.1%); and other reasons (8.5%), which were mainly a reaction to a negative situation in their life, or not knowing that the drug could be used in any other way. The influence of others was expressed in two different ways. Firstly, there was a desire on the part of some participants to be the same as their friends who injected, and secondly some participants were urged by their friends to try injecting. The economic imperative to inject was due to a shortage of money or drugs and therefore a need to use the available drug more efficiently by injecting rather than chasing/smoking or taking it orally.Participants were asked a series of questions about their life situation at the time of initiation into injecting drug use. One-third of participants 35.5%) were either unemployed or had dropped out of school knew other IDUs prior to their initiation into injecting Table . The meaAlmost one-quarter of participants (23.2%) had previously been in trouble with the police prior to their first injection, on an average of two occasions . Of those who had been in trouble with the police, 48.9% had been in prison (11.0% of the entire sample); on average 1.4 times .State differences in life situation at the time of initiation into injecting drug use were noted Table . ParticiAround the time of initiation into injecting drug use (as opposed to on the occasion of initiation outlined above) and during the last six months and the last week prior to the survey, use of multiple other drugs was commonplace Table .Information about injecting drug use during the last six months and the last week was collected. During the preceding six months 46.7% had injected only SP, 18.1% had injected only heroin, 23.1% had injected both, and 12.1% had injected neither. As some survey participants were recruited from drug substitution programs, their injecting drug use during the last week was atypical. Excluding the 56 (28.3%) participants who had not injected any drug in the last week, 67.6% injected SP and 43.0% injected heroin. Of those participants who had injected in the last week, the average number of injections was 16.9 . They spent an average of Rs149 per day (~USD 3.4) on injecting drugs .Participants were asked about injecting risk behaviours. The majority (81.2%) said they rarely or never used a needle used by someone else, but 12.5% did use a needle used by someone else at least weekly. Among those who have on occasions used a needle or syringe used by someone else, 77.7% always cleaned it before use, most commonly with water and/or saliva.A number of state differences in drug use history were observed Table . ParticiPrior to initiation into injecting, almost three-quarters of participants (72.4%) had heard of HIV and AIDS, 27.0% had heard of hepatitis B and 18.6% hepatitis C, but only 67.5% knew that diseases could be spread by unsafe injecting practices. The majority (87.5%) said they knew where to obtain new needles and syringes at that time. Most participants (68.5%) said that they were not worried about HIV infection the first time they injected. The most common reasons for not being worried were: lack of knowledge about HIV (41.6%); a new needle/syringe was used (37.2%); the focus was exclusively on obtaining the drug and getting high (9.5%); feeling confident that their friend was not HIV infected (5.1%); and the injecting equipment was cleaned prior to use (2.9%).Overall, 48.7% reported having been tested for HIV and only 5.2% for HCV infection. Of the 91 participants who had been HIV tested, 79 shared the result with the interviewer, and 19.0% of these were positive. Of the ten participants who had been tested for HCV, four were negative, three were positive, and three chose not to share this information. Only 10.0% of participants reported that they were vaccinated against hepatitis B.cf 35.0%, p < 0.01). The average number of people initiated was 5.0 . This means that 138 study participants went on to initiate 690 others into injecting drug use. Logistic regression modelling was used to identify variables independently associated with being an initiator of others (Table The majority of participants (69.3%) had helped someone else inject for the first time. This was unrelated to state, age, drug initially injected or length of time since first injection, but was associated with sex. Males were more likely to initiate others than females , have children , and to have either never attended or dropped out of schooling . They were less likely to be living with their parents , had a higher monthly income (p < 0.01), and were more likely to be obtaining money from partners and sex work . Consistent with anecdotal reports, females were more likely than males to have injected heroin the first time , and to have reported heroin use in the last six months , and the last week .Although the number of females in the study is too small to draw any meaningful conclusions about them, a number of sex differences were observed that are briefly described here. Compared with males, the females in this study were more likely to be married were with others at the time of initiation, and on average there was between three and four people present (including the person being initiated). The vast majority (85%) had at least one friend present. Other studies have identified social network factors as playing an important role in facilitating the transition to injecting ,9,15. Ma8% were wThe main reasons for deciding to inject at that point in time were curiosity about the high, pleasure seeking, the influence of others, and needing to use the drug more economically. These reasons are similar to those identified in other studies of transitions from non-injecting to injecting drug use ,12,13,15Almost 70% of participants had initiated another person into injecting, which is a lot higher than the proportions in other studies (47% and 37%) ,11. It dA number of limitations should be borne in mind when interpreting these results. The sample was not representative of IDUs from north-east India so the results should be generalised with caution. It may be the case that IDUs not in contact with NGOs and those from rural areas are different from the participants in this study in important ways that could be associated with initiation into injecting. Similarly, participants enrolled in oral substitution programs, who constituted up to one-quarter of the sample, may be different from other drug users. The small number of females in the study means that little can be said about them with confidence. More research is required to better understand the world of female IDUs in north-east India, especially given the recognised links between drug use and engagement in sex work. As the data were collected by peer workers from NGOs working in the area of harm reduction, social acceptability bias may have influenced some responses, especially those related to the safety of injections.These findings have a number of public health implications. As the majority of participants had previously used the drug they initially injected, it may be appropriate to target harm reduction messages to (non-injecting) oral/inhalant drug users. This could include messages delivered by peers regarding disease transmission and safe injecting practices, as well as messages to deter progression to injecting. Social change among IDUs in relation to needle and syringe re-use has been achieved, at least to some extent, so other changes are potentially possible. The idea of using social networks among community sub-groups engaged in HIV risk behaviours in order to change social norms has been described by others as a possible strategy for harm reduction -21.The proportion of participants who had participated in testing for blood-borne viruses was low, especially in the case of hepatitis C. While the availability of treatments is currently limited in this part of the world, the situation is gradually improving, and the more people that know they are infected with a hepatitis C, the more advocates for better access to treatment there will be, and the more others can be protected from infection.A number of state differences were observed declare that they have no competing interests.Michelle Kermode was primarily responsible for study conceptualisation and design, data collection, analysis and interpretation, and drafting and revising the manuscript. Verity Longleng and Chingsubam Bangkim Singh participated in data collection and analysis and revised the manuscript. Jane Hocking contributed to data analysis and interpretation, and drafting and revising the manuscript. Biangtung Langkham and Nick Crofts contributed to study conceptualisation and design, and drafting and revising the manuscript.
This study explored the Smoking Prevention and Cessation Partnership (SPCP) which builds upon a collaboration between two Danish municipalities targeted at the prevention of tobacco smoking. The aim of the study was to describe the processes of SPCP, to examine the difficulties this collaboration faced, and to assess how these experiences could be used to improve future partnership collaboration. We employed qualitative methodology comprising 12 semi-structured one-to-one interviews with SPCP’s stakeholders and an analysis of the partnership documents and reports. The findings suggested that the main potentials of the partnership were the personal relations between the members and stakeholders with the possibilities of the creation of new connections with other actors. Barriers to successful partnership building were the implementation of the new Local Government Reform as a competing task, and that the two municipalities were heterogenic in respect to organizational issues and working methods. Other impediments included the lack of continuity in leadership, the lack of clarity regarding the form of collaboration and roles, as well as different expectations of the stakeholders. We conclude that four factors remain critical for partnerships. The first is the clarity of the collaborative effort. Second, partnerships need to take into account the structural circumstances and culture/value systems of all stakeholders. Third is the impact of contextual factors on the development of the partnership; and the fourth factor is the bearing of personal/individual factors on the partnership e.g., personal engagement in the project. Early attention to these four factors could contribute to more effective partnership working. In Denmark, the percentage of adults who reported that they smoked everyday has decreased by more than half, declining from 47% in 1984 to 23% in 2008 . In linePartnership arrangements have been examined in many individual countries. For instance joint working efforts have been evaluated in the USA ; in the In 2002 the liberal government of Denmark established a commission for local government reforms in Denmark, which completed its work in 2004 . MeanwhiIn 2006, all municipalities in Denmark had an opportunity to apply for funding from The National Board of Health for developing and maintaining inter-municipality collaborative initiatives aimed at smoking prevention and cessation. Two municipalities in the west of Denmark (Varde and Esbjerg) applied together, and in 2007, the Danish SPCP was created as a collaborative model. SPCP’s stakeholders included health professionals and general practitioners, health care units, pharmacies, midwife centres, the health assistant education programme, outreach/public health nurses, citizens with chronic conditions, along with other partners such as private enterprises/businesses and other health or educational agencies . The purpose of this inter-municipal SPCP was to strengthen the health promotion efforts of both municipalities, particularly focussing on the prevention of tobacco smoking, and the improvement in implementation of smoking interventions through partnership working and exchange of experience. The SPCP was aimed at: (1) activities preventing the use of tobacco whilst motivating citizens not to start smoking ; (2) smoke-free environments; and (3) courses in quitting tobacco smoking (see below). However, during the course of the partnership, a new Governmental Act was instated that prohibited smoking in working places and on the job, in institutions and schools, in other educational institutions, on collective/communal means of transportation and in most restaurants (Act no. 512. Ad 6. June 2007). Hence, the implementation of this Act led to a shift in the aims of the partnership towards a focus predominantly on smoking cessation activities. SPCP’s objectives and criteria of success included: increased dialogue between both municipalities; joint working on non-smoking policies in private-sector work places; and, the gaining of experience in relation to municipal-level prevention of tobacco use. As such, SPCP was seen as the foundation for expanded future collaborations between both municipalities. This paper describes the evaluation of this inter-municipal “Smoking Prevention and Cessation” partnership.SPCP’s activities included courses in smoking cessation that were undertaken in almost all pharmacies in Denmark and were offered to either individual smokers or to groups of smokers. Beginning with the individual smoker’s needs, the pharmacy provided advice on smoking cessation. Each smoking cessation course span over 4–10 weeks duration and comprised five sessions of counselling, each about 1.5 to 3 hours. These sessions included testing of blood nicotine levels, measurement of blood and exhaled carbon monoxide levels, advice on smoking cessation and on choice and utilisation of nicotine replacements, in addition to scheduling a follow-up in order to document whether participants were progressing in the period of the smoking cessation. The sessions also involved support and advice to the smoker that further focussed on the effects of environmental tobacco smoke on colleagues at work or for the smoker’s family. At the end of any given course, the smoker’s support and future needs were discussed and agreed upon, and follow-up was established as to whether the smoker wished to be phone-contacted by the smoking cessation consultants 6 months later. Across Denmark, all the smoking cessation courses followed the same pattern with some minor variations, and in all cases the smoker had to pay the pharmacy for the course.The study was conducted in collaboration with two Danish municipalities. The purpose of the evaluation was to describe the processes of SPCP, to examine the difficulties that this collaboration faced and the reasons behind the difficulties, and to assess how these experiences could be used to improve future partnership collaboration in Denmark and elsewhere. The four specific objectives were to:describe the collaboration of the partnership’s stakeholders;assess the barriers, challenges and expectations among the various stakeholders;provide recommendations on how the partnership could be further developed to advance the aims of the SPCP; and,appraise whether such ‘obligatory’ collaboration between municipalities in reference to smoking prevention is at all feasible or desirable.A partnership is a formal collaboration between organisations, groups and agencies to reach a common goal. Guided by the published literature on partnerships, and in order to steer the evaluation, we employed the conceptual model published by El Ansari & Phillips . The modversus group concerns; and explore any divergent experiences and ‘outlier’ attitudes [The study collected information about the details and development of a collaborative effort between two municipalities, in addition to the collaboration between the municipalities and a private company and pharmacies that participated in implementing the smoking cessation courses. We employed a qualitative approach because the aim was to undertake an in-depth study of the processes of collaboration, the difficulties and promises associated with such collaborative efforts, and to assess how the participants’ experiences could be applied in order to enhance the efficiency of future partnership working. Hence the present study utilised individual (stakeholder) interviews. The interviews comprised in-depth conversations in order to gather qualitative information and the views of those individuals involved in a particular initiative or project, its context, implementation, results and impact. Such interviews can provide feedback on all aspects of a project’s inputs, activities, outputs, outcomes. Hence the research team employed this technique in order to: elicit the preliminary views of stakeholders; emphasize the individual ttitudes . The resThe first and third authors undertook 12 semi-structured one-to-one interviews with a range of strategic SPCP stakeholders e.g., health consultants, social workers and project leaders in the municipalities. These individual interviews included members of SPCP’s steering group (n = 2); former and present SPCP leaders (n = 2); other partners involved in the SPCP in both municipalities (n = 1); and smoking cessation consultants from both municipalities (n = 7). In line with El Ansari & Phillips , the intThe audiotapes were reviewed by the authors, and the data were transcribed and analyzed in the text using elements from grounded theory and the constant comparative technique . In the In addition the research team undertook a minor documentary analysis on the published information that was provided by the SPCP: partnership documents, pamphlets, brochures, statistics, evaluation reports and other information were all analysed for their content. The documentary analysis was conducted to achieve a contextual understanding of the policy and practice environment in the municipalities. In the documentary analysis, we systematically analyzed the content, the motivation, intent and purpose of documents employing content analysis as a technique ,28. The The data collection process was not straight forward as it was challenging to precisely identify the organisational participants who were actually actively participating in the SPCP. As with other partnerships elsewhere , membersAnalysis of the partnership documents and reports indicated that since January 2007 Esbjerg and Varde municipalities collected various statistics on the characteristics of SPCP participants. These included socio-demographic features and other aspects related to smoking , along with the number of cessation courses that were administered in both municipalities. Between 2007 and 2008, 213 smokers participated in the smoking cessation courses. Males comprised 43% of the participants, 85% were ≥45 years of age, 75% were employed and 70% were home owners.2) tests showed that factors significantly associated with quitting smoking after completing the course were older age (p = 0.03) and a longer duration of smoking (p = 0.002), while other factors like gender or employment status were not associated with quitting smoking.As regards the impact of the smoking cessation course, 66% (n = 141) of participants quitted smoking at the end of their course . Of those participants who quitted smoking at the end of their course, 43% maintained their smoking cessation for 6 months after the course. Chi-square .real partnership:Hence ‘external’ organisational factors influenced the setting up, initiation and actual implementation of SPCP. The reforms had substantial influence on duties and responsibilities that in reality it could have acted as a barrier to realising a real partnership between the municipalities. The result was two separate projects rather than one “You can’t say that is was a cooperation project, there are two projects; one project, but in practice we run them separately, we had some intentions about how we as municipalities, via this project and as neighbouring municipalities, could find some common cooperation norms, and we could transfer it to other activities, but we won’t get that, we won’t get that out of it” (respondent 1).Meanwhile, the smoking cessation consultants had high expectations about common collaborative smoking cessation activities that span the borders of both municipalities. However, such truly collaborative actions never came to light. This further draws attention to the context in which a partnership is being initiated and put into practice, as well as its timing in relation to other activities that are concurrently happening nationally or locally.Other challenges were the lack of clarity from the inception as to the extent of the cooperation, and hence the stakeholders’ expectations and roles:“From the beginning it should have been stated at which level the cooperation should be. Is it cooperation at the employees’ level, at leadership level or at a national level? Is it development or implementation? Or where do they wish to have cooperation?” (respondent 3).Collaboration requires much clarity, transparency and time. Such lack of clarity was questioned:“What is it they want with it and what effort will be put in this cooperation? Because cooperation takes time, it means that you have to give high priority to the employees who are involved in the partnership and give them permission to use time on it” (respondent 8).Conflicting messages contribute to lack of clarity. For instance, the smoking cessation consultants were trained in smoking cessation through SPCP, but their departmental leaders subsequently refused that the consultants use their time to conduct the courses:“Many of them, who were actually taking a course, got a message from their leaders, that they shouldn’t conduct smoking cessation courses. There were some restrictions about what they were allowed to do and what they were not allowed to do” (respondent 7).The two municipalities had different ‘ways of doing business’ that reflected the unique cultures of the distinctive agencies that participated in SPCP:“In Varde we have a general attitude: do something and then get forgiveness for it later, hence we start some activities and formalize them later. In Esbjerg all has to be formalized before starting and when everything is ok you can start up the activities, so it has been different workings methods” (respondent 1).As an administration, Esbjerg municipality is bigger than Varde, and exhibited more hierarchical structures. The result was that for SPCP planning, some decision-making required relatively more time in Esbjerg than in Varde:“It was very difficult to cooperate internally in Esbjerg municipality, because of their hierarchy, it was quite difficult to come through to those you should talk with and get answers and permission to do what you were supposed to do. So I think that in Esbjerg it was very difficult and it went very slowly, there is a lot of hierarchy, but in Varde it was much easier….I found out later that there was much more willingness and it was much easier to get through with the project in Varde, there was much more willingness, interest, there was not the same hierarchy to fight with” (respondent 2).Each municipality represented a political organisation and the priority of different areas within health ultimately depended on the leadership and politicians who would support the proposed areas of work. Such political-level decisions were evident in both municipalities and influenced SPCP’s activities. For example, in Varde a Health Centre was created to lead on the health promotion activities and hence SPCP activities. Conversely in Esbjerg, the Health Department at the municipality led the health promotion activities and therefore also the SPCP activities. However, such differences and variations could serve as valuable opportunities for mutual and cross-learning:“If we found a way, where the things are going very well in Varde, so of course we should learn from it in Esbjerg, but the problem is that it is working so different in Varde, that we can’t use in Esbjerg and vice versa” (respondent 4).Differences in stakeholders’ ways of working are not uncommon in partnerships. This was highlighted by an interviewee:“I don’t think that cooperation between Varde and Esbjerg will be in the near future, but Esbjerg can do it with another municipality and Varde with another, but those two municipalities are too different” (respondent 9).Hence, the impending structural conditions that were simultaneously implemented nationally could have posed challenges for both municipalities. Further, the working methods of the two municipalities were quite different:“Everything that could go wrong went wrong in this project, but I don’t think we could predict it, I don’t think so ... the structural reform that aggregated different municipalities in bigger units was a challenge ... it was a big change and it slowed down many of the decisions. And that two municipalities working together with so different working methods plus that we in the steering group have changed places, was also a challenge, but I’m happy that we keep up and didn’t give up” (respondent 1).Partnerships comprise many stakeholders who might not have traditionally worked together. In such joint initiatives effective leadership plays a pivotal role in communicating the vision of the partnership to the partners:“The first project leader was good to tell us about the project, I think it was really good. We have been told about the organization and we got all the project description. There were a lot of details in the project” (respondent 6).Successful partnerships are associated with good leadership. However, SPCP’s first leader subsequently left the partnership in order to take up another post and was replaced by another leader:“It didn’t work so well with the other project leader. I think people don’t burn the same way for this kind of project. I even don’t know if the new project leader wished to be the leader of the project or was pushed in it” (respondent 10).Interviewees commented that a continuity of the leadership was required and could have benefited SPCP:“The leaders of health department got sick and then resigned, the leadership in the department was missing and it was me who should carry out the collaboration” (respondent 2).“There were some meeting (network meetings for consultants) where we were both from Esbjerg and Varde, but then it happened that project leader is leaving, got another job....it happens, and then the new one is coming, but there was so long time without the project leader and I think that is was one of the reason that it didn’t work” (respondent 6).The continuity of the leadership in collaborative efforts is vital, as during periods when there was no project leader, there was no formalised cooperation between the two municipalities:“The weakness of project was that our “old” project leader has stopped” (respondent 6).“The leadership… I say it didn’t work, I don’t think it has something to do with the new project leader. It just went wrong when the first project leader stopped and it was a period of half year without anyone” (respondent 4).Partnership working effectively builds on the personal relations between individuals, stakeholders and members of the steering group. Such connections that contribute to garner trust, rapport and relationships were reported by interviewees:“Cooperation is a lot of personal relations, not that you see each other privately, but that you know each other a little bit and have trust to each other. This is what carries it out” (respondent 3).Without the mutual relationships and links, it would have been taxing to complete the SPCP, particularly given the multiple internal and external challenges associated with the national reforms:“The strong sides were the personal relations and the engagement which has been in the steering group and in the project leadership to accomplish the project in spite of all the odds we had, we will have something out of it; that was really good” (respondent 1).Another feature of the partnership was the possibilities to establish new connections with ‘new’ actors, which would not have been possible without the stakeholders working together as partners. For instance, as a direct result of SPCP, the municipalities forged contacts with agencies that had not previously been in direct links with the municipalities. These ‘new’ links included an independent boarding school for secondary students and one factory:“The project gave us possibility to have contact with an independent boarding school for lower secondary students” (respondent 5).“We got some economic help for some things, and to come out to the enterprises and do something, I don’t think we would have done it without the partnership” (respondent 5).Health and social care agencies are increasingly sharing information and resources with local authorities and governments in order to provide services that meet the needs of their populations . Such inAs regards our first objective, we described the collaboration of SPCP’s stakeholders . This DaHowever, about 6–8 months after SPCP was initiated, SPCP’s leader accepted another post elsewhere in Denmark. This resulted in a ‘quiet’ period (4–5 months) until a new project leader was appointed. In addition, the two municipalities had different organisational structures and working methods, which further delayed SPCP’s activities. Such procedural delays and/or financial constraints are obstacles for partnership working . CollectIn relation to objective two, the barriers included: whether SPCP was a ‘real’ inter-municipal partnership; the clarity of SPCP’s vision/purpose; the ‘culture’ and ways of working of the partner agencies; leadership issues; and, the development of the budding relationships, links and connections that were being established by these new partners. These findings supported the conceptual model proposed by El Ansari & Phillips , which hAs regards ‘real’ partnership, genuine partnerships require time, effort and resources. The groundwork preparation for a partnership , as wellSPCP members felt that the ‘culture’ and ‘ways of doing business’ of the participating agencies were also barriers to success. Differences in stakeholders’ values are not uncommon in partnerships ; are keyLeadership across organizational boundaries has generally received little attention . As regaWith reference to the relationships, the links and new connections that were forged as a consequence of SPCP are important to the partners. Hence an understanding of partnership theory and sociThe third objective was to provide recommendations on how the partnership can be further developed in order to advance SPCP’s aims. Based on our findings, we suggest the basis for an agenda for action for stakeholder individuals and organizations to include:Municipalities embarking on partnership efforts would benefit to initially explore the feasibility of such initiatives. Heterogeneities due to type of agencies and working methods requires early attention to develop fruitful partnerships in order not to miss opportunities for collaboration, experience exchange and the sharing of good practice. The time and resource challenges of creating a new structure (partnership) could be great, hence the economic support for such efforts should be appropriately gauged. Other national legislative or administrative reforms or local re-structuring that are implemented as the same time as the partnership effort should be considered in order to select an appropriate ‘time window’ of opportunity.Early attention and review of the clarity of the vision and mission of the partnership, where innovative and pragmatic approaches can be used to outline a realistic action plan and ensure that the participating organizations are ‘ready’ for the proposed interventions and goals. Clarity of the municipalities as regards the ‘level’ upon which they will collaborate is important.When training is provided to the partners in order to deliver certain interventions , their organizations, agencies and departments need to satisfy the partnership that they are committed to such goals and would provide the time and administrative support for those stakeholders to deliver the task/s they were trained for. The ‘conditions’ in which the partners will be delivering certain interventions require thorough study in order to generate guidelines.The leadership issues that are encountered in partnership arrangements need special attention, as well as the continuity of such leadership over time. Leadership would include the actual leaders of the partnership as well as the leaders of the participating agencies that comprise the stakeholders.In future, it could be productive to recruit external ‘facilitators’ with track record in partnership working to review a partnership’s proposals before activities are started, and provide hands-on advise and support to the budding partners.As regards the fourth objective, we assessed whether an example (SPCP) of ‘obligatory’ collaboration between municipalities in reference to smoking cessation intervention was at all feasible or desirable. In many instances, the availability of funding and resources act as stimuli for partnerships to be initiated and developed. This in itself need not be regarded as a disadvantage. However, a point to note is that a partnership (as a vehicle or intervention) is usually not employed as a first option to assist in solving public health and health promotion problems. Usually other solutions/interventions would have been implemented and would not have been successful in accomplishing the desired solutions. Hence, when partnerships are called upon as vehicles in health promotion, frequently the problems are then deeply entrenched and long standing; involving many stakeholders who might have traditionally not communicated or worked with each other; the solutions to the problems usually require behavioural change that requires time; and the problems would usually have exhibited resistance to other ‘solutions’ that had been tried in the past. In such situations, partnerships could be likened to national tertiary hospitals that usually receive nearly all the patients with serious, complicated and late conditions. Consequently, when such hospitals exhibit a higher mortality rate, that does not necessarily mean that their care provision is sub-optimal, but rather, it is a feature associated with the conditions of the patients they receive.per se was not provided at all. In health promotion, a common pitfall is that training is usually provided for the intervention (smoking cessation); but the vehicle of the intervention (partnership) itself is not viewed as a bone fide intervention that also merits experience, expertise and training. Partnership working is a science and art [Given the pre-condition that the partnership was being established while processes related to the structural reforms were simultaneously being undertaken in both municipalities at the same time, testing the ‘success’ of the partnership was not an explicit aim of this research, as times of re-structuring and organisational reforms are viewed as sub-optimal opportunity windows for such evaluations. Nevertheless, such assessments of partnership working that we undertook are critical in order to provide formative feedback, guidance and lessons of good practice to such ‘budding’ collaborative efforts. However, it is reasonable to conclude that this partnership initiative faced many challenges that contributed to its low success. Indeed all the characteristics outlined above would need to be considered before a partnership is dismissed as a ‘failed experiment’. Further, given the availability of funding as a driver, agencies and organisations are frequently and hurriedly ‘pushed’ into partnership arrangements, where funding bodies would expect to be able to measure tangible results within a short time frame. Tight time frames do not do justice to the use of partnership approach in health promotion where behavioural change is usually an ultimate aim. In addition, initial training in partnership working is rarely provided to potential stakeholders as a preparation for their collaborative effort, and in the case of SPCP, training in partnership working and art . Hence i and art which weThis study has limitations. It explored a partnership implemented in two particular municipalities, and hence caution needs to be exercised in generalizing the findings to other Danish municipalities or further afield. Furthermore, because of a change of SPCP’s leadership, the complete data about the smokers who participated in the smoking cessation course and those employees who were trained as smoking cessation consultants were not available. Such data, as an objective measure, would shed light about the effectiveness of the partnership. Similarly, the smoking cessation status was confirmed by the participants at the end of their smoking cessation course but was not independently bio-chemically validated, where such objective validation of the cessation status would have been useful. As our sample comprised 12 interviews, we were unable to indicate any individual participants’ role in SPCP in conjunction to their quotes, in order to preserve participants’ anonymity and confidentiality.On the macro-level, future research needs to explore how concurrent government reforms interact with and affect budding partnership initiatives, and whether a ‘minimal time window’ is desirable between such reforms and subsequent partnership efforts. On the micro-level, research needs to address the specific impacts that are strongly and early affected by any lack of continuity in leadership in collaborative efforts. While the implementation of smoke-free environments was boosted by a legislation on smoke-free public places and workplaces, one may speculate that other macro level measure, especially those related to the reduction of the demand for and supply of tobacco as suggested by the WHO Framework Convention on Tobacco Control would haSince the Ottawa Charter for Health Promotion communit
Technological advances, particularly, invention of computers, have revolutionized our way of working. Computer has become an integral part of our life. However, its use is not free from health hazards. Intensive computer work puts stress and strain on muscles, as well as joints, because of continuous and repetitive nature of movements. Individual factors, prolonged awkward postures, poor workstation design and psycho-social environment can lead to development of symptoms of musculoskeletal discomfort (MSD). If these symptoms are ignored and if no preventive measures are taken, cumulative trauma disorders such as myalgia, myofascial syndromes, nerve entrapment syndromes, tendonitis, epicondilitis and tenosynovitis can develop. In this talukas of Anand district - namely, Anand and Petlad - from May 2004 to January 2006. The sample size of 44 was calculated considering a prevalence of computer-related health problems as 20%, and 10% non-response rate. Thus 440 was the final sample size. Establishments and institutes in Anand and Petlad talukas, where computers are extensively used, were enlisted. These included banks, computer training centers and colleges running degree courses in computer applications. After seeking the permission of the heads of these institutes, all the staff and faculty members, as well as final year students, were enlisted. Four hundred forty subjects were selected randomly. Thirty-one did not participate in the study, making the nonresponse rate of 7.0%. Rest of the subjects (n = 419) were asked to fill up a pre-tested questionnaire, after obtaining their verbal consent. Other relevant information was gathered through personal inspection of workstation. Musculoskeletal discomfort (MSD) was considered when one or more of the following symptoms were reported by the respondents: neck or shoulder stiffness; neck or shoulder pain; tingling/numbness in hands, thumbs or fingers during work or many hours after stopping work; hand and wrist pain; backache; headache; leg cramps; leg stiffness; numbness in ankles and feet; swelling in ankles and feet; reduction in strength of hand and difficulty in grasping objects.The study was conducted in two Mean age of the subjects who participated in this study was 25 years, with a range of 18-55 years. Three-fourths of the subjects were young, with age of 15-25 years; and 279 (66.6%) were male. Majority (65.4%) of the respondents started using computers at a young age (16-20 years), and 236 (56.3%) individuals had been using computers for less than 5 years. About 41% of the respondents used to work on computers for about 21 to 40 hours in a week.et al. reported MSD prevalence of 25-76% among video display unit (VDU) operators in his study.. Armstrong et al. subjects enrolled in our study were doing one or more physical exercises like walking, running, etc. Such physical activity was not found to be associated with MSD in our study . A studyet al. showed tHabit of taking breaks for rest has been found to be associated with occurrence of MSD.47 Howeve4et al. did not observe such association in their study. subjects was normal, while 141 (33.7%) suffered from myopia, 4 (1.0%) from presbyopia and 5 (1.2%) from both. A significantly higher proportion of respondents with refractive error reported occurrence of MSD . StudiesThus, our study suggests that MSDs are a common problem among those who use computer intensively. It seems likely that long hours of working on computers and presence of refractive error may result in these problems. Future longitudinal studies should evaluate the relative importance of other individual factors like age, gender, habit of doing exercise and taking breaks. Results from such epidemiological studies can contribute to the development of appropriate hazard-prevention programs for workers who frequently use computers.
In the region of Västra Götaland in Sweden, prescribing guidelines, drawn up by 24 expert groups and determined by the regional board for drugs, are since 2006 available in the form of an annually published booklet. This study investigates, for the first time, the use of and attitudes towards this publication.A questionnaire was administered to doctors working in primary health care in the region of Västra Götaland in Sweden. Questions included characteristics of the responding doctor and use of the prescribing guidelines booklet, as well as attitude questions constructed as statements to which the responder should grade his level of agreement from 1 to 6 .th – 75th percentile) 80 (75–90)]. However, at renewal of a drug prescription, active change to a drug from the prescribing guidelines booklet occurs less often [median (25th – 75th percentile) 50 (20–70)]. The booklet also includes short therapy advice sections, which 231 doctors (42%) use every day and 191 (34%) use every week. The attitudes towards the prescribing guidelines booklet were generally positive. Doctors in privately run primary health care units and doctors running their own business were generally more negative and judged themselves to be less adherent to the prescribing guidelines booklet compared with doctors in publicly run primary health care units.Totally 603 filled-in questionnaires were returned (estimated response rate 60%). The majority of the doctors responded that they use the prescribing guidelines booklet, and when prescribing a drug for a new diagnosis, a drug from the booklet is chosen in most cases [median (25The prescribing guidelines booklet is frequently used and is generally appreciated, though differences exist between subgroups of users. Drugs are one of the keystones in the treatment of patients. Rational use of drugs requires adequate knowledge on benefits, risks and cost-effectiveness of drugs. Knowledge in the drug area is rapidly increasing and it may be difficult for a primary care doctor to be updated in all therapeutic areas. Lack of time for reading and evaluating scientific papers in favour of direct patient work may be one explanation. To determine the drug of choice on health economic bases may be an additional difficulty. Indeed, only a minority of doctors have been educated about drug costs . TherefoIn the region of Västra Götaland in Sweden, prescribing guidelines, drawn up by 24 expert groups and determined by the regional board for drugs, are since 2006 available in the form of an annually published booklet. The expert groups consist of one chairman, one clinical pharmacologist (in some cases these positions are held by the same person), one secretary (pharmacist) and 5–10 specialist doctors with knowledge and interest in drugs in their particular therapeutic area. The prescribing guidelines include lists of recommended drugs for common diagnoses based on evidence regarding safety, cost-effectiveness and clinical experience, as well as therapy advice on how to manage the different conditions. The prescribing guidelines are assembled into a booklet, which is updated every year and distributed to all doctors in the region. The guidelines are also available on the Internet.About 1,000 primary health care doctors work in the region of Västra Götaland. Like in the rest of Sweden, most primary health care doctors work in publicly run units, whereas a minority work in privately run units or have a business of their own. Publicly run units always bear the expenses for their prescribed drugs. Private units, on the other hand, have varying agreements concerning expenses for prescribed drugs, and are only occasionally responsible for their drug costs.To the best of our knowledge, no information concerning how the prescribing guidelines booklet is used and attitudes towards it is available. Attitudes towards, and the impact of, committees providing prescribing advice have been investigated previously in Sweden, but data have been published only in the form of reports written in Swedish. As for the international perspective, investigations have revealed that a majority of doctors agreed to several positive statements regarding prescribing guidelines, but at the same time a substantial minority agreed to a number of negative statements (see Discussion) ,8. The aAll primary health care units in the region of Västra Götaland in Sweden were included in the present questionnaire study. No exact number of doctors working in the primary health care is registered. Based on available information, the number of doctors was estimated to about 1,000. Questionnaires characteristics of the responder: gender, year of registration, level of doctor competence, and type of organization of the place of work , (ii) use of the prescribing guidelines booklet, including self-estimated percentage adherence to the lists of recommended drugs (i.e. percent of prescribing occasions at which the responder wittingly use these lists) as well as self-estimated use of the therapy advice sections, and (iii) attitudes towards the prescribing guidelines booklet. The latter questions were constructed as statements to which the responder should grade his level of agreement from 1 to 6 . An English translation of the questionnaire is available as an appendix.Statistical analyses were conducted using SPSS 14.0. Kruskal-Wallis test was used for comparisons between groups. To facilitate the interpretation of the results on the attitude questions for the reader, arithmetic mean values are shown in addition to median values, even though the results are not expected to be normally distributed. A P-value < 0.05 was considered significant. Percentages are based on the total number of responders to the particular question.Totally 603 doctors returned a filled-in questionnaire; estimated response rate 60% (see Methods section). The same response rate was estimated for doctors at publicly and privately run multi-doctor units, whereas only about 32% of doctors running their own business responded to the questionnaire.th – 75th percentile) year of registration was 1989 (1981–2000). In the subgroups doctors of publicly run units, doctors of privately run units and doctors running their own business, median year of registration was 1990, 1989 and 1980, respectively; specialists accounted for 67%, 87% and 100%, respectively; and males for 50%, 67% and 81%, respectively.Characteristics of the doctors are described in table Totally 571 doctors (97%) responded that they use the prescribing guidelines booklet, whereas 14 (2%) knew about its existence but do not use it. Six doctors (1%) did not recognize the existence of the prescribing guidelines booklet.th – 75th percentile) 80 (75–90)% when prescribing drugs for a new diagnosis and 50 (20–70)% as for active changes to a recommended drug upon renewal of a prescription. Results in subgroups of doctors are presented in table For all doctors, self-estimated adherence to the prescribing guidelines of the booklet was in median (25The use of the therapy advice sections of the prescribing guidelines in the booklet and on the Internet is described in figure Attitudes towards the prescribing guidelines booklet among the subgroups (i) doctors working at publicly run primary health care units, (ii) doctors working at privately run units and (iii) doctors having their own business are described in figure To investigate if demographic differences between doctors of publicly run units, doctors of privately run units and doctors running their own business could account for the above differences, a matched group of doctors from publicly run units was compared to male doctors running their own business (n = 25). This comparison showed differences almost identical to those between publicly run units and businesses of one's own presented above (data not shown), suggesting that these differences are not due to demographic confounders.Male interns currently working in primary health care judged the prescribing guidelines, and activities aiming at visualizing adherence to it, to trespass the freedom of the profession to a higher degree than their female equivalents . In contrast, no gender difference was found in residents or specialists in primary health care medicine.Attitudes towards the prescribing guidelines booklet among the subgroups (i) specialists, (ii) residents and (iii) interns differed concerning reasons for changing a not-recommended drug to a recommended one. The results are presented in figure The results of the present study indicate that the prescribing guidelines booklet is frequently used and adhered to by primary health care doctors. It was also deemed to reflect sound judgements concerning effects, safety and cost-effectiveness. The effort to draw up these guidelines and to administer them in the format of a booklet hence seems worthwhile. Access to producer-independent information is an important counterweight to drug information from drug industry representatives, since frequent visits by these have been shown to be associated with increased prescribing costs ,10, but The overall positive attitude towards prescribing guidelines found in this study is in agreement with the results of previous investigations. A systematic review of thirty studies revealed that about 70% of clinicians considered guidelines helpful sources of advice, good educational tools and intended to improve quality .The prescribing guidelines were generally not experienced to trespass the freedom of the profession; 22% of responders graded their level of agreement to ≥ 4 out of 6. This could be in accordance with a previous study, where 25% feared that guidelines can diminish clinical freedom and stifle innovation . In the No unambiguous reason for not changing a not recommended drug to a recommended one could be detected. Doctors in publicly run primary health care units were generally more positive to the prescribing guidelines and adhered to them more often. This is in accordance with previous data available only in Swedish. Financial incentives may be one explanation for this finding, as privately run units in Sweden more seldom are responsible for their prescribed drug costs. Indeed, an association between budgetary policies and prescribing performance has been shown ,12.Specialists in primary health care were more prone to actively change from a not recommended drug to a recommended one upon renewal of a prescription, when compared with residents and interns. An augmented comprehension of the difficulties in prescribing and appreciation of guidelines by age and experience may be one explanation for this finding. Another may be that less experienced doctors find it difficult to change prescriptions made by senior colleagues. Moreover, they may feel more stressed in the working situation, which could make them less tolerant to time-consuming activities that they do not find totally necessary. Indeed, residents avoided active changes to a recommended drug, based on the experience and potential risk of extra work, more often than specialists. Interns, however, ranked these as less important motives for avoiding active changes to a recommended drug, which could reflect their relative lack of experience, and that collegiate reasons may overshadow the risk of extra work in this group. Notably, less experienced doctors did not use the prescribing guidelines less frequently than more experienced colleagues when prescribing a drug for a new diagnosis, a situation in which recommendations could be time-saving rather than time-consuming.Gender differences as for attitudes to the prescribing guidelines booklet were seen only in interns, where male interns found it to trespass the freedom of the profession to a greater degree than female equivalents. This may be of principal interest, as interns were the only responders in the present study who have not decided to make a career in primary health care. Thus, it would be of interest to investigate if attitudes towards prescribing guidelines differ between doctors of different medical specialities and between genders within different specialities. Such studies are now planned by the undersigned.Prescribing guidelines in the booklet format seem to be used more frequently than Internet-based information. This may illustrate the importance of easy accessibility in the stressful clinical situation. Nonetheless, computer assistance has been shown to improve drug dosage and compThis study has several limitations. Firstly, the response rate was around 60%, yielding a possibility of substantial selection bias, where, possibly, individuals negative to the guidelines could have been less prone to respond. Doctors running their own business showed an even lower response rate, 32%, and may therefore be even more negative to the guidelines than indicated by the results of this study. Non-response could not be coupled to other demographic or geographic parameters, since the responders were anonymous. Secondly, the present study only allows conclusions regarding doctors' estimates on the prescribing guidelines and their adherence to it. Actual effects of the guidelines on prescribing patterns should be further explored, and such studies are planned by the authors. Thirdly, multiple analyses and several subgroup analyses have been performed in this study. This increases the probability of making type I errors , especially in small subgroups. Analyses of small subgroups, such as the gender analysis of interns, should only be seen as hypothesis generating.Regarding practical implications of this study for future prescribing guidelines work, the results may indicate a need for increased user-friendliness as for the Internet based guidelines, and need for intensified marketing of the guidelines booklet towards doctors working outside the public health care system.The prescribing guidelines were judged to be frequently used and are generally appreciated, though differences exist between subgroups of users.MABA, AM and SMW are involved in the expert groups drawing up the prescribing guidelines. AM is editor of the prescribing guidelines booklet.MABA conceived the study, drafted the questionnaire, carried out acquisition and analysis of data and revised the manuscript. MS carried out acquisition and analysis of data. AM revised the manuscript. SMW performed statistical analyses and drafted the manuscript. All authors contributed to the design of the study, and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Questionnaire: Questionnaire concerning use of and attitudes towards the prescribing guidelines booklet in primary health care doctors in the region of Västra Götaland.Click here for file
Dengue is a major health problem in tropical and subtropical regions. In Colombia, dengue viruses (DENV) cause about 50,000 cases annually, 10% of which involve Dengue Haemorrhagic Fever/Dengue Shock Syndrome. The picture is similar in other surrounding countries in the Americas, with recent outbreaks of severe disease, mostly associated with DENV serotype 3, strains of the Indian genotype, introduced into the Americas in 1994.The analysis of the 3'end (224 bp) of the envelope gene from 32 DENV-3 strains recently recovered in Colombia confirms the circulation of the Indian genotype, and surprisingly the co-circulation of an Asian-Pacific genotype only recently described in the Americas.These results have important implications for epidemiology and surveillance of DENV infection in Central and South America. Molecular surveillance of the DENV genotypes infecting humans could be a very valuable tool for controlling/mitigating the impact of the DENV infection. Flavivirus, transmitted by Aedes mosquitoes and constitutes a major concern in public health, infecting millions of people per year in tropical and subtropical areas throughout the world. DENV causes a wide spectrum of clinical manifestations in humans, ranging from a flu-like illness, known as Dengue Fever (DF), to the more severe Dengue Haemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS).Dengue viruses (DENV) belong to the genus DENV are enveloped viruses with a positive sense ssRNA of about 11 kb coding a single open reading frame for three structural and seven non-structural proteins .Although four DENV serotypes can be differentiated by immunofluorescence, it does not provide information about epidemiologic origin and phylogenetic relationship between strains from different geographic regions. In fact, studies of evolution and molecular epidemiology of DENV have demonstrated the occurrence of genotype clusters within each serotype -9. For tIn Colombia, the four serotypes of DENV have been involved in epidemics, although DENV-1 and DENV-2 have had the higher circulation rate since 1971. Moreover, since the time when the first case of DHF was described, at the end of 1989, these two serotypes have been particularly associated with severe disease. DENV-4 was first detected in 1984 and since then has been sporadically isolated from mild cases of DF.. DENV-3 was first reported in Venezuela in 1999, and was subsequently detected in Peru and Ecuador in 2000 and Brazil in 2001. In Colombia, 24 years after it had disappeared, DENV-3 was again detected in the state of Santander in 2001 .In the Americas, DENV-3 circulation was reported in the 1960's and 1970's, and all sequenced strains were clustered within genotype IV or American genotype . After tIn the present study, we attempted to amplify historical Colombian strains of DENV-3 isolated in 1977, but it could not be achieved, maybe due to poor samples, or improper maintenance or storage during this time. The recovery of these samples could enrich the basal clade of genotype IV, or might help in explaining the presence of an Asian genotype (genotype I) in Colombia at present if it had been circulating in the past, a very difficult hypothesis to corroborate.E gene of some strains has been sequenced, and the topology results are newly confirmed and a strain from Putumayo, Colombia (352_Putu02), a state along the border, offers hard support for this idea. Finally, the entry of genotype III into the Americas was first reported in Panama and Nicaragua in 1994 [Since DENV-3 genotype III has been present in northeastern and southwestern Colombia since early 2002, different routes of introduction are possible. First, The Venezuelan origin is supported by high similarity of sequences and circulation of this genotype in Venezuela in August of 2001, when the largest epidemic caused by DENV there since the 1989 DENV-2 epidemic ended . The Ven in 1994 , so anotC-prM and partially the E gene [E gene, without recent closely-related sequences available on GenBank to date. Moreover, the related sequences corresponding to Asian strains were isolated in 1973 in Japan as an imported case and in 1980 in Guangxi, China (GenBank: AB111085 and AF317645). Samples that clustered in this lineage are located in a basal branch into genotype I, with high bootstrap support (86%) and mean distance between clades of 5%, estimated with Tamura-Nei model to be classified as a fifth different genotype, referred to as genotype V in [DENV-3 genotype I was recently described in the Americas from nine cases in Brazil, as a result of phylogenetic analysis using two fragments corresponding to e E gene . Here, wThe presence of DENV-3 genotype I only in Colombia, and its close relation with Asiatic strains from 1973 and 1980, suggests that strains circulating in Colombia during the 1970's would have not been of genotype IV, like other American strains from that period, but, perhaps a strain of Asiatic origin that had been circulating without detection for over 25 years until 2002. This speculation needs more data to support it, because there is no evidence for genotype circulation in Colombia in the past, and explaining possible silent circulation without causing outbreaks for more than twenty years could be a challenge.The presence of the Southeast Asia/South Pacific genotype has recently been detected not only in Colombia, but also in Brazil .DENV-3 genotype IV was last reported in Puerto Rico in 1977 (as corroborated by sequencing) , but to E gene of strains corresponding to both DENV-3 Colombian genotypes. The results of the phylogenetic reconstruction .Intra-serotype recombination has been detected in natural populations of DENV ,25-29. NAs known, the potential for causing severe disease has been described for all four serotypes of DENV, and the main factors considered to explain its pathogenicity are host genetic susceptibility, antibody dependent enhancement and differences in virulence among strains . It is ein vitro [Determinants of virulence have been located in three genomic regions and havein vitro , so the in vitro ,34. The Although the origin of genotype I is uncertain, the co-circulation with genotype III could have epidemiologic implications if it has intra-serotype antigenic variation related with differential generation of protective antibodies and immune response . It is iThe relevance of these results is the detection of two different genotypes in the same country, one of them of Asiatic origin, only recently described in the Americas . The resThe strains included in the study, with locality, year and GenBank accession numbers, are listed in table C6/36 cells cultured in Dulbecco's modified Eagle's medium (DMEM), were infected with 0.15 ml of samples and incubated for 10 days at 28°C, washed with PBS, removed by hitting the culture tubes manually and seeded on slides. Cells were then fixed with acetone and the indirect immunofluorescence procedure was carried out incubating the cells with serotype-specific monoclonal antibodies for 60 minutes and then washed with PBS and incubated for another 60 minutes with a commercial secondary antibody conjugated with fluorescein isotyocianate.® LS , and a final volume of 15 μl was recovered in these cases.Aliquots of 140 μl of serum or supernatants of cell cultures were placed into 540 μl of AVL buffer with Carrier RNA and used to extract the viral RNA with QIAamp Viral RNA Minikit as indicated by manufacturer. RNA obtained in 60 μl of AVE buffer was stored at -70°C and used in the RT-PCR. Alternatively, the total RNA of some samples was extracted by the use of TRIZOLE/NS1 of 776 bp, and nested-PCR to amplify an internal region of 350 bp.The RT-PCR and nested-PCR have been previously described . When viProducts of RT-PCR or nested-PCR were purified using QIAquick PCR Purification Kit . Sequencing reactions on both strands were performed with 10 pmol of the primers used for the second round of amplification, and the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit , and analysed using an ABI model 377 automated sequencer .Four sequences were obtained for each sample, two sequences with sense and two with antisense primer. Editing and consensus obtaining were performed with the SeqMan module of Lasergene .E gene (nucleotides 1256 to 1479). The portion of the NS1 gene amplified with the nested-PCR was excluded from the analysis due to the absence of this portion in the majority of reported sequences.Sequences on GenBank corresponding to different lineages of DENV-3 were downloaded and aligned with the consensus sequences obtained in this study, using Clustal W software . AdditioAlignment of the sequences obtained in the present study n = 32) Table and homo Table aThe strain 359_Caqu03 was completely identical to 363_Caqu03, 366_Caqu03, 367_Caqu03, 368_Caqu03, and 464_2003; strain 449Meta05 was identical to 456_Guav05 and 461Guav05; strain 352_ Putu02 to 484_Putu02; strain 221_Guaj02 to 233_Guaj03, 517_Caqu03, 518_putu04, 247_Guav05 and 530_Guav05; and finally, strain 375_SAnd03 was identical to 389_Guaj03, 395_NSan04, 417_Guav04, 429_Huil04, 535_Huil04 and DV06_Ant05.E gene. Initially, the neighbour-joining algorithm was used with 10000 bootstrap replicates and the Tamura-Nei model of nucleotide substitution with MEGA 3.1 software [The phylogenetic trees were estimated for the 224 bp fragment, corresponding to the 3' end of the software . Maximumsoftware . For selThe more important finding of this work is the co-circulation of genotype III of DENV-3, widely distributed, and the recently reported genotype I, never before described in the Americas, in three Colombian states. Co-circulation of different genotypes in an area could be related with the current association between DENV-3 infection and severity of disease. Moreover, intra-serotype antigenic variation related with differential generation of protective antibodies and immune response could be one of the reasons for the high epidemiological impact of DENV-3 in the Americas.The authors declare that they have no competing interests.JAUC contributed to the experimental design, carried out the experiments and phylogenetic analysis, and drafted the manuscript. JAM contributed to the experimental design, carried out the experiments and provided a critical review of the manuscript. AT conceived the study, its experimental design and provided a critical review of the manuscript. GJR contributed to the experimental design and provided a critical review of the manuscript. CD participated in the experimental design, contributed to the interpretation of data and the critical review of the manuscript. JCGG conceived the study, participated in its design and coordination and finalised the manuscript. All authors read and approved the final version of the manuscript.E gene corroborating the presence of two different lineages. The Tamura-Nei nucleotide substitution model was used to estimate distance matrix. Sequences obtained in present study marked with circles and boxes correspond to genotype I and III, respectively. Bootstrap values major of 50% were maintained in the tree supporting clustering in genotypes after 1000 pseudo-replications. Horizontal branch lengths are drawn to scale.Neighbor-joining phylogenetic tree of the DENV-3 Click here for file
Acute and transient psychotic disorders (ATPD) have been characterized by the development of florid psychotic symptoms within 2 weeks and complete remission of symptoms. Although there are no definite guidelines, these are usually treated by antipsychotic medication.This preliminary study examined the effectiveness of olanzapine in paediatric ATPD.In this 6-week open trial of olanzapine in paediatric ATPD, the patients were rated weekly on the Brief Psychiatric Rating Scale (BPRS), Clinical Global Impression (CGI) Scale and Dosage Record Treatment Emergent Symptom Scale (DOTES).n=14, 60.9%), increase in appetite , weight gain and drowsiness . No patient developed extrapyramidal symptoms.Twenty-three patients were included in the study. The mean olanzapine dosage was 12.7±3.9 mg/day (range 5–20 mg/day). All the patients showed significant improvement in 6 weeks. The results showed a significant decrease (p< 0.0001) in scores of BPRS (mean at baseline 46.2±7.0 to 21.4±3.9 at week 6). Severity of illness (CGI) decreased from 4.7±0.8 to 1.6±0.9 in 6 weeks. Also, global improvement (CGI) showed marked improvement in 14 (60.9%), good improvement in 8 (34.8%) and minimal improvement in 1 (4.3%) patient. Some common side-effects were dryness of mouth (Olanzapine was safe and effective in paediatric ATPD. Acute and transient psychotic disorders (ATPD) have been characterized by the development of florid psychotic symptoms within 2 weeks and complete remission of symptoms.26There are few studies on the use of antipsychotic medication in ATPD in children. An open trial of haloperidolAll children aged 6–16 years consecutively attending the child psychiatry department and presenting with symptoms suggestive of ATPD were assessed initially by a psychiatry postgraduate resident and then jointly with VA or PS. Children—both inpatients and outpatients—with a first episode of ATPD fulfilling the ICD-10VA assessed each patient again in detail, and a consensus diagnosis (VA and PS) was made using the ICD-10 criteria.The patients were started on olanzapine 5 mg/day in the first week. Thereafter, the dosage was increased or decreased every week by 2.5–5 mg up to a maximum of 20 mg/day depending upon the clinical improvement or adverse effects. Medications were administered to the patients daily directly under the supervision of one parent to ensure compliance. Concomitant lorazepam 2–4 mg was given during the day or at night for sedation, if needed. Trihexyphenidyl was given for EPS, where necessary.t-test (two tailed) for comparison of baseline scores to scores at the end of weeks 1 to 6. Intent-to-treat analysis was done. The last observation of each patient was carried forward if the patient had at least 2 weeks of follow-up.Statistical analysis was done using the paired Twenty-three children with a mean age of 14.0±1.3 years (range 11–16 years) were included in the study. Twenty-two (5 inpatients and 17 outpatients) completed the trial while 1 was lost to follow-up after 3 weeks. Twenty patients were studying while 3 were illiterate. A family history of ATPD was present in the mothers of 2 patients and of mania in the sister of 1 patient. The mean duration of illness prior to inclusion in the study was 15.0±10.6 days. Eleven patients (47.8%) were diagnosed as having acute polymorphic psychotic disorder without symptoms of schizophrenia (F23.0), 5 (21.7%) with other ATPD (F 23.8), 3 (13.1%) with acute polymorphic psychotic disorder with symptoms of schizophrenia (F 23.1), 3 (13.1%) with acute schizophrenia-like psychotic disorder (F 23.2), and 1 (4.3%) with other acute predominantly delusional psychotic disorders (F 23.3).Mean doses of olanzapine were 9.7±1.0, 12.1±2.9 and 12.7±3.9 mg/day at the end of week 2, 4 and 6, respectively. The dose in 1 patient was decreased from 15 mg/day to 12.5 mg/day after 4 weeks due to drowsiness. Concomitant lorazepam was needed in 11 patients (47.8%) a dosage of 2.5±0.9 mg/day in the first week. Nine children (39%) required lorazepam at a dosage of 2.2±1.1 mg/day in the second week. In the next 1–2 weeks, lorazepam was tapered off and it was given to 6 (26%) and 3 patients (13%) in week 3 and 4, respectively. Only 1 patient required trihexyphenidyl. This patient was previously prescribed haloperidol and she developed severe EPS without any improvement in her ATPD. Haloperidol was tapered off in 1 week and she was started on olanzapine. To manage EPS, trihexyphenidyl 6 mg/day was added in the first 2 weeks of the trial and then stopped.22 =18.15, p<0.0001) as compared to the baseline. Significant improvement was observed as early as the end of week 2 . Severity of illness on the CGI scale also showed significant improvement at the end of week 6 as compared to baseline. A significant reduction in severity was seen at the end of week 2 . The ratings of individual patients on the CGI scale showed marked improvement in 14 (60.9%), good improvement in 8 (34.8%) and minimal improvement in 1 (4.3%) patient (this patient was lost to follow-up after 3 weeks) at the end of 6 weeks.)All the patients showed improvement on olanzapine. The ratings of BPRS at the end of week 6 showed an=14, 60.9%), increase in appetite , weight gain and drowsiness . Other adverse effects were headache , dizziness constipation , blurring of vision , and decreased appetite . No patient developed EPS. The adverse effects were usually mild. Only 2 patients (8.6%) developed moderate drowsiness and increased appetite. A significant increase in weight was observed at the end of the study (mean at baseline 32.7±4.6 kg to 34.0±5.3 kg at the end of the study) in 8 male and 4 female patients. Weight gain was 1 kg in 4 patients, 2 kg in 2 patients, 3 kg in 4 and 5 kg in 2 patients. It was gradual over 6 weeks (average weight gain 6.6%). Clinical assessment did not warrant repeat haematological laboratory investigations during or at the end of the study in any patient.All the children tolerated olanzapine well. Adverse effects were seen in 19 patients (82.6%). Some common adverse effects were dryness of mouth and with risperidonen=12, 4–6 mg/day; 75% response). Also, olanzapine was well tolerated by all the adolescents in this study. No patient developed EPS and concomitant lorazepam was needed in only 47.8% of patients at a dose of 1–4 mg/day for the initial 1–2 weeks. One may argue that the improvement in these children (47.8%) occurred because of lorazepam. On comparing the improvement beyond week 2 (mean BPRS score 35.6±5.8) in these children (n=11), there was a significant improvement at week 4 and at week 6 as compared to week 2. So, it can be confidently said that the improvement in these children was because of olanzapine as they improved significantly beyond week 2.The remission rate 95.7%) in our study is comparable with or even better than the remission rates in acute psychosis treated with haloperidol% in our et al.In the studies on haloperidolLimitations of this study are like those of any open and uncontrolled trial. However, the use of placebo in children with ATPD with florid symptoms poses an ethical dilemma. In the risperidone study, 42.8% patients dropped out in the placebo group as they became unmanageable despite being given lorazepam.This preliminary study shows that olanzapine may be a good first-line antipsychotic drug in paediatric ATPD because of its effectiveness and tolerability. It has a better side-effect profile and patients require less concomitant medication. Also, it has a convenient dosing schedule.This suggests that further randomized, controlled trials on larger samples are required to confirm the efficacy and safety of olanzapine in paediatric ATPDs.
Decreased heart rate variability (HRV) is associated with a higher risk of mortality. Overall, postmenopausal women have lower levels of HRV than premenopausal women, which may be additionally complicated by lifestyle related behaviors such as physical inactivity and obesity. Though cardiorespiratory exercise training increases HRV, little is known regarding the exercise dose necessary to promote this improvement.Our primary aim was to measure HRV in post-menopausal women following 6-months of exercise training. We examined supine resting HRV in 373 post-menopausal women (45–75 y) after 6-months of randomly assigned and double-blinded administered exercise training exercise training at 50%, 100% and 150% of the NIH Consensus Development Panel's recommended minimal physical activity level. This corresponded to 4, 8, or 12 kcal/kg per week (KKW) of energy expenditure. At baseline, we observed no significant differences in HRV or hormone replacement use between treatment groups. However, we did observe that Caucasian women and those taking antidepressant medications had lower levels of baseline HRV. After 6-months of exercise intervention, we observed a dose dependent increase in all parasympathetically derived time and frequency domain measurements across exercise groups after adjustment for age, ethnicity, antidepressants, and baseline rMSSD . For example, the parasympathetic index rMSSD was greater than control (23.19±1.0) for the 4-KKW , 8-KKW , and 12-KKW groups at follow-up.Moderate intensity exercise training exercise is sufficient to improve HRV in previously sedentary postmenopausal women in a dose-dependent manner, as 4-KKW is insufficient to improve parasympathetic indices of HRV, while 12-KKW conferred no greater improvement than 8-KKW.NCT 00011193Clinicaltrials.gov Menopause represents a key transition period in a woman's health. A fundamental etiology associated with menopause is the intricate link between estrogen metabolism and the autonomic nervous system. Autonomic nervous system balance can be assessed non-invasively via the use of heart rate variability (HRV). While a thorough review of HRV can be obtained elsewhere,Current recommendations from the National Institutes of Health (NIH) Consensus Development Panel recommend that women attain a minimal level of moderate intensity activity such as walking briskly for 30 minutes We have recently published a complete description of the DREW design, methods, and primary outcomes for our current cohort. These two papers detail how we derived the sample size for our main outcomes, the randomization sequence and allocation procedures, blinding techniques, recruitment and adverse events 2), and had a systolic blood pressure of 120.0 to 159.9 mm Hg. As previously reported, there was no difference in this cohort at baseline or following our intervention for fasting measures of cholesterol, triglycerides, and glucose After an initial run-in period, we randomized 464 postmenopausal women (45–75 y) to 1 of 3 exercise training groups or a non-exercise control for a 6-month intervention period. Study participants were sedentary , overweight or obese , an electronic, rate-independent ergometer. Participants cycled at 30 Watts (W) for 2 min, 50 W for 4 min, followed by increases of 20 W every 2 min until they could no longer maintain a pedal cadence of 50 rpm. Breath-by-breath respiratory gases were measured using a Parvomedics True Max 2400 Metabolic Measurement Cart. Volume and gas calibrations were conducted before each test. Gas-exchange variables were averaged every 15 s. Heart rate was measured directly from the ECG monitoring system. Ratings of perceived exertion (RPE) were obtained using the 20-point Borg scale. Fitness was defined as the mean of two exercise test assessments at both baseline and 6 months. The reproducibility of the two fitness tests (VO2abs) was examined and characterized by an intraclass correlation of 0.88 at both baseline and follow-up testing.2max for each exercise modality. We used a computer-controlled exercise training management that allowed us to input of relevant data points on each woman . The computer then provided us with the appropriate power output (PO) for the cycle ergometer, and the correct speed and grade for the treadmill. By knowing the exact PO for the cycle ergometer and the treadmill, the total kilocalories expended each minute and the time needed to reach the target energy expenditure for the exercise session or for the week can be calculated. The duration of each individual session depends on the number of visits required to reach the target KKW. To monitor the participant's caloric expenditure over the course of 6 months, we created a weekly tracking report, which was used to track and make calculate caloric adjustments. The number of calories expended per session was adjusted each week, within the limits of the study design so that the total number of calories expended is equal to the total number prescribed per week for the 24-wk program. This report also averaged the number of visits per week so that we can determine whether the participants are exercising at least two, but no more than four, sessions each week.To assess potential changes in non-supervised physical activity, all randomized participants wore a step counter to record their daily steps. Exercisers removed their step counter during supervised exercise sessions. Cardiovascular exercise training consisted of having women expend 4, 8, or 12 kcal/kg per week (KKW). During their exercise training sessions, women alternated exercise training using a recumbent cycle ergometer or treadmill. The target training intensity for each exercise training session was 50% of each woman's VODuring exercise, HR is monitored every 3–6 min using a Polar HR monitor (Polar Vantage XL and Polar Vantage NV). The target HR was used to monitor each woman's performance during each session to ensure that she was exercising at the proper intensity. Monitoring HR allowed us to control exercise intensity and document the specific amount of exercise done during each session. As women improved their fitness, they worked at gradually higher PO and spent less time to expend the required KKW. We contacted participants if they missed a scheduled session so that arrangements could be made to bring them back on schedule as soon as possible. As previously reported, the non-exercise training control group maintained their current level of activity during the trial period and showed no significant increase in physical activity We examined HRV between 06:30 a.m. and 11:00 a.m. for 25 minutes in a semi-dark room (22–23°C) following a 12-hour fast. Participants abstained from consuming caffeine-containing products and alcoholic beverages for 12 hours and heavy exercise for 48 hours. We controlled respiration rate using a metronome at a fixed rate of 12 breaths/min (0.2 Hz). HRV measurements were conducted at the same time (±1 hour) of day for each assessment period. We used a two-channel ECG signal detected by a Polar Heart Rate Monitor and transmitted online to a PC through Polar Advantage Interface receiver. QRS timing was fixed at 1 ms. The computer program labeled each QRS complex, and the resulting signal was filtered to eliminate ectopic beats and artifact edited and replaced with an average value. Segments containing >15% artifact ectopy were interpreted as premature beats and were excluded from our data analysis For our primary outcome measurement, we examined the parasympathetic nervous system by calculating the square root of the mean of the sum of the squares of differences between adjacent R–R intervals (rMSSD). rMSSD is considered to be a stable measure of parasympathetic modulations in heart rate s. All reported P values are two-sided (P<0.05). All analyses were performed using SAS version 9.1 .We used a generalized linear model (GLM) to analyze the influences of the differing doses of exercise training on HRV characteristics. We adjusted all our outcomes among the randomization groups for select specified covariates including baseline HRV, race, and the use of antidepressant medication. When our GLM demonstrated a significant overall statistical effect and a significant treatment group effect, we further explored pairwise comparisons between the exercise training groups vs. the control group using a Dunnett-Hsu post-hoc assessment. The Dunnett-Hsu test allows for specific multiple pair-wise comparisons while still protecting against type I statistical errors. Between group differences at baseline and follow-up in prevalence were examined using Chi-square tests. We also used a subgroup analysis to compare dose-response effects across predefined baseline groups with significance of interactions relationships between variables were tested using a Spearman correlation analysis and denoted as rP<0.0001) and antidepressant medication use (P<0.002). Post-hoc assessments revealed that African American women had statistically higher levels of rMSSD vs. Caucasian women (P<0.0001). Spearman correlations characteristics for various physiologic variables and HRV indices are presented in s = −0.1; P<0.05), VLF , and body weight .We have presented a schematic CONSORT flowchart our HRV analysis in P<0.0001), but not for treatment group effect (P = 0.10) after 6 months of exercise training. Thus, we found no specific exercise training effect despite a trend for a reduction in resting HR. For our time domain HRV measurements, we observed a significant main statistical effect for rMSSD and SDNN and for treatment group . Post hoc comparisons revealed that rMSSD for the 8-KKW and 12-KKW exercise training conditions were greater than the control after 6 months (P<0.05). However, our post-hoc assessment only showed a significant statistical difference in SDNN for the 8-KKW treatment versus control (P<0.05).We present the HR, time, and frequency domain analyses in Ln, LFLn, VLF, and PT components and for treatment group . Post hoc assessment revealed that the HF index of HRV denoting parasympathetic tone was significantly greater versus the control group for the 8-KKW and 12-kcal KKW treatment conditions . Post hoc comparisons for LF, VLF, and PT showed each HRV index to be greater than the control group for the 4-KKW, 8-KKW, and 12-KKW treatment conditions . No statistical effects were noted for the LF∶HF ratio.For the frequency domain analysis, we observed a significant main statistical effect for the HFThe primary finding from our study is that moderate intensity exercise training improves HRV characteristics in previously sedentary, overweight or obese postmenopausal women. To our knowledge, this is the largest clinically controlled exercise training trial examining this question and the only study examining the dose response of exercise training and HRV. Of particular interest is that these effects appear to be dose-related regarding parasympathetic tone as only women exercising at or above the NIH recommendation exhibited a positive change in these HRV characteristics. The same pattern was present for SDNN; however, SDNN represents mixed sympathetic and parasympathetic signaling which makes the interpretation of this index more difficult. These findings have important implications for women entering into and completing menopause.Normal aging influences a variety of indices related to CV circulation including the adrenoreceptor responsiveness and baroflex sensitivity Previous cross-sectional studies helped to define the question evaluated our the current study. In a recent report, Reland et al. categorized older women into three groups: low activity , moderate activity , and high activity and found that women in the high activity group who participated in chronic physical activity demonstrated an increase in parasympathetic indices of HRV 2max) was insufficient to improve HRV in eight postmenopausal women max (∼80–85% HRmax). From these studies, one could surmise that intensity and study length are important factors for examining HRV changes in women; however, the results of these trials may create confusion regarding the effectiveness of exercise training for improving HRV in women.Earlier literature describing the use of exercise training to modulate HRV in women is limited and difficult to interpret due to variances in study protocol participant age, length, small sample sizes and the exercise training intensity used for the intervention. Davy et al reported that 12 weeks of aerobic exercise at 70% of maximal heart rate that a significant change in HRV does accompany longer periods of exercise training consistent with the NIH recommendation and (2) a greater level of energy expenditure may not be necessary to achieve greater improvements in HRV. In essence, our findings suggest a “threshold effect” regarding aerobic exercise training and improvements in HRV. Nonetheless, we caution readers not to misinterpret that 4-KKW is ineffective for improving HRV as several indices of HRV were increased at the 4-KKW. Unfortunately, the interpretation of these results is more difficult to explain regarding the parasympathetic influence associated with HRV. SDNN and LF power are thought to represent mixed sympathetic and parasympathetic signaling and the physiologic characteristics surrounding VLF are still a matter of debate We believe that improvements in HRV via exercise training are likely to reduce the risk of mortality for several reasons. In animal studies, dogs documented with a high mortality risk due to the previous occurrence of ventricular fibrillation during acute myocardial ischemia were randomly assigned to 6 weeks of either daily exercise training or cage rest. After exercise training, indices of HRV increased by 74% and all animals were better able to survive an experimental ischemic challenge after the exercise training period A primary strength of the DREW study is that it used a well-controlled dosage of exercise, with a high level of adherence. Moreover, the exercise dose used in our study is easily obtainable in sedentary women. The DREW study has limitations because its sample is limited to sedentary postmenopausal women. We do not know if the results will apply to other women or men. An interesting finding from our study is that women exercising at a higher caloric expenditure (12-KKW) did not experience greater improvements in HRV than women exercising at 8-KKW. Finally, our findings should not dissuade women from exercise training at a lower dose of they are not able to achieve the minimum level of exercise training necessary to improve HRV. Given the global effects of aerobic exercise training, HRV represents only one measure of CV risk, which may ultimately show improvement accompanying longer periods of exposure.Checklist S1CONSORT Checklist(0.23 MB PDF)Click here for additional data file.
A case-control study of 689 breast cancer patients seen at Tata Memorial Hospital during the period 1980-84 was carried out. During the same period 711 females who attended the hospital without a history of benign breast lesions or gynaecological complaints were selected as controls. Patients were interviewed by trained investigators to collect data on reproductive factors, menstrual history, tobacco smoking and chewing habit, dietary practices (vegetarian and non-vegetarian diet) and alcohol consumption. Cases and controls were stratified into four age groups and three places of residence . The adjusted relative risk (RR) for unmarried women compared with married women was 2.3. Nulliparous women had a 2.2-fold higher risk than parous women. Late age at marriage (30 years and above) and late age at first pregnancy (30 years and above) showed excess risks of 2.5 and 5.4 compared with women married at the age of 14 years and age at first pregnancy of < or = 14 years. Three or more pregnancies was associated with a 40-50% reduction in risk (P < 0.01). Non-vegetarian diet, literacy status and a history of stillbirth and abortion did not emerge as significant risk factors for breast cancer in our study. These findings, in a low-risk population, were consistent with those reported from high-risk populations.
These manifold functions impose significantconstraints on the C-peptide structure that are conservedin evolution. After cleavage of proinsulin, the intactC-peptide is stored with insulin in the soluble phase of thesecretory granules and is subsequently released in equimolaramounts with insulin, providing a useful independentindicator of insulin secretion. This brief review highlightsmany aspects of its roles in biosynthesis, as a prelude toconsideration of its possible additional role(s) as a physiologicallyactive peptide after its release with insulin into thecirculation in vivo.The C-peptide links the insulin A and B chains in proinsulin,providing thereby a means to promote their efficientfolding and assembly in the endoplasmic reticulum duringinsulin biosynthesis. It then facilitates the intracellulartransport, sorting, and proteolytic processing of proinsulininto biologically active insulin in the maturing secretorygranules of the
Thirty-one patients with a diagnosis of carcinoma of unknown primary site were treated in Aberdeen Royal Infirmary between 1997 and 2001 with MCF. In total, 136 cycles of MCF were delivered (median of 5 cycles per patient). Toxicity was acceptable, with 19% grade 3 or 4 neutropenia, 16% grade 3 or 4 thrombocytopenia and 13% grade 3 or 4 nausea and vomiting. No cases of neutropenic sepsis were seen and there were no treatment-related deaths, however, six patients developed thrombotic complications. The overall response rate was 27% . Median time to progression was 3.4 months (95% CI 1.1–5.6 months) and median overall survival was 7.7 months (95% CI 5.7–9.8 months). Survival at 1 year was 28%, and at 2 years, 10%. MCF is a tolerable regimen with comparable toxicity, response rates and survival data to most platinum-based combination chemotherapy regimens in use for this devastating disease.Carcinoma of unknown primary site remains a common clinical diagnosis, accounting for between 5 and 10% of all cancer patients. Numerous combination chemotherapy regimens have been used in the management of carcinoma of unknown primary site, resulting in response rates of 0–48%. We present the results of a single centre phase II study of the use of the combination of mitomycin C 86, 1238–1242. DOI: 10.1038/sj/bjc/6600258www.bjcancer.comCancer Research UK© 2002 The diagnosis of carcinoma of unknown primary site accounts for 5–10% of all new patients referred to oncology clinics . Of thesThe most common metastatic sites in ACUP are lymph nodes, lung, liver and bone. Intensive investigation rarely identifies a primary site, and if successful, seldom alters treatment , after adjusting for age and number of metastatic sites. Single agent chemotherapy studies in patients with ACUP show response rates for 5-fluorouracil (5-FU) and cisplatin of 0–16% and 19% respectively cancers, of cisplatin in lung, breast, ovarian and upper G-I cancers, and of 5-FU in breast and G-I cancers, thereby producing a combination with potential activity against the main primary tumour sites responsible for ACUP. In addition, the three agents give rise to generally non-overlapping toxicities.Patients were eligible for the study if there was a cytologically or histologically confirmed diagnosis of carcinoma of unknown primary. Patients were required to be aged between 18 and 75, chemo-naïve, have a WHO performance status of ⩽2 and have adequate haematological, renal and liver function.All patients were evaluated clinically by medical history and physical examination. Baseline investigations included full blood count, serum biochemistry and tumour markers . Plain chest radiographs and abdominopelvic CT scans were performed, with mammography in women. Further investigations, such as chest CT scan, pelvic ultrasound, endoscopy, barium studies and bone scintigraphy were performed dependent on the patient's symptoms or signs.All pathology was analysed centrally. Where adenocarcinoma was diagnosed on light microscopy, immunohistochemistry was performed on the pathological specimen for CEA and PSA in men, and for CEA, Ca125 and hormone receptors in women. Where poorly differentiated tumour was diagnosed on light microscopy and immunohistochemistry, appropriate stains were used to confirm carcinoma and exclude haematological malignacies, melanoma, germ cell tumours and sarcoma .−1 and/or bone only disease in a male), ovarian or primary peritoneal cancer , or germ cell neoplasm , or if they had nodal disease only which was localised to a single lymph node region.Patients were excluded if tumour markers, radiology and/or the clinical scenario were in keeping with primary prostatic cancer was delivered on day 1 of every alternate cycle. Cisplatin was delivered on day 1 of each cycle. 5-FU was delivered as a continuous infusion (300 mg m−2 day) throughout treatment, via a tunnelled catheter and portable pump. Prophylactic warfarin (1 mg daily) was given to reduce the incidence of line-associated thrombosis. Prophylactic anti-emetic therapy consisted of 8 mg dexamethasone and 8 mg ondansetron pre-treatment, and thereafter 2 mg dexamethasone t.d.s and 8 mg ondansetron b.d. for 3 days and was altered as required. Chlorhexidine mouthwash was supplied to all patients.The MCF regimen was delivered every 21 days for a maximum of six cycles. Mitomycin C (7 mg m9 per litre and platelets ⩾100×109 per litre) was required, otherwise chemotherapy was delayed for 1 week or until the myelosuppression had resolved. Renal function was monitored by calculating creatinine clearance prior to each cycle, to ensure a clearance of ⩾60 ml min−1. For values below 60 ml min−1, the total dose of cisplatin per cycle was reduced to the GFR value in mg, and below 40 ml/min, cisplatin was omitted.Prior to each cycle of therapy (whether mitomycin C was due or not), adequate haematological function , time to progression and overall survival. Response was assessed at each cycle by clinical examination, tumour markers and CXR if appropriate. CT scans were repeated after 3 and 6 cycles of chemotherapy. Although WHO response criteria were used, it was not possible to confirm responses after 1 month because of resource limitations. Time to progression and overall survival were defined as the time from the first cycle of therapy to the date of documented progression or death, respectively.The response rates for previous platinum- or taxane-based regimens in carcinoma of unknown primary lie between 19 and 50% see . AssuminMedian time to progression and median overall survival were estimated by the Kaplan Meier method using SPThirty-one consecutive eligible patients with CUP were recruited into the study at Aberdeen Royal Infirmary between April 1997 and January 2001. The patient characteristics are listed in Table 1A total of 136 cycles of MCF chemotherapy was delivered, with a median of 5 cycles per patient (range 2–6). Twenty-two cycles were delayed, most by only 1 week. The reasons for delay were myelosuppression in 14 cases, stomatitis in four cases, and grade 3 vomiting, grade 4 constipation, grade 2 diarrhoea and unexplained jaundice in one case each.Dose reductions were instituted for mitomycin C in two patients (due to neutropenia), for cisplatin in four patients and for 5-FU in 12 patients .The delivered dose intensity for each drug was calculated by averaging the mean dose received per week for the entire treatment course for each patient, and the results are compared with the intended dose intensities in Table 2No treatment-related deaths were observed within the study. There were 12 emergency admissions in 11 patients. The reasons for admission were thrombotic complications in four cases, and one case each of urinary retention, rigors with no other evidence of infection, grade 3 vomiting, grade 4 thrombocytopenia and grade 4 anaemia. The remaining three admissions arose as a consequence of disease progression rather than therapy, two with bowel obstruction and one with obstructive uropathy.All patients were assessable for toxicity and the data are summarised in Table 35-FU related toxicity was common, with 48, 33 and 22% of patients experiencing grade 1 or 2 stomatitis, diarrhoea and palmar-plantar syndrome respectively, however, severe toxicity was rare.No patients developed significant chemotherapy-related nephrotoxicity, although the calculated creatinine clearance fell by between 10 and 20% in five patients from the start to the end of chemotherapy. In no case did calculated renal function fall by more than 20%.A total of six Hickman line complications occurred in four patients. There were three episodes of subclavian vein thrombosis, two episodes of line infection and one pneumothorax.Thirty patients had measurable disease and were included in the response assessment. After six cycles of MCF, eight patients had responded to chemotherapy, one complete response (3%) and seven partial responses (23%), giving an overall response rate of 27% (95% CI 11–42%). In total, 63% of patients progressed during their chemotherapy. Of the 10 patients who had stable disease after three cycles, two subsequently achieved a partial response (both of whom had shown a minor response after three cycles), three maintained stable disease and five had progressed by completion of treatment. Of the eight patients who had a partial response after three cycles, two had progressed by the end of the sixth cycle; in view of the lack of a confirmatory 1 month scan, the initial responses of these two patients were not included in the overall response rate.All eight patients who achieved a response after six cycles of chemotherapy had a histological diagnosis of adenocarcinoma (as opposed to poorly differentiated carcinoma), six with liver involvement and one each with node only and peritoneum only disease.Table 4Survival data were available for all 31 patients. After a median of 25 months follow-up (range 7–53 months), the survival data are mature. Median time to progression is estimated as 3.4 months (95% CI 1.1–5.6 months) and median survival as 7.7 months (95% CI 5.7–9.8 months (Figure 1−2 of 5-FU may be preferable as a starting dose.The MCF regimen was found to be generally well tolerated in patients with carcinoma of unknown primary, with grade 3 or 4 toxicity rates which are very similar to those reported for the same regimen when used in gastric carcinoma (A response rate of 27% was seen. Of the eight patients who had stable disease with no evidence of even a minor response after three cycles of MCF, five had progressed by the sixth cycle and only three maintained stability, raising the suggestion that in those patients without any demonstrable reduction in tumour size after three cycles, MCF should be discontinued.vs carboplatin and etoposide (The 27% response rate and 7.7 month median survival observed with MCF are broadly similar to other cisplatin-based regimens (see Table 5More important than response rates and median survival data in CUP, where the majority of patients do not respond to chemotherapy, are longer term follow-up data. In our study, 28% of patients survived 1 year, and 10% survived 2 years. This is in keeping with two previous cisplatin-based studies in which 1-year survival is reported as 20–25% and indeWhile the addition of a taxane to chemotherapy for CUP may well be advantageous, heterogeneity in patient characteristics in phase II studies makes this extremely difficult to demonstrate convincingly at present. This problem is exemplified by the very impressive 15 month median survival quoted for me-FAM (The heterogeneity of these tumours continues to be the problem, and perhaps the future for the management of cancers of unknown primary lies in improved molecular profiling and better targeted therapy.−2 day of 5-FU is recommended. The advent of capecitabine may allow the replacement of continuous infusion 5-FU with this oral antimetabolite in due course, removing the potential complications associated with Hickman lines. The role of the taxanes in this heterogeneous disease requires evaluation in a randomised study and our future plans include a comparison of MCF with a taxane based regimen.MCF appears to be an active regimen in good performance status patients with carcinoma of unknown primary, although in view of the 5-FU-related toxicity observed in this study, a dose of 250 instead of 300 mg m
The isoxazole ring adopts an envelope conformation, with the N atom displaced by 0.672 (2) Å from the plane of the other ring atoms. The thiophene ring is oriented with respect to the succinimide and phenyl rings at dihedral angles of 40.03 (12) and 5.21 (13)°, respectively. The dihedral angle between the succinimide and phenyl rings is 39.38 (12)°.The crystal structure of the title compound, C Å b = 10.9803 (6) Å c = 11.1069 (9) Å V = 1558.22 (17) Å3 Z = 4 Kα radiationMo −1 μ = 0.22 mmT = 296 K 0.65 × 0.46 × 0.27 mm Stoe IPDSII diffractometerX-RED32; Stoe & Cie, 2002T min = 0.889, T max = 0.935Absorption correction: integration (6395 measured reflections3332 independent reflectionsI > 2σ(I)2899 reflections with R int = 0.051 R[F 2 > 2σ(F 2)] = 0.037 wR(F 2) = 0.099 S = 1.04 3332 reflections221 parameters1 restraintH atoms treated by a mixture of independent and constrained refinementmax = 0.22 e Å−3 Δρmin = −0.22 e Å−3 ΔρAbsolute structure: Flack 1983, 1451 FrFlack parameter = −0.13 (8) X-AREA (Stoe & Cie, 2002X-AREA; data reduction: X-RED32 (Stoe & Cie, 2002SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEP-3 for Windows (Farrugia, 1997WinGX (Farrugia, 1999Data collection: 10.1107/S1600536808020102/hk2484sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536808020102/hk2484Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:
Introduction: Well leg compartment syndrome is a rare complication that can occur after prolonged surgery in lithotomy position. We stress the importance to recognize high-risk patients for this complication and finding ways to reduce this risk. We also emphasize high level of suspicion of at risk patients and early recognition of signs and symptoms to enable early diagnosis and treatment. Methods: Presentation of a case report of bilateral well leg compartment syndrome with review of literature and discussion of the pathophysiology, risk factors and treatment of this condition. Result: This is a possible complication of surgery associated with significant morbidity and mortality. Published papers have suggested possible ways to reduce this risk and achieve early diagnosis. Conclusion: Clinicians should be aware of the aware of the risk factors for developing well leg compartment syndrome and in patients and have high index of suspicion when assessing them. Further studies looking at the risks, benefits and feasibility of suggestions on ways to reduce this risk is required. Compartment syndrome of the lower leg occurs when increased osteofascial compartmental pressure leads to decreased tissue perfusion to the leg. It can develop after prolonged surgery in the lithotomy (Lloyd Davies) position. When a patient is in this position, lower leg compartment systolic pressure falls. If this decrease in systolic blood pressure falls below the perfusion pressure, tissue ischemia occurs.The patient was a 44-year-old woman with ulcerative colitis. She was admitted for proctectomy, pouch formation, and ileostomy. The patient was a nonsmoker and led an active lifestyle. She had a body mass index of 26.6.The surgery was carried out under general anesthesia, with thoracic epidural analgesia. The patient had antiembolic stockings throughout the procedure without intermittent pneumatic compression. She was placed in a lithotomy (Lloyd Davies) position for the procedure with Lloyd Davies stirrups and no head-down tilt (lithotomy-Trendelenburg position). The patient was maintained in this position throughout the procedure. The procedure lasted 7 hours with 980 mL of blood loss during the procedure, and the patient's arterial blood pressure was maintained at 90/55 mmHg or above throughout the procedure.In the recovery unit, the patient complained of severe cramping-type pain in both legs but there were no swelling or tenderness to palpation. The pain was initially attributed to the surgery and the patient's analgesics were increased. Her epidural analgesia continued for 2 days before it was changed to patient controlled analgesia morphine infusion. Two days later, on examination, patient's left leg was swollen, tender, and hard. Posterior tibial and dorsalis pedis pulses were present in both legs. Doppler ultrasound scan carried out on the left leg showed thrombosis of left popliteal and calf veins. Patient was infused with heparin. On evening of day 2 postoperative, the patient's left leg was found to be cold and mottled with her calf swollen and tender. There was paresthesia in her left leg and foot. Left dorsalis pedis and posterior tibial pulses were absent but present in her right leg. Plasma creatine kinase levels was found to be 35,000, but the renal function remained within normal limits. Although compartment pressures in both legs were not measured, a clinical diagnosis of compartment syndrome was made. Emergency bilateral fasciotomies were carried out with 2 incisions to release all 4 compartments in both legs.The patient was later transferred to the plastic surgery unit for the management of fasciotomy wounds. On examination postfasciotomy, the patient had sensory deficit in the distribution of superficial and deep peroneal nerves bilaterally. She had reduced power on plantar flexion and was unable to dorsiflex and evert both feet. The patient was taken to the operation theatre for debridement and exploration of fasciotomy wounds Figs and 2. ICompartment syndrome is a well-known complication posttrauma in the legs. It is, however, rare to develop compartment syndrome after surgery. The overall incidence of well leg compartment syndrome in patients after major pelvic surgery in lithotomy position is estimated at 1 in 3500 cases.Diagnosis of compartment syndrome is usually made clinically with patient complaining of paresthesia, pain out of proportion to the injury, pain on passive stretch, and tight calves.Compartment pressure can be measured with different pressure catheters. These catheters have fine bore and their use is associated with minimal morbidity.1It is uncommon for deep vein thrombosis to cause acute compartment syndrome. Massive iliofemoral thrombosis causing compartment syndrome is noted with phlegmasia cerulea dolens. Distal venous obstruction is an unusual cause of compartment syndrome.The treatment of compartment syndrome is surgical decompression of the compartments immediately. Fasciotomy is carried out to release the pressure in all 4 compartments in the leg. It is important to recognize and treat compartment syndrome early because delayed treatment in patients leads to increased morbidity and mortality.8The common options for the closure of fasciotomy wounds include split-thickness skin graft and direct closure. Meshed split-thickness skin graft secured with foam vacuum suction dressing after excising all devitalized tissues has been recommended.4The cause of well leg compartment syndrome is multifactorial. It is important that we recognize these risks and carry out appropriate steps to prevent patients from developing it. Raza et alWell leg compartment syndrome is an acute condition associated with significant morbidity and mortality.
We hypothesized that foetal administration of SQ-29,548,a putative thromboxane receptor blocker, would preventfoeto–placental vasoconstriction produced by the thromboxanemimic U46619. Arterial blood gases, continuousmonitoring of maternal and foetal heart rates and bloodpressures were performed in chronically catheterized pregnantewes. Foetal blood flows and vascular resistance weredetermined with radioactive microspheres. SQ-29,548effectively blocked the expected vasoconstrictive effects ofthromboxane. However, prolonged infusion of SQ-29,548resulted in significant decreases in umbilical–placentalblood flow and foetal mean arterial pressure. This wasaccompanied by a respiratory acidemia. Potential therapyfor the vasoconstrictive disorders of pregnancy with SQ-29,548awaits further investigation of its intrinsic vasoactiveproperties in the umbilical–placental vasculature.
The median delivered dose-intensity was 98% (range 79–102%) of the planned dose for both drugs. There were seven complete responses and 15 partial responses, for and overall response rate of 58% . Responses were even seen in three patients with hepatic metastases. The median time to progression was 6.9 months, and the median overall survival was 10.4 months. Two patients who achieved CR status remain free of disease at 4 and 3 years respectively. Grade 3–4 granulocytopenia occurred in 27 patients, resulting in five episodes of febrile neutropenia. There was one toxic death in a patient with grade 4 granulocytopenia who developed acute abdomen. Grade 3–4 thrombocytopenia was rare (one patient). Other grade 3–4 toxicities observed were anaemia (three patients), vomiting (five patients), diarrhoea (four patients), peripheral neuropathy (two patients) and non-neutropenic infections (seven patients). Docetaxel plus cisplatin is an effective and well-tolerated regimen for the treatment of advanced urothelial cancer, and warrants further investigation.A multicentre phase II trial was undertaken to evaluate the activity and toxicity of docetaxel plus cisplatin as first-line chemotherapy in patients with urothelial cancer. Thirty-eight patients with locally advanced or metastatic transitional-cell carcinoma of the bladder, renal pelvis or ureter received the combination of docetaxel 75 mg mBritish Journal of Cancer (2002) 86, 326–330. DOI: 10.1038/sj/bjc/6600121www.bjcancer.comThe Cancer Research Campaign© 2002 Chemotherapy is the treatment of choice for patients with locally advanced and metastatic urothelial cancer. The combination of methotrexate, vinblastine, doxorubicin and cisplatin (M-VAC) has been the most widely used regimen, with reported response rates of 36 and 78% transitional-cell carcinoma of the bladder, renal pelvis or ureter, not curable with surgery, were eligible. Patients with mixed tumours including transitional-cell carcinoma were considered eligible, whereas those with pure squamous, adenocarcinoma, or small-cell carcinoma were not. Patients must not have received prior chemotherapy for advanced disease, although prior adjuvant or neoadjuvant chemotherapy was allowed if this was completed more than 6 months before study entry. Patients were required to have bidimensional measurable disease and no previous radiotherapy of the indicator lesion. Patients were also required to be 18 years or older, with a Karnofsky performance status of 60 to 100. Other inclusion criteria were as follows: normal baseline haematologic parameters, creatinine clearance of 60 ml min−2, diluted in 250 ml of 5% glucose, as a 1 h infusion. Cisplatin 75 mg m−2 was infused in 500 ml of normal saline over 30–60 min, with adequate pre- and post-hydration and mannitol. Both drugs were given on day 1 and repeated every 3 weeks. Premedication included dexamethasone, 8 mg orally b.i.d., the day before and four consecutive days following chemotherapy. Antiemetic treatment consisted of intravenous ondansetron or granisetron in combination with dexamethasone 20 mg on day 1. Cycles were not started unless the granulocyte count was >1500 mm−3 and platelets were >100 000 μl−1. Prophylactic use of growth factors (G-CSF) was not routinely recommended. However, if grade 4 granulocytopenia or febrile neutropenia was present, prophylactic Lenograstim, 263 μg day−1 over 10 days, was administered in subsequent cycles. The docetaxel dose was reduced to 55 mg m−2 if patients experienced grade 4 thrombocytopenia, febrile neutropenia despite prophylactic administration of G-CSF, or grade 2 hepatotoxicity. Doses of both agents were reduced by 25% if patients experienced grade 2 peripheral neuropathy. Patients were taken off the study if creatinine clearance decreased to less than 50 ml min−1. Patients received treatment for a maximum of six cycles unless they developed progressive disease or experienced excessive toxicity as judged by the investigator or the patient.Docetaxel was administered at a dose of 75 mg mNational Cancer Institute common toxicity criteria were used to analyze toxicity. Response evaluation was performed every three cycles using standard WHO criteria. Even patients receiving just one course were considered evaluable for response and toxicity assessment. Patients were followed for survival and disease progression every 3 months until death or loss to follow-up. The method of Kaplan–Meier was used to estimate duration of response, time to progressive disease and overall survival. The planned sample size was 35 patients, using a two-stage sequential design, with the assumption that the regimen would not be of interest if it had a response rate of less than 30% but would be of considerable interest if it had a response proportion of 50% or more .From February 1997 to August 1999, 38 patients were entered into the study at seven centres. All patients were eligible for the study, and all assessable for toxicity and response. Pre-treatment characteristics of the patients are summarized in Table 1A total of 166 cycles of chemotherapy were administered. The median number of cycles received per patient was six . Of the 38 patients, 35 (92%) received at least two cycles of chemotherapy. The median delivered dose-intensity was 98% of the planned dose for both drugs. Fifteen patients received G-CSF in a total of 48 cycles of chemotherapy. G-CSF was administered prophylactically in 42 cycles, and as a treatment of febrile neutropenia in six cycles. Overall, patients were ultimately withdrawn from therapy due to progressive disease (nine patients), adverse events (six patients), stable disease (three patients), or request (one patient). Twenty-two of the 38 assessable patients obtained an objective response, for an overall response rate of 58% , 41–74%), with seven patients obtaining a radiological complete response and 15 a partial response . An additional four patients (10.5%) had stable disease, and 11 patients had progressive disease. Responses were seen in three out of the six patients who had received prior adjuvant chemotherapy. Fifteen of the 22 patients who achieved a response had locally advanced or metastatic lymph node disease exclusively. However, five patients had visceral metastases, including three with liver and two with lung disease, and two additional patients had soft tissue metastases. The median duration of response was 10.5 months . The median time to progressive disease was 6.9 months , and the median overall survival time for all patients was 10.4 months . At the time of the report, two patients who had achieved CR status remain alive and free of recurrence at 48 and 36 months respectively. Both had exclusively locally advanced and lymph node disease before the chemotherapy. One of them underwent salvage cystectomy after chemotherapy, and no viable tumour was found at pathological study.Table 2The initial experience with docetaxel in the treatment of urothelial cancer demonstrated single-agent activity in a series of patients previously treated with chemotherapy (The present study evaluated docetaxel in combination with cisplatin which is generally considered to be the most active agent against urothelial cancer. Prior phase I studies showed the feasibility of this combination and its activity on different tumours (The toxicity of this regimen was generally acceptable and manageable. The most common toxicity was haematological, mainly granulocytopenia. However, most episodes of granulocytopenia were brief and did not cause clinical repercussion, since only 13% of the patients experienced febrile neutropenia. Nevertheless, there was a toxic death associated with acute abdomen and severe granulocytopenia. This rare and serious complication has been reportedly associated with different chemotherapy schedules, including docetaxel (Recently, two studies that used the same regimen in bladder carcinoma have been reported (Table 3Two additional studies have evaluated docetaxel in other combinations in the treatment of urothelial cancer. One of them evaluated the three-drug combination with cisplatin and epirubicin (In the last few years, several clinical trials aimed at identifying polychemotherapy regimens with new drugs active against urothelial cancer have been initiated. The objective has been to find combinations that demonstrate improved efficacy and a better toxicity profile compared with that of M-VAC. The most extensively studied drugs have been gemcitabine . HoweverA few trials with three-drug regimens containing two new drugs have also been performed in recent years. The combination of paclitaxel, gemcitabine and cisplatin (In summary, the present study shows that the association of docetaxel and cisplatin is an effective regimen in the treatment of advanced urothelial cancer. The results also show that a small number of patients, who would otherwise succumb to disease, can achieve long-term disease-free survival after this chemotherapy regimen. Although the toxicity of this regimen was not insignificant, it was tolerable considering that the severe toxicity observed was in general reversible and manageable. These results, added to the other recent studies that included docetaxel in their treatment regimens, indicate the interest in this drug in the treatment of urothelial cancer as an alternative to conventional therapeutic regimens. Further studies aimed at determining more accurately the potential of docetaxel are warranted together with investigation into new strategies that will introduce it into three-drug regimens or in combinations without cisplatin.
To the Editor: Deaths caused by new members of the genus Morbillivirus, family Paramyxoviridae (Phoca vitulina) in 1988 died in 1990 and 1991 (Delphinus delphis ponticus) from the Black Sea died in 1994 because of infection with dolphin morbillivirus (DMV) (Tursiops truncatus) in the Gulf of Mexico from 1987 through 1994 (Phocoena phocoena) in European waters in 1988 (Porpoise morbillivirus) and endangered Mediterranean monk seals (Monachus monachus) in 1997 . After these epidemics, the viruses disappeared and no marine or terrestrial reservoirs have been identified.Lagenorhynchus albirostris) was found stranded on the North Friesian coast of Germany. The animal was humanely killed and a complete necropsy was performed. The main lesion was a nonsuppurative meningoencephalitis with neuronal degeneration and few eosinophilic cytoplasmic inclusion bodies characteristic of a viral disease. Lungs showed suppurative and interstitial pneumonia. Paraffin-embedded sections of brain were examined for morbillivirus antigen by using an immunoperoxidase technique. We used various monoclonal antibodies that recognize different morbilliviruses. Tissues from a seal infected with phocine distemper virus and a dog with canine distemper were used as positive controls. Tissues from a white-beaked dolphin that underwent an autopsy in 2006 were used as negative controls. In the diseased dolphin, morbillivirus antigen was found exclusively in neurons and glial cells of the brain , panel BHistologic changes in the dolphin resembled those of distemper in seals (Our findings indicate that DMV is still circulating in some marine mammals. Similar to infections in terrestrial hosts, morbillivirus infections may occur in marine mammals in cycles without overt clinical disease in susceptible animals, as documented for harbor seals (White-beaked dolphins are found in moderate and subarctic waters of the Atlantic Ocean between the eastern coast of North America and northern Europe. They may migrate hundreds of kilometers within days. Therefore, these dolphins may play a role as a reservoir and vector for this morbillivirus, which is infectious for harbor porpoises, bottlenose dolphins, and other cetacean species (
Branchiostoma floridae, which diverged from other chordates just after duplication of the ancestral SR. The B. floridae genome contains two SRs: BfER, an ortholog of the vertebrate estrogen receptors, and BfSR, an ortholog of the vertebrate receptors for androgens, progestins, and corticosteroids. BfSR is specifically activated by estrogens and recognizes estrogen response elements (EREs) in DNA; BfER does not activate transcription in response to steroid hormones but binds EREs, where it competitively represses BfSR. The two genes are partially coexpressed, particularly in ovary and testis, suggesting an ancient role in germ cell development. These results corroborate previous findings that the ancestral steroid receptor was estrogen-sensitive and indicate that, after duplication, BfSR retained the ancestral function, while BfER evolved the capacity to negatively regulate BfSR. Either of two historical mutations that occurred during BfER evolution is sufficient to generate a competitive repressor. Our findings suggest that after duplication of genes whose functions depend on specific molecular interactions, high-probability degenerative mutations can yield novel functions, which are then exposed to positive or negative selection; in either case, the probability of neofunctionalization relative to gene loss is increased compared to existing models.Gene duplication is the predominant mechanism for the evolution of new genes. Major existing models of this process assume that duplicate genes are redundant; degenerative mutations in one copy can therefore accumulate close to neutrally, usually leading to loss from the genome. When gene products dimerize or interact with other molecules for their functions, however, degenerative mutations in one copy may produce repressor alleles that inhibit the function of the other and are therefore exposed to selection. Here, we describe the evolution of a duplicate repressor by simple degenerative mutations in the steroid hormone receptors (SRs), a biologically crucial vertebrate gene family. We isolated and characterized the SRs of the cephalochordate B. floridae, a cephalochordate that diverged from vertebrates ∼500 million years ago, just after the ancestral SR duplicated. One retained the ancestral gene's estrogen receptor–like functions, while the other evolved a new function as a competitive repressor of the first. Either of two historical mutations is sufficient to recapitulate evolution of this function by disabling the receptor's response to estrogen, but leaving its DNA-binding capacity intact. Our results suggest that, for the many genes that function by specifically interacting with other molecules, simple mutations can yield novel functions that, beneficial or deleterious, are exposed to selection.Most genes evolved by duplication of more ancient genes. Under existing models of this process, mutations that compromise one copy have no effect on the other; as long as one copy remains intact, such “degenerative” mutations are shielded from selection. Because degenerative mutations are common, most duplicates are expected to be disabled before new functions can evolve. The great functional diversity of genes is therefore somewhat puzzling. Here, we reconstruct how simple degenerative mutations produced a new function in the steroid hormone receptors (SRs), a gene family crucial to reproduction and development. We characterized the two SRs of The vast majority of genes in eukaryotic genomes are hierarchically organized in gene families and superfamilies, because they were generated by a serial process of gene duplication and divergence All major models of duplicate gene evolution to date assume that the two genes' products do not interact with each other physically or functionally. Mutations in one copy therefore have no effect on the functions of the other.In the classic model In the second model–Duplication, Degeneration and Complementation (DDC) In the third model, an increase in the dose of a gene's product due to duplication yields an immediate selective advantage; thereafter, purifying selection purges degenerative mutations and conserves the ancestral function in both copiesGiven the high probability of degenerative mutations relative to gain-of-function mutations, it remains unclear how large numbers of duplicate genes have evaded nonfunctionalization long enough to evolve new functions. A key omission from the major models is their assumption that the products of duplicate genes do not interact, either through direct physical contact or by competing for other molecular partners. Gene duplication is recognized as providing raw material for the evolution of elaborate molecular interaction networks But molecular interactions may affect the fate of gene duplicates much more directly, and two types are of particular interest . First, Steroid hormone receptors (SRs) exemplify both types of functional interaction described above The SR gene family evolved by duplication and acquisition of novel functions from a single ancestral SR gene (AncSR1). AncSR1 is as ancient as the protostome-deuterostome divergence, based on discovery of a steroid receptor gene in mollusks Strongylocentrotus purpuratus and the urochordate Ciona intestinalis, but all SRs were lost completely in these lineages The evolutionary events by which this neofunctionalization process occurred are obscure. It was complete by the time of the vertebrate ancestor, some 470 million years ago, because agnathans, the most basal vertebrate taxon, contain one ortholog each for the ERα/ERβ, the GR/MR, and the AR/PR pairs of jawed vertebrates Branchiostoma floridae contains the set of cytochrome P450 (CYP) genes required for the biosynthesis of the sex steroids testosterone, progesterone, and estradiol, and the synthesis of these hormones in B. floridae has been experimentally established B. floridae, characterize their functions, and analyze their evolution.Cephalochordates therefore have the potential to provide key information about the early evolution of the kSR gene after duplication of AncSR1 B. floridae. Coding sequences of both genes were determined using RACE (rapid amplification of cDNA ends) on RNA extracted from B. floridae adults, and full-length coding sequences were then isolated using the polymerase chain reaction. One of the B. floridae receptors has high amino acid identity to the human ERs, particularly in the DNA-binding domain, and much lower similarity to the AR, PR, GR, and MR. The other has approximately equal similarity to the ERs and the other SRs superfamily have evolved by partial degeneration to function primarily as repressors of the transcriptional activity of paralogous NRs. Some have lost their capacity to activate transcription but retain their ability to bind DNA, so–like BfER–they compete for the binding sites of other receptors, whose activity they downregulate. Others have lost the capacity to bind DNA but retain the ability to form dimers with other NRs and thus inhibit their activity ArabidopsisParamecium genomes are under strong purifying selection As for the cases in which degenerative mutations are deleterious, it is not possible to directly determine the historical importance of selection against duplicate repressors in shaping genome evolution, but there are reasons to believe it may have played a significant role in delaying nonfunctionalization. Most protein families–enzymes, transcription factors, hormones, neurotransmitters, growth factors, to name a few–depend upon specific molecular interactions for their functions. In many of these families, separate domains, independent molecular surfaces, or even specific sets of residues mediate interactions with different partners or other aspects of function. It is therefore reasonable to expect that after duplication a large class of mutations would compromise specific aspects of function without abolishing all interactions, thereby producing competitive repressors. It is also likely that a nontrivial fraction of these alleles would be deleterious. If these assumptions are correct, then purifying selection would have played a role of general importance in purging degenerative mutations after gene duplication, maintaining duplicates for longer periods of time, and extending the temporal window during which neofunctionalization can occur. Indeed, it has been observed that duplicate genes involved in signal transduction have been preferentially retained in Not all degenerative mutations in genes whose functions depend on interactions are likely to produce repressor alleles. Those that radically reduce expression, impair protein folding or stability, or increase the tendency for a protein to aggregate are expected to yield alleles compromised in their ability to interact with any and all partners. These fully nonfunctional alleles would have no effect on their duplicate's function and would be sheltered by redundancy as predicted by the classic and DDC models' neutral scenarios. Only those mutations that affect modular domains or molecular surfaces that control distinct subfunctions within the coding sequence have the potential to eliminate one aspect of a protein's functions without abolishing its interactions with at least some molecular partners. It is therefore likely that purifying selection would be partially relaxed after duplication–in contrast to the situation for unduplicated genes, in which all mutations that compromise function, including those that also generate novel functions, would be exposed to the constraining influence of selection. Ohno's idea that functional diversity can evolve by random exploration of sequence space after gene duplication may strain credulity in its original conception of a purely neutral setting and an inexorable tendency toward “entropic decay.”Branchiostoma floridae genome database by tblastn search using as queries exons from the conserved DBD and LBD of human steroid receptors, as well as the inferred sequence of the ancestral steroid receptor B. floridae sequences were used as queries to reciprocally search the human protein database using blastx, and those that recovered SR family members as best hits were retained. Using this technique, two B. floridae sequences were identified as likely SR orthologs.Steroid receptor orthologs were identified in the From the two partial SR sequences recovered, primers were designed for RACE (Rapid Amplification of cDNA Ends). Amphioxus RNA was extracted from gravid individuals collected in October 2003 using the RNeasy kit . The isolated RNA was reverse transcribed using Thermoscript and oligo dT primers or PowerScript reverse transcriptase with gene specific primers. Both 5′ and 3′ sequences were obtained by RACE using the SmartRace method (Clontech) and Phusion polymerase . Products were cloned into TOPO TA cloning vector (Invitrogen) and multiple clones were sequenced (accessions EU371730 and 371729). Numerous synonymous polymorphisms were found in both receptor genes.B. floridae estrogen related receptor ERR . To classify human proteins into paralogous groups, we used a single-linkage clustering algorithm based on reciprocal best hits in a BLASTp search of each protein in the human genome against all proteins in the human genome. To identify orthology relationships, BLASTp searches were performed between each human protein sequence and all proteins in the B. floridae genome, and between each B. floridae protein and all proteins in the human genome. Orthology relationships were inferred for reciprocal best hits between a human paralog group and a B. floridae protein. If multiple amphioxus proteins were reciprocal best hits for a human paralog group, the paralog group was split accordingly. A sliding window analysis was then performed between each human chromosome and all amphioxus scaffolds to group orthologs into conserved syntenic regions. A sliding window size of 100 genes was used. B. floridae scaffold 42 (100% of scaffold length), and 6 megabases of human chromosome 6 (3.5% of chromosome length). Non-orthologous genes that fall between regions of conserved synteny are not shown.Synteny relationships were investigated using the human genome and the Full-length amphioxus SR cDNAs were amplified using specific forward and reverse primers designed at start and stop codons and were cloned into the mammalian expression vector pcDNA3 . Fusion constructs were prepared by amplifying the DBD or the LBD and subcloning them into pCMV-AD or pSG5-DBD (gift of D. Furlow), respectively. Site-directed mutagenesis was performed using QuickChange II . All clones were verified by sequencing.Reporter gene transcription assays were performed in Chinese Hamster Ovary (CHOK-1) cells grown to 90% confluence then harvested with trypsin (Invitrogen) and transferred to a 96-well plate containing phenol red-free αMEM supplemented with 10% dextran-charcoal-stripped fetal bovine serum and no antibiotics . For LBD assays, cells were transfected using lipofectamine and Plus reagent (Invitrogen) with 1 ng receptor plasmid, 100 ng pFRluc reporter plasmid , and 0.1 ng pRLtk reporter plasmid for normalization in Optimem (Invitrogen). For DBD assay, cells were transfected in Optimem with 4 ng receptor plasmid, 2 ng of reporter pGL3-4(EREc38)-luc or SRE-luc , 0.1 ng normalization reporter pRLtk (Promega), and 95 ng of pUC19 DNA as filler for transfection efficiency. Four hours later, the transfection mixture was removed and replaced with antibiotic-free αMEM with 10% fetal bovine serum; cells were incubated overnight. LBD assays were then incubated with hormones or vehicle control in triplicate at each dose for an additional 24 hours. Cells were lysed and assayed for luminescence using Dual-Glo Luciferase System (Promega) on a Perkin-Elmer Victor3 plate reader. To calculate normalized luciferase activity, firefly luciferase luminescence was divided by Renilla luciferase luminescence. Dose-response relationships were calculated using nonlinear regression using Prism software .−6 M. Co-transfection of BfER and BfSR full-length plasmids was performed using identical conditions to individual full-length transcriptional assays except that cells were transfected with varying concentrations of each receptor , and treated with estradiol at 10−6 M. In screens of full-length receptor sensitivity to a broad panel of hormones, 5 ng receptor and 50 ng reporter were transfected. All experiments were done in triplicate and repeated at least twice with the same results.Transcriptional activity of full-length receptors was assayed by transfecting CHO-K1 cells using reporter plasmids pGL3-4(EREc38)-luc or SRE-luc. Full length human GR in pcDNA3 (gift from B. Darimont) was used as the positive control on SREluc and treated with cortisol at a concentration of 10CHO-K1 cells were transfected with plasmids containing full-length BfER alone (4 µg), BfER (1 µg) and BfSR (4 µg), or BfSR alone (4 µg) as described above, treated with 1 µM estradiol for 4 hours and harvested in TEGDK buffer glycerol, 1 mM dithiothreitol) with 1% protease inhibitor cocktail . Cells were lysed with four freeze-thaw cycles and centrifuged at 10,000×g for 20 min at 4 C. Protein was quantitated using the Bradford protein assay . Protein from BfER-transfected cells (2 µg), BfER+BFSR-transfected cells (5 µg), or BfSR-transfected cells (10 µg) was prepared and incubated with 10 ng biotinylated ERE- probe according to the manufacturer's protocol . Reactions were separated on a 5% native polyacrylamide gel in 1× tris-borate EDTA buffer for 3 hours at 90 V at 4 C. The long run was required to separate the sizes of BfER/ERE and BfSR/ERE complexes; in this time, unbound labelled probe migrated off the end of the gel. Gel contents were transferred to Biodyne B membrane for 30 min at 300 mA. Chemiluminescent detection of biotinylated DNA was performed using the Panomics EMSA kit.vasa was used as a positive control and to identify putative germ cells; vasa was identified in the B. floridae genome database using the human sequence as a query and amplified as described below. B. floridae cDNA fragments of BfER, BfSR, and BfVasa were amplified using the following primers: er-forward 5′- GCTAGTGCCTTTGACAAGTC -3′ and er-reverse 5′- CAGACACCTGGTCAGTGAG -3′ (corresponding to nucleotides 661–1501 of the BfER open reading frame), sr-forward 5′- AACTCATAGTGAGCCCCACC -3′ and sr-reverse5′- CTGCAGAGTTCTCCACTGC -3′ (nucleotides 875–1724), and vasa-forward 5′- GAGCCAACGTGGCGAAGG -3′ and vasa-reverse 5′- CATCAGGGCCTCCTCTCTC -3′ (1–849 nt). The Vasa sequence has been deposited with accession EU371731. Amplicons were cloned into pCR4-TOPO vector (Invitrogen) and used to synthesize digoxigenin-labeled riboprobes (Boehringer-Mannheim). In situ hybridizations on cryo-sections were performed as described previously [101]. In situ hybridizations with different probes were performed on adjacent sections in alternate slides to facilitate comparison among the expression patterns of different genes. Negative control hybridizations were performed without riboprobe to identify any endogenous alkaline phosphatase activity.Gravid amphioxuswere collected in March, 2007 and fixed in 4% paraformaldehyde, 0.5 M NaCl, 1 mM MgSO4, 2 mM EGTA and 0.1 M MOPS. Fixed animals were dehydrated in a stepwise series of PBS∶methanol and stored in methanol at −20°C. Samples were re-hydrated in PBS, embedded in agar, and cross-sectioned in a cryostat at 16 mm. The germ-cell marker Figure S1Amino acid sequences of steroid receptor DNA-binding domains.(0.31 MB PDF)Click here for additional data file.Figure S2Amino acid sequences of steroid receptor ligand-binding domains.(0.25 MB PDF)Click here for additional data file.Figure S3Mixture-model maximum likelihood phylogenetic analysis.(0.14 MB PDF)Click here for additional data file.Figure S4Synteny analysis of BfER- and BfSR-containing scaffolds with human chromosomes.(0.12 MB PDF)Click here for additional data file.Figure S5Reporter expression assay of full-length BfER and BfSR.(0.24 MB PDF)Click here for additional data file.Figure S6Complete results of maximum likelihood phylogenetic analysis with likelihood ratio statistics.(0.48 MB PDF)Click here for additional data file.Figure S7Complete results of maximum likelihood phylogenetic analysis with chi-square support statistics.(0.41 MB PDF)Click here for additional data file.Figure S8Complete results of Bayesian phylogenetic analysis.(0.40 MB PDF)Click here for additional data file.Table S1Gene names, species, and accession numbers of sequences used in phylogenetic analyses.(0.03 MB PDF)Click here for additional data file.
The skull base surgery is one of the most demanding surgeries. There are different structures that can be injured easily, by operating in the skull base. It is very important for the neurosurgeon to choose the right approach in order to reach the lesion without harming the other intact structures. Due to the pioneering work of Cushing, Hirsch, Yasargil, Krause, Dandy and other dedicated neurosurgeons, it is possible to address the tumor and other lesions in the anterior, the mid-line and the posterior cranial base. With the transsphenoidal, the frontolateral, the pterional and the lateral suboccipital approach nearly every region of the skull base is exposable.In the current state many different skull base approaches are described for various neurosurgical diseases during the last 20 years. The selection of an approach may differ from country to country, e.g., in the United States orbitozygomaticotomy for special lesions of the anterior skull base or petrosectomy for clivus meningiomas, are found more frequently than in Europe.The reason for writing the review was the question: Are there keyhole approaches with which someone can deal with a vast variety of lesions in the neurosurgical field?In my opinion the different surgical approaches mentioned above cover almost 95% of all skull base tumors and lesions. In the following text these approaches will be described.These approaches are:1) pterional approach2) frontolateral approach3) transsphenoidal approach4) suboccipital lateral approachThese approaches can be extended and combined with each other. In the following we want to enhance this philosophy. In the 1970 s Yasargil laid to There are many variants for the pterional approach. Mainly it is a trepanation which permits access to the frontal and to the temporal lobe as well as the Sylvian fissure Figure . After aYasargil also described a technique that allows access to the same area: his skin incision is done until the temporal fascia is reached. He then creates a triangular galea flap and seperates the muscle after additional horizontal incision in order to protect the frontal branch of the facial Nerve. In our opinion the rate of nerve injury is minimal also if one is using the first technique. The advantage of the Yasargil Technique can be seen if one wants to combine the pterional approach with the subfrontal approach. The muscle can be retracted much easier downwards with this technique. In my opinion the rate of atrophic changes of the temporal muscle postoperatively is a little bit higher. We are using Palacaos to fill the burr holes. Other authors are using selected bone dust.A 51-year-old man underwent an operation for a craniopharyngioma, through a pterional approach from the right side six months before. He was presented with a pituitary infarction. He has been treated with Testosterone (Testogel), Desmopressin (Minirin) and Hydrocortisone. He suffered from a progressive visual disturbance for 8 weeks. A bitemporal hemianopsia was diagnosed.We diagnosed a recurrence of the craniopharyngioma Figure . Besides64-year-old woman presented with unknown symptoms of vertigo and diplopia. Further there were no clinical deficits. The MRI showed a left space-consuming lesion latero para- and retrosellar of approximately 2 × 2,5 cm size Figure .There were normal findings post-operatively with occasional diplopia, while the wound was healing per primam. Pathology revealed a meningothelial tumor of WHO grade I.66-year-old patient, presented with a history of six seizures, suffers from a known retrochiasmal lesion with a growth in the MRI examinations.We addressed the tumor via the pterional approach from the right side Figure . The chiThere were no neurological deficits and the healing of the wound was by first intention. In contrast the patient showed fluctuating vigilambulism with nausea, vertigo and electrolyte imbalance. The patient was prescribed a long-term hydrocortisone substitution and Desmopressin (Minirin) for a short period of time.The frontolateral or unilateral subfrontal approach provides exposure of the anterior cranial base. It allows addressing olfactory groove meningiomas (OGMs) by a minimally invasive procedure and it is also one of the traditionally used approaches on OGMs ,10. FranThis approach allows different skin incisions, which depend on the patient's anatomy and physiognomy. The osteoplastic trepanation above the pterion and above the temporal muscle follows a curved skin incision or an eyebrow skin incision Figure . The treA 47-year-old patient with bitemporal hemianopsia and a 6-weeks history of progredient headache was presented. The patient manifested no further neurological deficits.Neuropathological findings revealed parts of an epithlial cyst inflammatory cell infiltration in connective tissue stroma. The MRI showed a cystic lesion above the pituitary gland Figure . We perfThe further course was without any complications and the wound healing was per primam.Visual field defects were on the decrease. Furthermore, we couldn't recognize any hormonal failure.The transsphenoidal route was first used by the Egyptians in order to remove the brain. The pioneering work of Harvey Cushing and OskaThe classic technique begins with the patient in supine position. An x-ray machine is positioned laterally to control each step of intervention. In microsurgery the surgeon stands at the tip of the head whereas in pure endoscopic pituitary surgery the neurosurgeon stands at the shoulder of the patient. We would like to concentrate on the microsurgical approach in our review article. After incising the septal mucosa Figure and reveThe posterior cranial base can be explored through the lateral suboccipital route. Processes in the posterior fossa such as acoustic neurinoma surgery can be carried out through the lateral suboccipital approach. The unilateral suboccipital approach was popularized by Woolsey (1903) and with great contributions by Krause (1905) ,22. AfteWe normally use a semi-sitting position for the suboccipital lateral route Figure . The opeAttention should be paid to the variable emissary veins, which lead to the sigmoid sinus. First the dura should be opened under microscopic magnification in a triangular shape in the region of the pars horizontalis to gain cerebrospinal fluid and in order to relax the cerebellum.Then the dura has to be opened near the sinus via a curved skin incision that connects with the previous dural opening.By applying the transsphenoidal, the unilateral subfrontal, pterional and lateral suboccipital approach, the surgeon can expose almost any skull base involvement. The diameters of exposure can be modified by combining different approaches. The introduction of microsurgical techniques in the 1960 s ,32 is poComplex pathologies at the skull base may need tailored approaches, which mainly have the base on one of the mentioned techniques. Extension of approaches can also be done in an interdisciplinary fashion with colleagues from other faculties like ENT or maxillofacical surgery, which is recommended by the author. While ENT can help accessing the skull base, e.g., in acoustic neuroma surgery or by thThe authors declare that they have no competing interests.MS obtained clinical photos, coordinated, and drafted the manuscript. RP drafted the manuscript and participated in its coordination and design. JT, CL, AH and KB provided critical review of the manuscript for important intellectual content. All authors read and approved the final manuscript.A written informed consent was obtained from the patients for publication of the case reports and accompanying images. Copies of the written consents are available for review by the Editor-in-Chief of this journal.
The aim of the present study was to investigate the impact of the time interval from collapse to return of spontaneous circulation (CPA-ROSC) in cardiac arrest patients and the types of patients who will benefit from therapeutic hypothermia.Four hundred witnessed adult comatose survivors of out-of-hospital cardiac arrest of cardiac etiology were enrolled in the study. The favorable neurological outcome was defined as category 1 or 2 on the five-point Pittsburgh cerebral performance scale at the time of hospital discharge. A matching process based on the propensity score was performed to equalize potential prognostic factors in the hypothermia and normothermia groups, and to formulate a balanced 1:1 matched cohort study.P < 0.05) in the hypothermia group (n = 110) than in the normothermia group in patients with CPA-ROSC of 15 to 20 minutes (64% vs. 17%), 20 to 25 minutes (70% vs. 8%), 25 to 30 minutes (50% vs. 7%), 35 to 40 minutes (27% vs. 0%) and 40 to 45 minutes (29% vs. 2%). A similar association was observed in a propensity-matched cohort, but the differences were not significant. There was no significant difference in the rate of favorable neurological outcome between the hypothermia-matched group and the normothermia-matched group. In the patients whose CPA-ROSC was greater than 15 minutes, however, the rate of favorable neurological outcome was higher in the hypothermia-matched group than in the normothermia-matched group . In multivariate analysis, the CPA-ROSC was an independent predictor of favorable neurological outcome .The rate of favorable neurological outcome was higher (The CPA-ROSC is an independent predictor of neurological outcome. Therapeutic hypothermia is more beneficial in comatose survivors of cardiac arrest with CPA-ROSC greater than 15 minutes. Cardiac arrest has a poor prognosis and is a major cause of unexpected death in developed countries. Despite cardiopulmonary resuscitation (CPR), only a few patients fully resume their former lifestyle, mainly because of anoxic brain injury ,2. Mild The time interval from collapse to return of spontaneous circulation (ROSC) has been reported to be a strong independent predictor of neurological outcome in comatose survivors of cardiac arrest ,10-14. WWe retrospectively enrolled witnessed adult (>18 years of age) OHCA patients with cardiac causes transported to Hiroshima City Hospital, who achieved ROSC and who were comatose between September 2003 and January 2010. All OHCA patients were treated in accordance with an advanced cardiac life support protocol, and the patients who met the criteria for hypothermia treatment were treated with MTH as reported previously : age 18 Before 2006, an assessment of cardiac arrest complicated by ischemic heart disease was made in patients treated with and without MTH. In patients with suspected acute coronary syndrome, emergency coronary angiography or percutaneous coronary intervention, or both, were subsequently performed. After 2006, routine emergency coronary angiography was performed in patients treated with MTH.The present study was approved by the local ethics committee on human research and is conducted in accordance with the guidelines of the Declaration of Helsinki. All data were collected within the normal daily care routine in an anonymous fashion. The institutional review board therefore waived the need for informed patient consent.MTH was induced in comatose survivors using a surface cooling mattress and the administration of physiological saline (4°C) as reported previously . Before The primary endpoint was a favorable neurological outcome, which is defined as category 1 (good performance) or category 2 (moderate disability) on the five-point Pittsburgh cerebral performance scale; the other categories are 3 (severe disability), 4 (vegetative state), and 5 (death) at the time of hospital discharge .Continuous variables are presented as medians (with interquartile ranges), and categorical variables are presented as numbers and percentages. Differences between groups at baseline were analyzed using the Mann-Whitney U test for continuous variables and a chi-square test or Fisher's exact test for categorical variables as appropriate. The rate of favorable neurological outcome was plotted against the CPA-ROSC every 5 minutes for patients treated with and without MTH. We used Fisher's exact test to access differences in the rate of favorable neurological outcomes every 5 minutes between the groups.To detect favorable neurological outcome, we constructed receiver-operating characteristic curves for the CPA-ROSC.The threshold for performing MTH was set high early in the study period. For example, we induced MTH only in comatose survivors whose initial rhythm indicated ventricular fibrillation. Because the patients were not randomly assigned to receive MTH or normothermia, potential confounding and selection biases were accounted for by developing a propensity score. The propensity for MTH or not was determined without regard to the outcome using a multivariate regression model. For this model, we chose variables we thought might increase the propensity for MTH over normothermia therapy, which included age, Pittsburgh overall performance category score before cardiac arrest, initial rhythm, the time interval from collapse to start of CPR and the CPA-ROSC. This model yielded a concordance statistic of 0.80, which indicated good discrimination. A propensity score was then calculated from the logistic equation for each patient, which indicated the probability that a patient would be treated with MTH. All study patients were pooled and sorted according to their propensity scores in the ascending order and a propensity-matched cohort was formed .A logistic regression model was used to examine the association between the CPA-ROSC and favorable neurological outcome in an unadjusted model, in a model adjusted for age, gender, and initial rhythm, and in a model adjusted for age, gender, Pittsburgh overall performance category score, initial rhythm, time interval from collapse to start of CPR, hypertension, diabetes mellitus, history of heart disease, emergency coronary angiography, primary percutaneous coronary intervention, use of intra-aortic balloon pump, use of extracorporeal life support, admission after 2006 and propensity score.The JMP statistical package and R version 2.9.2 were usen = 400) with cardiac causes were enrolled in the study. The baseline clinical characteristics, treatment and findings for study patients are shown in Tables A flow diagram of the study patients and their outcomes is depicted in Figure P < 0.001) and 30-day survival were higher in the hypothermia group than in the normothermia group. The CPA-ROSC in patients with a favorable neurological outcome was longer (P < 0.001) in the hypothermia group than in the normothermia group .The outcomes of the study patients are shown in Table P < 0.01), 20 to 25 minutes , 25 to 30 minutes , 35 to 40 minutes and 40 to 45 minutes . There was a trend toward a higher rate of favorable neurological outcome in the hypothermia group than in the normothermia group in patients in whom the CPA-ROSC was 30 to 35 minutes . The rate of favorable neurological outcome was similar in patients with a CPA-ROSC less than 15 minutes.The rate of favorable neurological outcome was plotted against the CPA-ROSC every 5 minutes Figure . In the Receiver-operating characteristic curves of the CPA-ROSC to detect favorable neurological outcome are shown in Figure P = 0.94; Figure P < 0.001) in patients in the hypothermia-M group than those in the normothermia-M group .The propensity score-matching process selected 79 patients from the hypothermia group (hypothermia-M group) and 79 patients from the normothermia group (normothermia-M group). The mean ± standard deviation propensity score was 0.41 ± 0.22 in the hypothermia-M group and was 0.41 ± 0.22 in the normothermia-M group (P = 0.86). In patients whose CPA-ROSC was more than 15 minutes, however, the rate of favorable neurological outcome was higher in the hypothermia-M group than in the normothermia-M group .The rate of favorable neurological outcome was plotted against the CPA-ROSC every 5 minutes in the propensity-matched cohort Figure was obseP < 0.001). After adjustment for age, gender, and initial rhythm, this association was still significant . Finally, after adjustment for all of the above-mentioned variables, the association between the CPA-ROSC and favorable neurological outcome remained significant .In the full cohort, we tested the association between the CPA-ROSC and favorable neurological outcome in multiple models. In an unadjusted model, there was a significant association between the CPA-ROSC and favorable neurological outcome (every 1 minute: odds ratio = 0.90, 95% CI = 0.88 to 0.92, We showed that the CPA-ROSC is a strong independent predictor of favorable neurological outcome in the present MTH era, and that MTH prolongs the maximum CPA-ROSC to obtain a favorable neurological outcome. MTH is more beneficial in OHCA patients with a CPA-ROSC longer than 15 minutes in terms of neurological outcome.As there is not enough blood flow to maintain the metabolism of organs in cardiac arrest patients, despite chest compression, the organs experience ischemic damage from the time of collapse to ROSC . FurtherThe International Liaison Committee on Resuscitation has issued guidelines for treatment with MTH . BecauseIn our study, the CPA-ROSC of 65 minutes in the hypothermia group and of 45 minutes in the normothermia group had a negative predictive value of 100%. These values may be the present points of no return for CPR in the present clinical settings. Our study showed that the shorter the CPA-ROSC, the higher the ratio of favorable neurological outcome. Cardiac arrest patients should be treated in a manner that achieves ROSC as soon as possible to obtain a better outcome.Our study was not double-blind or randomized and had the inherent limitations of any single-centre retrospective investigation. The present study was prone to biases related to unmeasured factors. We did, however, use multivariate and propensity analyses to carefully match patients in an effort to eliminate bias. It was difficult for us to estimate the time of collapse of cardiac arrest patients correctly, so we enrolled only the witnessed cardiac arrest patients in this study. CPR guidelines were changed during the study period, which might affect the outcomes. We attempted to adjust for this effect with the additional covariate of admission after 2006 . AlthougThe CPA-ROSC is an independent predictor of neurological outcome in comatose survivors of OHCA. A CPA-ROSC longer than 15 minutes in the normothermia therapy group and longer than 30 minutes in the hypothermia therapy group was associated with low rates of favorable neurological outcome. MTH prolongs the CPA-ROSC, which may help comatose survivors of cardiac arrest obtain favorable neurological outcomes, and is more beneficial in patients whose CPA-ROSC is greater than 15 minutes.• MTH is more beneficial in patients whose CPA-ROSC is greater than 15 minutes.• Neurological outcome in patients treated with normothermia and hypothermia were similar in patients with a CPA-ROSC less than 15 minutes.• The CPA-ROSC is an independent predictor of neurological outcome in comatose survivors of cardiac arrest.• A CPA-ROSC greater than 15 minutes in the normothermia therapy group and of 30 minutes in the hypothermia therapy group was associated with low rates of favorable neurological outcome.CI: confidence interval; CPA-ROSC: time interval from collapse to return of spontaneous circulation; CPR: cardiopulmonary resuscitation; MTH: mild therapeutic hypothermia; OHCA: out-of-hospital cardiac arrest; ROSC: return of spontaneous circulation.The authors declare that they have no competing interests.All authors participated in the design and coordination of the study and draft of the manuscript. All authors read and approved the final manuscript.
Patients with diabetic polyneuropathy (DPN) are often confronted with ulceration of foot soles. Increased plantar pressure under the forefoot has been identified as a major risk factor for ulceration. This study sets out to test the hypothesis that changes in gait characteristics induced by DPN related muscle weakness are the origin of the elevated plantar pressures.Three groups of subjects participated: people diagnosed with diabetes without polyneuropathy (DC), people diagnosed with diabetic polyneuropathy (DPN) and healthy, age-matched controls (HC). In all subjects isometric strength of plantar and dorsal flexors was assessed. Moreover, joint moments at ankle, knee and hip joints were determined while walking barefoot at a velocity of 1.4 m/s. Simultaneously plantar pressure patterns were measured.Compared to HC-subjects, DPN-participants walked with a significantly increased internal plantar flexor moment at the first half of the stance phase. Also in DPN-subjects the maximal braking and propelling force applied to the floor was decreased. Moreover, in DPN-subjects the ratio of forefoot-to-rear foot plantar pressures was increased. Body-mass normalized strength of dorsal flexors showed a trend to be reduced in people with diabetes, both DC and DPN, compared to HC-subjects. Plantar flexors tended to be less weak in DC compared to HC and in DPN relative to DC.The results of this study suggest that adverse plantar pressure patterns are associated with redistribution of joint moments, and a consequent reduced capacity to control forward velocity at heel strike. Diabetic foot ulceration is one of the major complications of diabetes, leading to high morbidity, poor quality of life and high costs . The patMuscle weakness -10 and aIn normal walking, the centre of pressure (CoP) under the foot transfers gradually from the heel to the forefoot. Several authors found that in diabetic patients with neuropathy the pressures under the forefoot are relatively increased compared to those under the heel ,21. HoweEight diabetic patients with polyneuropathy (DPN), ten diabetic controls without neuropathy (DC) and ten healthy, age-matched controls (HC) participated Table . ClinicaSubjects visited the laboratory twice, for a dynamometer test and for a gait test. To estimate whether subjects could safely participate in the tests, blood glucose concentration was measured before and after each measurement.To determine strength of plantar and dorsal flexors of the right leg, subjects performed in a dynamometer maximal, isometric, voluntary contractions at 25 combinations of knee and ankle joint angles. Each contraction lasted about 3 seconds. To prevent fatiguing, three minutes of rest were allowed between subsequent contractions. Contractions were performed at five knee and five ankle joint angles that covered the whole range of joint motion of each individual. At each combination of knee and ankle joint angles one contraction was performed. The range of joint motion at knee and ankle did not differ between subject groups.As a measure of plantar sensitivity the vibration perception threshold >25 V was determined on the hallux .2; Novel GmbH, Munich, Germany). The pressure platform, which was flush with the walkway surface, was placed on top of the force platform. Since gait velocity influences plantar pressures [For gait analysis markers were attached to the skin over the head of the fifth metatarsal bone, the heel of the foot, the lateral malleolus, the epicondyle of the femur and the greater trochanter of the right leg. A 2D, 50 Hz video system registered the marker positions in the sagittal plane. The spatial resolution of the video system was 3.85 mm/pixel; spatial accuracy of marker position is approximately 25% of this: 1 mm. A pair of electromyography (EMG) electrodes was placed halfway on the line connecting the greater trochanter and the sacrum, over the belly of gluteus maximus to record the activation of the underlying muscle tissue. Subjects walked barefoot across a 12-meter walkway with halfway an embedded force plate and a pressure platform and theGait velocity was calculated as the average horizontal velocity of the greater trochanter marker. Stride time was determined as the time between the onsets of two consecutive EMG-bursts of the gluteus maximus.The leg was modeled as three rigid segments . Position data of the markers were used to determine the position of segments, and subsequently -by differentiation- (angular) velocity and acceleration.During walking, movement is caused by moments of force generated by muscles around ankle, knee and hip joint Figure . Based oOnly when the GRF was >300 N, the point of application of the GRF could be measured validly. Consequently, inverse dynamics could not be performed for the entire stance phase. In the graphical data presented, this results in graphs ranging typically between 10 and 90% of the stance phase.As we were especially interested in changes in gait pattern due to diabetic polyneuropathy and not due to differences in body mass, GRF data and joint moment curves were normalized for stance phase duration and body mass; average curves were calculated for each participant. For each curve the minimal and maximal values were determined. In addition, for the fore-aft component of GRF the braking and propulsive impulses were assessed. For the vertical component of GRF and the support moment curves, which both have a typical M-shape, the maximal values at both peaks and the minimal value between the peaks were determined. The ankle joint moment reached a plateau between approximately 30 and 50% of the stance phase; therefore, the value at 40% of the stance phase was also determined to evaluate the level of this plateau. Finally, the area under the ankle joint moment curve was calculated, which represents the joint impulse. This impulse is a measure for the amount of work generated around the ankle joint.Using a PRC-mask, peak plantar pressures and the plantar pressure time integrals were calculated for ten anatomical areas of the foot . PressurThe non-parametric Kruskal-Wallis test was applied to investigate whether muscle strength and gait performance variables differed between the groups. P = 0.05 was chosen as level of significance. If this test revealed a significant effect of the groups on a variable, Mann-Whitney U-tests were applied as a post hoc test. For the latter test the Bonferroni-correction was adapted to correct the level of significance; p-values smaller than 0.017 were considered significant.Body-mass-normalized dorsal flexor moment tended to be lower in both diabetic groups compared to healthy controls p = 0.128). The average normalized dorsal flexor moments were 0.57, 0.47 and 0.47 Nm/kg . For normalized plantar flexion strength a non-significant trend (p = 0.153) was observed: 1.07, 0.83 and 0.58 Nm/kg in the HC, DC and DPN patients, respectively . In addition, no differences were observed in cadence or stride length (data not shown). The self-selected velocity was for all subjects lower than the imposed velocity. The ratio of the imposed velocity over the self-selected velocity was larger in diabetic polyneuropathy participants than in healthy and diabetic controls , indicating that the DPN participants had to walk relatively fast in the test condition.The body-mass-normalized braking force differed between groups (p = 0.041); Figure In DPN-subjects the plantar flexion moment during the first half of the stance phase: at 40% of the stance phase was approximately 30% higher than in HC subjects than DC and HC participants. Simultaneously, a relative forward shift of the pressure under the foot was found. The question is now, is a redistribution of joint moments the cause of increased plantar pressures? It has been suggested that limited mobility at the ankle joint, claw/hammer toe deformity and increased maximal plantar flexor moments are causes of increased plantar pressure -40, but Interestingly, the changes in plantar flexion moment occurred during the first half of the stance phase. At a normal walking speed, the GRF is the major determinant of the external ankle joint moments figure . As illuThe stance phase of gait is characterized by subsequent braking and propulsive parts. Consequently, velocity is reduced in the first half of the stance phase in normal gait and increases again during the second half. As mentioned, the changes in joint moments occurred especially in the first half of the stance phase, suggesting that the changes in joint moments represent a limited ability to brake in the first half of the stance phase. This was confirmed by the significantly lower braking (and propelling) peaks of the horizontal component of the ground reaction force Figure . Meier ei.e. conductive velocity, or decreased proprioceptive or sensory information. In DPN-participants sensitivity was less compared to both other groups, as a result of this it can be assumed that proprioception in DPN will be disturbed and that consequently proper activation of muscles might be hampered. Also adaptations in more proximal muscles cannot be excluded as the origin of the adapted gait. In previous publications it has been suggested that disturbed gait performance is associated with delayed activity of dorsal flexors like tibialis anterior [i.c. vastus medialis [The other side of the cascade of relationships that we wanted to investigate relates to the question whether the redistribution of joint moments has been caused by muscle weakness. In this study only strength of lower leg muscles was measured; a non-significant decrease of plantar and dorsal flexor strength was found. Based on this it cannot be concluded that muscle weakness, operationalized as maximal strength, caused the adaptations in gait dynamics. Other factors that contribute to muscle function, like rate of force development and level of activation, might have contributed too. Changes in the activation of muscle might arrange from affected nerve function, anterior and kneemedialis .2 was used. If the sensor area is larger than the area of relevant pressure peaks, the real pressure will be underestimated. As shown among others by Cavanagh and Ulbrecht [With respect to the accuracy of the technical equipment the determination of the point of application of the GRF seems to be most critical. Its accuracy (8 mm) was considerably smaller than relevant differences in joint moment arms (2–3 cm) that were found between the groups. So this accuracy can be considered to be sufficient. To assess plantar pressure pattern a pressure plate with a resolution of two sensors/cmUlbrecht such a tIn conclusion, people with diabetic polyneuropathy had a higher forefoot-to-rear foot ratio of plantar pressures than diabetic and healthy controls. It was suggested that adapted plantar pressure patterns are associated to a redistribution of joint moments around the ankle joint during walking. A hypothesis has been presented relating muscle weakness and adapted joint moments to changes in plantar pressure patterns. In addition, a trend to relatively reduced lower leg muscle strength in people with diabetes was found. This might be one of the factors underlying adaptations in gait dynamics.The authors declare that they have no competing interests.HHCMS contributed to the conception and design of this study, also he took care of data analysis and initial interpretation of data and wrote the first draft of the manuscript. NCS, TLHdL and KM were involved in the conception of the study and in the critical revision of initial versions of this manuscript. PJBW lead the data-acquisition and set up analysis and data processing. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:
Different molecular therapies like the EGFR-inhibiting antibody cetuximab have come into clinical practice. Cetuximab is EMEA-approved for metastatic colorectal cancer and advanced squamous-cell head and neck cancer. Administration is said to be safe and well tolerated with common, usually mild dermatologic side effects.en-bloc oesophagectomy was performed. Few days later the patient died due to gas exchange dysfunction and circulation instability after a previously unseen combination of drain-erosion of the stomach with subsequent pleurisy and air leak of the left main bronchus.We present the case of a patient with fatal complications after oesophagectomy and neoadjuvant chemotherapy including cetuximab for squamous-cell esophageal cancer. A transthoracic So far we have never observed this fatal combination of drain erosion of the stomach with fibrinous pleurisy and unmanageable progressive tracheal defect before. The role of cetuximab in the multifactorial aetiology of damages of stomach and trachea after oesophagectomy remains unclear since we are not able to link the complication directly to cetuximab or definitely exclude it as a sole surgical complication. Clinicians should be aware of the possibility of fatal side effects and careful recording of all complications is necessary in ongoing and planned studies to obtain more evidence about safety and tolerance of targeted therapies. Oesophageal cancer represents the sixth leading cause of cancer-related death in the world. Despite recent advances in surgical critical care medicine and combined modality therapies 5-year overall survival rates 10–14%) are unsatisfactorily low 0–14% are. Next toIn the last decade different molecular therapies have changed the field of research, trying to inhibit or modulate targets of signal transduction pathways. One of those that made it into clinical practice is the epidermal growth-factor receptor (EGFR) inhibiting chimeric antibody cetuximab . This monoclonal antibody blocks EGF and TGF-α binding to the extracellular domain of EGFR, which results in cell-growth inhibition, induction of apoptosis and decreased production of EGF .Cetuximab is EMEA-approved for second line treatment of EGFR-expressing metastatic colorectal cancer refractory to irinotecan-based chemotherapy and locally advanced squamous-cell head and neck cancer with concomitant radiotherapy. Many solid tumors including esophageal cancer overexpress EGFR, predicting poor survival, poor response to therapy as well as higher probability for disease progression and resistance to therapy -10. ThisGenerally EGFR-antibodies or EGFR tyrosine kinase inhibitors are said to be safe and well tolerated without systemic side-effects of chemotherapy. Common dermatologic side effects of cetuximab in a considerable number of patients are acneiform eruptions, xerosis, eczema, fissures, teleangiectasia, hyperpigmentation, hair changes and paronychia . More seIn this report we present a case report with fatal postoperative complications after neoadjuvant chemotherapy including cetuximab for squamous-cell esophageal cancer and discussion of the literature.A 52-year old man was diagnosed with squamous-cell esophageal cancer of the lower third. Pretherapeutical investigations included endoscopical biopsy, CT scan, endosonography and mediastinoscopy with lymph-node biopsy. These investigations showed a locally advanced stage T4N1 cancer.2 and 5-FU 1000 mg per m2. After the first chemotherapy cycle the patient developed grade 3 mucositis and esophagitis combined with an infection of the port-a-cath system, which had to be removed. This intense toxicity gave us reason to search for a dihydropyrimidin-dehydrogenase-deficiency. The result of the genetic testing was negative. Because of the toxic esophagitis and mucositis the patient refused to undergo the planned radiotherapy. From the second cycle continous 5-FU was replaced by oral capecitabine because of the port-a-cath infection and cetuximab was added as an alternative to radiotherapy after informed consent in a compassionate use setting. The EGFR-testing had shown a strong overexpression in all tumor cells. The treatment consisted of an intravenous standard loading dose of 400 mg per m2 after administration of diphenhydramine and ranitidine and continued with 250 mg per m2 once weekly for four weeks. After five weeks the patient developed disseminated pustules with generalized deeply infiltrated erythematous plaques highly indicative for a severe acute generalized exanthematic pustulosis (AGEP) as shown in figure The patient was scheduled for two cycles of neoadjuvant radiochemotherapy with cisplatin 100 mg per men bloc oesophagectomy five weeks after the last cetuximab application. Replacement of the esophagus was performed with an orthotopic gastric tube and cervical esophago-gastrostomy. On the third postoperative day a leakage from the right thoracic drain was observed and the following immediate revision operation showed an erosion of the stomach, which was most likely caused by a thoracic drain positioned in the vicinity of the gastric tube. The anastomosis 3 cm above the defect was completely intact with macroscopic sufficient blood circulation. Because of an additional fibrinous pleurisy we had to conduct a disconnection esophagostomy, catheter-gastrostomy and -jejunostomy for early enteral feeding. After four days the patient developed an airleak of the left main bronchus just below the tracheal bifurcation. At the time of the first bronchoscopy we found a 5 mm ulcer where the tracheal tube cuff was located during the operation until extubation on the first postoperative day. Within four days the defect's size increased by fourfold as shown in figure Reevaluation showed nearly no remission of the tumor as well as stable disease in the suspect lymph nodes. Because of lack of response and the intense toxicity the patient wanted to stop the neoadjuvant therapy and proceeded to transthoracic en bloc transthoracic oesophagectomy with paraesophageal, paratracheal, aorto-pulmonary and paracardial lymphadenectomy including lymph nodes of the splenic, hepatic and celiac arteries followed by cervical esophagogastrostomy under general anesthesia with separate intubation. The operation time was 4.5 hours and the blood loss approximately 600 ml. The reoperation consisted of a disconnection esophagostomy, catheter-gastrostomy and -jejunostomy. The tracheal leakage was treated by a 12 French Dumont silicone stent.The patient's squamous-cell esophageal cancer of the lower third (35 – 38 cm from incisor teeth) was treated by Histologic tumor studies were done by haematology and eosin staining and EGFR-overexpression was measured by EGFR Pharm Dx kit K 1494 (Dako Cytomation).The oesophageal tumour was staged as ypT3 N1(9/25) MX R0 on final pathology. After reoperation the histologic examination of the resected specimen revealed extensive ischemic necrosis of the gastric mucosa with erosions and microabscesses, oedematous submucosa and the muscular and serosal layer next to the defect in good order.The autopsy showed massive fibrinous pleurisy, mediastinitis, left-sided bronchitis and bronchopneumonia in addition to the 2 cm tracheal defect of the membranous part of the left main bronchus. Histological complete tracheal wall destruction was observed with fibrin, necrotic areas and focal formation of granulation tissue without evidence of residual tumour figure .So far we have never observed this fatal combination of drain erosion of the stomach with fibrinous pleurisy and unmanageable progressive tracheal defect. Tracheal fistulae with airleaks are rare but life threatening complications after esophageal resections one to thirty days after oesophagectomy. The reported incidence of tracheal lesions is about 4 percent and about a third of the patients dies during the postoperative course, mostly because of unhealed lesions at the bifurcation or in the left main stem bronchus . IschemiPreoperative radiochemotherapy predisposed to this complication significantly and also in our patient neoadjuvant chemotherapy including cetuximab was administered. In the interdisciplinary tumorboard review a multifactorial event was discussed with cetuximab possibly playing a role. Other predisposing factors such as tracheal ischemia after extended mediastinal lymphadenectomy and damage of the membranous part after perforation of the stomach with subsequent mediastinitis have certainly contributed to the lethal outcome.Since the reassessment of EGFR-expression in the gastric and tracheal tissue was not possible one can only speculate about a possible contribution of cetuximab in addition to surgical complications after extensive lymphadenectomy.et al., described the important role of EGF in restitution of gastric mucosae in guinea pigs, that was significantly inhibited by an anti-EGFR antibody [et al., demonstrated that EGF inhibits acid secretion, exerts a trophic effect on gastroduodenal mucosa, protects gastric mucosa against injury, mediates mucosal adaptation and accelerates gastroduodenal ulcer healing by stimulating cell migration and proliferation [Yanaka antibody . Tarnawsferation . Regardiferation and seveferation .et al., hypothesised that EGFR ligands and other growth factors mediate bronchial epithelial repair in sheep [et al., demonstrated in human airway epithelial cells that EGF induced epithelial repair via cell migration after mechanical injury [et al., showed that EGF-promoted wound closure in airway epithelial cells was retarded by a selective EGFR tyrosine kinase inhibitor (AG 1478) [Considering the bronchial system Barrow in sheep . White el injury . PuddicoAG 1478) . These pAG 1478) .th to 6th leading cause of death in the US causing 106.000 fatalities per year [et al., have demonstrated that voluntary reporting identifies only about 6% of ADRs in the clinical routine and improvement is essential [Adverse drug reactions are suspected to be the 4per year althoughper year . Cullen ssential . Linkingssential . The totssential .et al., found tracheal complications in 4% after 785 esophagectomies [et al., found no evidence that induction therapy adversely influences the incidence of postoperative morbidity or mortality after oesophagectomy in 155 patients [The role of cetuximab in ischemic damages of the stomach and trachea after ooesophagectomy is unclear. While Bartels y alone) . Kelley patients .Preclinical evidence exists for a possible correlation between EGFR-antibody therapy and impaired EGFR-mediated repair mechanisms in gastric and airway epithelial cells. The widespread, in general safe and well-tolerated use of cetuximab in metastatic colorectal and locally advanced squamous-cell head and neck cancer has to lead to further investigations in neoadjuvant settings also in other cancers such as esophageal cancer. In our patient with severe postoperative complications the etiology of this complication is likely to have been multifactorial, with cetuximab possibly playing a role. Although the suspected increase of risk for complications after oesophagectomy needs to be confirmed in prospective trials before final conclusions can be made, clinicians should be aware of the possibility of these side effects. Careful recording of all complications is necessary in ongoing and planned studies to obtain more evidence about safety and tolerance of targeted therapies since these new biologic agents may be not as safe as we have initially assumed.The author(s) declare that they have no competing interests.KM & HA Conception and design, writing of manuscriptWE, OF: Help in preparation of draft and editing of manuscript, data analysis and interpretation.AK, CP, TM: Managed the case and helped in preparation of draftKM, LA, TM: Collection and assembly of dataAll authors read and approved final manuscript for publication.
Saccharomyces cerevisiae contain introns, which is in sharp contrast to the genome-wide five percent intron-containing genes. It is well established that introns carry regulatory sequences and that the transcription of RP genes is extensively and coordinately regulated. Here we test the hypotheses that introns are innately associated with heavily transcribed genes and that introns of RP genes contribute regulatory TF binding sequences. Moreover, we investigate whether promoter features are significantly different between intron-containing and intronless RP genes.More than two thirds of the highly expressed ribosomal protein (RP) genes in We find that directly measured transcription rates tend to be lower for intron-containing compared to intronless RP genes. We do not observe any specifically enriched sequence motifs in the introns of RP genes other than those of the branch point and the two splice sites. Comparing the promoters of intron-containing and intronless RP genes, we detect differences in number and position of Rap1-binding and IFHL motifs. Moreover, the analysis of the length distribution and the folding free energies suggest that, at least in a sub-population of RP genes, the 5' untranslated sequences are optimized for regulatory function.Our results argue against the direct involvement of introns in the regulation of transcription of highly expressed genes. Moreover, systematic differences in motif distributions suggest that RP transcription factors may act differently on intron-containing and intronless gene promoters. Thus, our findings contribute to the decoding of the RP promoter architecture and may fuel the discussion on the evolution of introns. In this study, we investigate three hypotheses. First, introns are innately associated with heavily transcribed genes . Second,p-value: 0.166) [see Materials and Methods in Additional file p-value: 1.479e-09; Fig. The average mRNA abundance of intron-containing and intronless RP genes is not significantly different when measured with SAGE predictions derived from 5'SAGE experiments . In a rep-value: 0.00761). The most pronounced difference is observed for the longest 5'UTRs, which may form a special group. To investigate this further, we used the Vienna package to compute the folding free energy (ΔG) of the first 50 bases of each RP mRNA including the 5'UTR [p-value: 0.00205) but not the transcription rate .We estimate the lengths of the 5'UTRs as distance between the translation start codon (ATG) and the predicted TSS from the 5'SAGE experiments, excluding introns. There is no strong dependence of the UTR length on the mRNA expression level, although the three most highly expressed genes, RPL38, RPL41A and RPL41B have short UTRs Fig. . Althoughe 5'UTR . Among the 5'UTR . MoreoveFor factors that are known to regulate RP gene transcription and those that have been predicted by genome-scale experiments, we select position weight matrices (PWM) to represent the binding specificity and scan the region from 600 bp upstream of the TSS to 600 bp downstream of all the genes of our three sets for potential binding sites using T-Reg [see Additional files RPS22B. In contrast, we did not detect Rap1 sites in six of the 33 intronless genes referring to Zhang et al (2005) are listed.Click here for fileRP gene transcription factors. Transcription factors that are enriched on RP gene promoters in ChIP-Chip experiments.Click here for fileWeight matrices for TFBS search. This file contains the weight matrices for the transcription factors that have been used for promoter scans for potential binding sites.Click here for filePromoter regions of RPS29A and B. A snapshot from the Genome Browser illustrates a typical the structure of RP gene promoters.Click here for fileRegression table. This file contains the data table that was used for linear regression analysis.Click here for fileGenome wide Arr1 motif scan. The yeast ORF's (SGD) including 1000 bases upstream of the ATG are scanned for Arr1 binding motifs.Click here for fileProfile of GC base content. The profile of GC base content is specifically optimized in the highly expressed RP genes.Click here for fileMotif hit distributions. Distributions of binding site motifs for several transcription factors.Click here for file
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Although a wide range of therapeutic options is available, the efficacy of these methods and the prognosis of patients with HCC remain very poor. This study was conducted to evaluate the efficacy and safety of viscum fraxini-2 in patients with chemotherapy–naïve, advanced hepatocellular carcinoma. 23 patients with unrespectable HCC who had received no prior systemic chemotherapy with objectively measurable tumors were enrolled on this study. The mistletoe preparation for the study is an aqueous injectable solution. It contains one milliliter of viscum fraxini in dilution stage–2 which is equivalent to 10 000 ng/ml injection ampoules. 2 ampoules of viscum fraxini–2 were administered subcutaneously once weekly. As assessed by conventional imaging criteria, 3 (13.1%) patients have achieved complete response, 2 (8.1%) patients have achieved a partial response. 9 (39.1%) had progressive disease while 9 (39.1%) patients didn't have evaluation of response due to early death. The median overall survival time for all patients was 5 months (range 2–38 months), for those who achieved a CR was 29 months (range 12–38 months) and, for those who achieved a PR was 6.5 months (range 6–7 months). The median progression free survival for all patients was 2 months (range 1–38 months), for those who achieved a CR, it was 29 months (range 8–38 months) and for those who achieved a partial response, it was 5 months (range 4–6 months). No hematologic toxicity has been encountered. The spectrum of non-hematologic toxicity was mild. The WHO toxicity criteria grade 3–4 were 34.8% drug related fever, 13.1% erthyma at injection site and 17.4% pain at the site of injection. No drug related discontinuation or toxic deaths have occurred. Viscum fraxini-2 seems to be particularly promising in patients with advanced HCC, it shows antitumor activity and low toxicity profile. Further studies in combination with other active agents are clearly warranted. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Although it is far less common in western countries, it is the most common malignant tumor in areas of Africa and Asia . EventuaThe role of chemotherapy in the treatment of patients with HCC remains controversial. Numerous single chemotherapeutic agents and drug combinations have been given to HCC patients in an attempt to alter their predictably short survival time. Unfortunately, the activity of a single agent is limited, with only a few drugs showing a response rate >10%. Moreover, combination chemotherapy has proven equally disappointing, because additional drugs have resulted in increased toxicity without any increased efficacy compared with single–agent doxorubicin therapy . Therefon=8), tumor remission (n=1), overall quality of life (n=3), and quality of life in relation to side effects during cytoreductive therapy (n=3) and it iα-fetoprotein >400 ng/ml with a hepatic tumor highly suggestive of HCC by imaging studies; (2) unresectable tumor and patient was not a candidate for either transcatheter arterial chemoembolization (TACE) or percutaneous ethanol injection (PEI); (3) bi-dimensional measurable disease; (4) no previous systemic chemotherapy; (5) age between 16 years and 75 years; (6) performance status 0–3 WHO, 7) within normal renal, cardiac and hematological profile.The eligibility criteria included: (1) pathology proven primary HCC or α-fetoprotein, triphasic liver computed scan (CT) and Child class evaluation were performed before treatment. The mistletoe preparation for the study is an aqueous injectable solution. It contains one milliliter of viscum fraxini in dilution stage–2 which is equivalent to 10 000 ng/ml injection ampoules. 2 ampoules of viscum fraxini–2 were administered subcutaneously once weekly. Patients were seen on a weekly basis during treatment for history taking and physical examination. A complete blood count was determined every week. Renal and liver functions and α-fetoprotein levels were examined every 4 weeks. The tumor was assessed by CT every 8 weeks.Prior to entry into the study, all patients provided a complete history and physical examination, including performance status, concurrent nonmalignant diseases and therapy. Laboratory studies included a complete blood cell counts, differential count, biochemical liver and renal function tests, electrolyte, chest x-rays, Determination of the tumor response followed standard response criteria established by the World Health Organization (WHO) . Extensive one lobe affection was present in 15 patients (65.2%). Distant metastasis were present in 4 patients (17.4%) (2 bone metastasis and 2 lymph node metastasis). Main portal vein thrombosis was detected in two patients (8.7%) while ascites was present in 4 patients (17.4%).Base line patient characteristics and clinical features are summarized in According to conventional radiological response criteria, 3 patients (13.1%) achieved complete response. The first patient achieved CR after 4 months from starting the treatment and remained disease free for 4 months. The second and the third patients achieved CR after 6 months and they are still living disease free for more than 29 and 38 months, respectively patients remained alive including two patients with CR and one patient with slowly progressive disease. The median overall survival time for all patients was 5 months (range 2–38 months), for those who achieved a CR was 29 months (range 12–38 months) and, for those who achieved a PR was 6.5 months (range 6–7 months). The median progression free survival for all patients was 2 months (range 1–38 months), for those who achieved a CR, it was 29 months (range 8–38 months) and for those who achieved a partial response, it was 5 months (range 4–6 months). The Kaplan- Meier actuarial overall survival and PFS curves for all patients are shown in Figure 2All patients were evaluated for toxicity. Drug related fever developed in 8 patients (34.8%). Erythma at injection site developed in 3 patients (13.1%). 4 patients (17.4%) suffered pain at site of injection. 3 patients had to reduce the dose to one ampoule on the subsequent treatment courses. Anti-inflammatory and analgesics were used in only one patient due to severe pain and erythma at injection site. There were no drug related discontinuation or toxic deaths.Hepatocellular carcinoma is one of the most common cancers worldwide . ResectiThe antitumor activities of a number of chemotherapeutic agents have been evaluated in HCC patients, but most yielded poor results and probably are associated with severe side effects. Doxorubicin remains the most active drug against HCC, with a single agent tumor response rate of about 10–20%. However, its toxicity outweighs its benefit , inadequViscum fraxini is an aqueous extract of mistletoe. Controversy exists as to how the plant extracts exert their postulated dual activity of cytotoxicity towards tumor cells and stimulation of immune cells (γ) and tumor necrosis factor-α (TNF-α) by T cells and macrophages respectively with a good impact on survival. While most of Phase II trials of treatment of HCC confirmed that there is no effective drug for HCC and all single systemic anticancer agents produced a response rate of less than 10% , Viscum In conclusion, Viscum Fraxini-2 is active in HCC with anti-tumor activity and low toxicity profile. Further studies in combination with other active agents are clearly warranted.
No definitive data are available regarding the value of switching to an alternative TNF antagonist in rheumatoid arthritis patients who fail to respond to the first one. The aim of this study was to evaluate treatment response in a clinical setting based on HAQ improvement and EULAR response criteria in RA patients who were switched to a second or a third TNF antagonist due to failure with the first one.This was an observational, prospective study of a cohort of 417 RA patients treated with TNF antagonists in three university hospitals in Spain between January 1999 and December 2005. A database was created at the participating centres, with well-defined operational instructions. The main outcome variables were analyzed using parametric or non-parametric tests depending on the level of measurement and distribution of each variable.Mean (± SD) DAS-28 on starting the first, second and third TNF antagonist was 5.9 (± 2.0), 5.1 (± 1.5) and 6.1 (± 1.1). At the end of follow-up, it decreased to 3.3 , 4.2 and 5.4 . For the first TNF antagonist, DAS-28-based EULAR response level was good in 42% and moderate in 33% of patients. The second TNF antagonist yielded a good response in 20% and no response in 53% of patients, while the third one yielded a good response in 28% and no response in 72%. Mean baseline HAQ on starting the first, second and third TNF antagonist was 1.61, 1.52 and 1.87, respectively. At the end of follow-up, it decreased to 1.12 , 1.31 and 1.75 , respectively. Sixty four percent of patients had a clinically important improvement in HAQ (defined as ≥ -0.22) with the first TNF antagonist and 46% with the second.A clinically significant effect size was seen in less than half of RA patients cycling to a second TNF antagonist. Treatment with TNF antagonists has improved the outcome of rheumatoid arthritis (RA) patients . In bothTNF antagonists as a group have similar efficacy in RA, although their effectiveness differs in other rheumatic diseases. Moreover, case series and nonrandomized, open-label observational studies in RA indicate that some patients may fail to respond to one TNF inhibitor but will respond to another. This is partially supported by data showing that TNF antagonists differ in their pharmacokinetics and mechanisms of action . NeverthThe aim of this study was to evaluate in a clinical setting the clinical response based on evaluation of HAQ and EULAR response criteria in RA patients with an insufficient response or loss of efficacy to the first TNF antagonist who were switched to a second or third one.This was an observational, prospective study of a cohort of 417 RA patients treated with TNF antagonists in three university hospitals in Spain between January 1999 and December 2005. A database was created at the participating centres, with well-defined operational instructions. Patients who had participated in clinical trials were excluded.Patients had been systematically evaluated at the initiation of therapy and every three months thereafter. Patients switching between TNF antagonists or switching to rituximab were evaluated on starting therapy and every 3 months thereafter. Evaluations included painful and swollen joint counts, visual analogue scales of pain, global health assessment by the patient and the physician, ESR, C-reactive protein (CRP), Health Assessment Questionnaire (HAQ) and DAS-28 score. DAS-28-based EULAR response was estimated. Data on the reason for switching to a second TNF antagonist were recorded.Descriptive statistics with central tendency and dispersion measures were calculated. The main outcome variables were analyzed using parametric or non-parametric tests depending on the level of measurement and distribution of each variable. A p-value < 0.05 (two tailed) was considered significant. Survival analysis was performed using Kaplan-Meyer curves.The study was conducted according to good clinical practice as applicable to epidemiological studies, which ensures that the design, implementation and communication of data are reliable, and that patients' rights, integrity and data confidentiality are protected. The study protocol was approved by the Ethics Committee of the Hospital Universitario Virgen Macarena which considered that informed consent was not required due to the retrospective nature of the analysis of anonymous data.The initial TNF antagonist was infliximab (INF) in 238 cases (57%), etanercept (ETA) in 141 (34%), and adalimumab (ADA) in 38 (9%). Eighty-three patients had switched to a second TNF antagonist and 18 to a third TNF antagonist. Mean patient follow-up was 21.4 + 15.6 months, and TNF exposure was 443 patient-years for INF, 200.2 patient-years for ETA and 31.7 patient-years for ADA. Switching in 48 cases (58%) was due to inefficacy, in 24 cases (29%) to adverse events, and in 11 cases (13%) to other reasons, primarily the doctor's or patient's decision.Relevant clinical data on the 417 patients (82% women) are presented in table Mean DAS-28 on starting the first TNF antagonist was 5.9 ± 2.0 and decreased to 3.3 ± 1.6, at the end of follow-up. The improvement was statistically significant for the whole group as well as for the three TNF antagonists considered independently. DAS-28 on starting the second TNF antagonist was 5.1 ± 1.5 and decreased to 4.2 ± 1.5 at the end of follow-up . DAS-28 on starting the third TNF antagonist was 6.1 ± 1.1 and decreased to 5.4 ± 1.7 at the end of follow-up . The results are shown in table The DAS-28-based EULAR response level for the first TNF antagonist was good in 42% of patients and moderate in 33%. For the second anti-TNF, good response was achieved in 20% of cases and 53% failed to respond. For the third TNF antagonist, 26% of cases had a good response and 72% failed to respond . The reasons for discontinuation were inefficacy (40%), adverse events (40%) and other (20%). Retention rates with the second TNF antagonist were 70%, 60% and 47% at 12, 24 and 36 months, respectively. Twenty-five patients discontinued the biologic, most commonly due to inefficacy (77%). Only 9 of the 18 patients switching to a third TNF antagonist retained the biologic at 6 months. Six stopped the biologic due to inefficacy.Fifty-four patients had a severe adverse event during treatment with the first TNF antagonist. Infusion reaction was the most frequent adverse event, occurring in 16 patients treated with INF, followed by urticaria or severe skin rash in 9 patients, and upper respiratory tract infection in 8. Other less frequent adverse events were congestive heart failure, tuberculosis, herpes zoster infection, acute pancreatitis, cutaneous vasculitis and lupus-like syndrome. Seven patients had severe adverse events while treated with the second TNF antagonist Table .In the present study we describe relevant outcomes in clinical practice in RA patients failing to respond to one TNF antagonist and switching to another, in comparison with RA patients who retain the first antagonist. Our data show that a number of patients switching between TNF antagonists may attain a significant response, yet a large proportion of patients have marginal or no clinical improvement. Improvement is generally negligible in patients switching to a third biologic. Interestingly, patients switching to a third TNF antagonist were younger, more frequently female, and had a higher prevalence of erosions despite the fact that disease duration was similar to the other patients. This group probably represents a subset of patients with more severe disease which is resistant to these biologics.Up to September 2008, there were 35 reports in the literature -42 on swMost patients included in previous publications were women , with an average age of 52 years (range: 32–68), and a disease duration of 12 years (range: 3–27). When reported at baseline, mean DAS was 5.6 (range: 2.4–6.8), and mean HAQ 1.7 (range: 1.5–1.9). This baseline information is no different from what was found in our study. Of note, few publications report on DAS-28 effect size, DAS-based EULAR response and HAQ improvement in patients switching between TNF antagonists in comparison with patients who retain the first antagonist. A value of -0.22 in HAQ is considered the minimum clinically important difference (MCID) in studies of responsiveness . The sizThree studies ,15,20 haA significant treatment effect size with a second TNF antagonist in RA patients failing to respond to the first one is limited to less than half of those treated. Furthermore, RA patients who are refractory to TNF-antagonist treatment have a poorer response to rituximab, abatacept and tocilizumab than patients who are naive to biological drugs. Evidence that the efficacy of cycling between TNF antagonists is similar or superior to switching to the new biologics is largely missing. The results reported by other authors as well ADA: Adalimumab; DAS: Disease Activity Score; ETA: Etanercept; EULAR: European League Against Rheumatism; HAQ: Health Assessment Questionnaire; INF: Infliximab; LF: Leflunomide; MCID: Minimum Clinically Important Difference; MTX: Methotrexate; RA: Rheumatoid Arthritis; TNF: Tumor Necrosis Factor.DRM, BH, VNC, SM and MB declare that they have no competing interests. FNS and JJGR have participated in Advisory Boards and received lecture fees from Abbott, Bristol-Mayer-Squib, Roche, Schering-Plough and Wyeth.FNS and JJGR conceived, designed and coordinated the study, and prepared the draft of the manuscript. DRM, VNC, SM and MB collected the data and reviewed the data analyses. BH performed the statistical analysis and contributed to the design of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:
What is the underlying mechanism behind the fat-tailed statistics observed for species abundance distributions? The two main hypotheses in the field are the adaptive (niche) theories, where species abundance reflects its fitness, and the neutral theory that assumes demographic stochasticity as the main factor determining community structure. Both explanations suggest quite similar species-abundance distributions, but very different histories: niche scenarios assume that a species population in the past was similar to the observed one, while neutral scenarios are characterized by strongly fluctuating populations. Since the genetic variations within a population depend on its abundance in the past, we present here a way to discriminate between the theories using the genetic diversity of noncoding DNA. A statistical test, based on the Fu-Li method, has been developed and enables such a differentiation. We have analyzed the results gathered from individual-based simulation of both types of histories and obtained clear distinction between the Fu-Li statistics of the neutral scenario and that of the niche scenario. Our results suggest that data for 10–50 species, with approximately 30 sequenced individuals for each species, may allow one to distinguish between these two theories. One purchases 100 wineglasses and 100 pairs of pants. After one year, 10 glasses and 10 pants survive. What can be said about the relative quality of the survivors? Well, clothes “die” as a result of accumulated wear; the surviving items are of better quality. The breaking of a wineglass is an external, random event: here the survivors are not the best, but the luckiest. To tell apart the superior from the fortunate, one should examine the development over time: the number of surviving items decays exponentially with time for the glasses and follows a sigmoid curve for the pants. An ongoing argument among macroecologists deals with similar issues. Adaptive theories suggest that the frequent species are the fittest, while the neutral theory explains the observed frequencies as a result of demographic stochasticity, assuming all species to have the same fitness. The histories suggested by the two scenarios are clearly different, but how can one probe the prehistoric abundance of species? In fact, past abundance is reflected in current genetic variance within a population. Here, we present a new technique, based on the Fu-Li F-statistic, which allows one to distinguish between niche and neutral scenarios and to resolve this important debate. One of the most interesting peculiarities of mother nature is the large variance in abundance of otherwise similar species. In the tropical rainforest, for example, there are differences of 4–5 orders of magnitude in the observed abundance of tropical trees The simplest explanations for this phenomenon are based on “niche theory” Another theory that gained much popularity in the last decade is the neutral theory of species abundance. It assumes time evolution of species abundance, while the adaptive theories assume such a process in the fitness/resource space. Thus, if niche-based theories are correct, the real-time stochastic birth-death process is biased towards the observed (present) frequency Within the framework of the neutral model, demographic stochasticity may be described as a multiplicative random walk along the abundance line. Multiplicative random processes are known for many years as the underlying mechanism behind fat-tailed statistics, e.g., firm size distributions Confronting the different models on the basis of current community structure data poses a very difficult statistical problem In this work we present an experimental method that extracts these differences and allows one to distinguish between the two scenarios. It requires the collection of a large amount of genetic data from the current population, in particular noncoding DNA from either haploid or diploid sequences. Intuitively, the genetic diversity of these sequences should reflect the history of the species abundance; one expects different results for a more or less fixed population (as suggested by the niche theory), than for a strongly fluctuating population with bottlenecks and high prevalence times . Here we quantify this concept, explain how to distinguish between the two scenarios, and demonstrate our results in a numerical experiment using “DNA sequences” obtained from simulated data with different histories.Our technique is limited by two time scales. The sequence mutation time sets its resolution, as no reliable conclusion may be drawn on the basis of only a few mutations. The abundance history may be recovered for timescales that are much larger than the typical time needed for a single mutation to appear in the whole sequence. The time to the most recent common ancestor sets, of course, the maximal timescale. For an almost fixed population (niche scenario) of size A similar idea, utilizing the differences in assumed history to distinguish between the two hypotheses, was suggested by Ricklefs environmental stochasticity. We do assume, however, that these fluctuations either conserve the species abundance ratio or are relatively weak. If environmental fluctuations cause rapid shifts in the relative species frequency, the conceptual meaning of the “niche theory” becomes unclear and the difference between the two scenarios is not so interesting. Throughout this work we therefore assume that the effect of environmental stochasticity is weak and yields only minor corrections to the niche/neutral predictions. In the final section we return to this issue, and discuss in detail the various types of environmental stochasticity, together with their identification using genetic polymorphism data.Before we discuss the polymorphism analysis itself, let us add an important comment. Restricting our considerations to “pure” adaptive/neutral histories, like those demonstrated in We tried a number of methods in order to distinguish between the genetic polymorphism of the two scenarios, and found that the most efficient one is Fu & Li F-statistic average internal branch length. Under the correct scaling, these lengths should be the same, if the assumptions of the Kingman Coalescent Model are fulfilled. Therefore, in the Wright-Fisher process, for example, the value of the F-Statistic is zero. In a growing population, this value is negative, and for a shrinking population it is positive.The Fu-Li F-statistic compares the sum of the lengths of the external branches to the recent past, which affect the external branch length, to the features of the far past, affecting the internal branches. Thus, it emerges as a suitable technique for distinguishing between the two scenarios. In the niche scenario, the population in the past is similar to the population in the present, so the statistic should be approximately zero. For a neutral scenario, the population in the present differs from the past population; in most cases, the population in the present is larger than the population in the past . We must add that if this effective population size is somehow known this method may be superior because the sample size needed from each species is small (10 or less).The average number of lineages as a function of time, The weak point of this method lies in the fact that the goodness of fit changes with the population size and again requires an a-priori knowledge of the population's effective size.An interesting idea is to use the abundance statistics of “genetic families” in order to learn about the species abundance. We defined a “family” as all the individuals with exactly the same genome (any mutation is an establishment of a new family). If the population is fixed, the family size distribution should obey the same abundance statistics of the neutral theory, as this is simply a neutral process with mutations.It turns out, however, that there is almost no difference between the niche and the neutral histories in the family size distribution. The reason for this is that the family size distribution depends only on the close history (recent times), and in this period the two scenarios admit the same population size. We hope to discuss this issue in a separate work.As we have mentioned above, the “pure” niche/neutral scenarios considered here are an idealization that may be true in some cases , but in other cases one should expect large fluctuations in the abundance of a species due to environmental stochasticity. Indeed, for certain ecosystems (like phytoplankton) some degree of environmental fluctuations has been suggested in order to explain, in the framework of niche theory, how the system overcomes the competitive exclusion principle large, fast and independent environmental fluctuations. If the fitness of a species varies tremendously in time, and is uncorrelated with the fitness of other species, and if the correlation time of these fluctuations approaches zero, the fitness differences are averaged out and the adaptive dynamics is equivalent to the neutral one. Niche theories are meaningful only if (at least) one of the three conditions mentioned above is not satisfied: the environmentally-induced fitness fluctuations should be either weak, slow, or correlated.One fundamental observation is that all theories become practically neutral in the limit of If the effect of environmental stochasticity is weak, the corrections to the predictions of the “pure” theory are relatively small. In that case one may use our Fu-Li technique, expecting only small deviations from the predictions for the idealized case. In other occasions, however, one can find evidence for large perturbations that affect the ecosystem. Here the timescale is important: one may try to relate observed quantities to the predictions of an adaptive theory only if the characteristic time needed for the ecosystem to reach demographic equilibrium is much shorter than the typical period between environmental shifts. The Fu-Li statistic in that case will fit our predictions if the genetic time horizon, two competing niche scenarios. One can imagine an adaptive ecosystem that is subject to uniform (correlated) pressure, such that the abundance of all species shrinks or grows in the same proportions . On the other hand, the pressure may be uncorrelated (niche-selective), not affecting all species in the same manner, in which case the abundance ratio is time-dependent Even if these conditions are not satisfied and the Fu-Li statistic (as well as the species abundance ratio) fails to follow the pure scenario predictions one may still uses other polymorphism based methods. The basic challenge, now, is to discriminate between the neutral scenario suggested by As suggested above, polymorphism data may be used in order to calculate Most of the empirical tests suggested for the niche-neutral debate rely on snapshots, such as via comparison of the predicted and the observed species abundance ratio. Some authors did consider historic abundance data We have gathered our data from a simulation of the Wright-Fisher model with discrete generations. We initiate the system with
Perivascular epithelioid cell tumor (PEComa), other than angiomyolipoma (AML), clear cell sugar tumor (CCST), and lymphangioleiomyomatosis (LAM), is a very rare mesenchymal tumor with an unpredictable natural history. The uterus is the most prevalent reported site of involvement of PEComa-not otherwise specified (PEComa-NOS). To the best of our knowledge, about 100 PEComa-NOS have been reported in the English Language medical literature, of which 38 were uterine PEComa-NOS. These reported cases of uterine PEComa-NOS have usually shown clinically benign behavior, but 13 tumors, three of them associated with tuberous sclerosis complex (TSC), exhibited local aggressive behavior and four of them showed distant metastases.We report the case of a 59-year-old woman, who presented with renal and pulmonary lesions seven years after the initial diagnosis of uterine leiomyosarcoma. Left nephrectomy and right middle lobe wedge resection were performed. Histological and immunohistochemical analysis of the renal and pulmonary lesions, in addition to retrospective re-evaluation of the previous uterine tumor, led to the final diagnosis of malignant uterine PEComa with late renal and pulmonary metastases. All three lesions had the typical histological appearance of PEComa-NOS showing a biphasic growth pattern with continuous transition between spindle cells and epithelioid cells, often arranged around vascular spaces. Immunohistochemically, the tumor cells of both phenotypes in all three lesions stained for melanocytic (HMB-45 and Melan-A/MART-1) and myoid markers.The findings indicate that despite the small number of reported cases, PEComas-NOS should be considered tumors of uncertain malignant potential, and metastases to other organs might become evident even several years after the primary diagnosis. The World Health Organization defines perivascular epithelioid cell tumors (PEComas) as "mesenchymal tumors composed of histologically and immunohistochemically distinctive perivascular epithelioid cells (PECs)" . In 1991To the best of our knowledge, about 100 PEComas-NOS have been reported in the English Language medical literature, of which 38 were uterine PEComas-NOS -17. ThesA 59-year-old woman with a past medical history of hysterectomy and bilateral salpingo-ophrectomy for uterine leiomyosarcoma seven years prior presented to our institution for her regular periodic computed tomography (CT) scans of the abdomen, chest and pelvis at 6-month intervals as tumor follow-up investigations. These CT scans revealed several coalescing rim-enhancing nodules in the upper and middle poles of her left kidney and a solitary rim-enhancing nodule (measuring 1.5 cm) in her right middle lobe of lung, which were absent on her previous CT scans six months earlier. The clinical impression was metastasis from her previous uterine leiomyosarcoma. No additional nodules were identified in the brain, gastrointestinal (GI) tract, liver, spleen, pancreas, bladder and bones on subsequent staging magnetic resonance imaging (MRI) of the brain, abdomen, chest and pelvis, and positron emission tomography (PET) scan. The patient did not have any stigmata or family history of TSC, and had no history of melanoma. General physical examination was unremarkable. Her hemogram, urine and blood biochemical analyses were within normal ranges.A left radical nephrectomy and video-assisted thoracoscopic surgical wedge resection of the right middle lobe solitary nodule were performed. Grossly, her resected left kidney revealed a sharply demarcated and lobulated firm gray-tan nodular tumor (7.5 × 6.2 × 5.4 cm) occupying the upper and middle poles, and showed focally hemorrhagic areas on cut surface Figure . The intHistologically, the renal tumor was biphasic and composed mainly of medium to large epithelioid cells and focal areas of spindle/elongated cells around numerous small blood vessels Figures . The medMicroscopic examination of the pulmonary tumor (not shown) exhibited the same morphological features as the renal tumor described above Figures . The intImmunohistochemically, both the epithelioid and spindle neoplastic cells of the renal tumor were strong and diffusely (>80% of the neoplastic cells) positive for desmin (cytoplasmic and membranous) {Ventana, Tucson, Ariz} Figure , HMB-45 On the basis of the above histopathological and immunohistochemical features, a definitive diagnosis of malignant uterine PEComa with late renal and pulmonary metastases was rendered. The patient elected not to have any adjuvant chemotherapy or radiotherapy. Five follow-up CT scans of the brain, abdomen, chest and pelvis, and PET scans performed at 3-month intervals after surgery revealed no nodules. She is alive with no evidence of local recurrence or distant metastasis after 15 months of follow-up, and scheduled to have regular periodic CT scans of the abdomen, chest and pelvis at 3-month intervals as her tumor follow-up plan.In this report, we describe a case of unusual epithelioid tumors in the kidney and the lung seven years after the initial diagnosis of uterine leiomyosarcoma, with pathological and immunohistochemical features entirely compatible with those of a PEComa. PEComas are characterized by epithelioid to spindle cells with eosinophilic to clear cytoplasm, an intimate relationship with blood vessels, and demonstrate positive immunostaining for markers of both melanocytic and myoid differentiation . PEComasPEComas are of interest primarily because of their immunoreactivity with melanocytic and myoid markers. They are also almost always negative for S-100 protein and cytokeratins. Folpe and colleagues recentlyNot unexpectedly, the uterine tumor of the case herein presented was initially misdiagnosed as a uterine leiomyosarcoma at an outside institution seven years prior. Myomelanocytic marker expression was found to be prominent in both components of all the three lesions in the case herein presented. Controversy exists regarding the minimum criteria for the diagnosis of malignant PEComa ,19. In sIn addition to epithelioid smooth muscle tumors (epithelioid leiomyosarcoma and epithelioid leiomyoma), the other important differential diagnosis of PEComa include malignant melanoma, clear cell sarcoma of tendon and aponeuroeses (melanoma of the soft parts), alveolar soft part sarcoma, endometrial stromal sarcoma with clear cell features, uterine tumor resembling sex cord tumor, carcinoma , paraganglioma, angiomyolipoma, and any other tumor with focal or prominent clear cell change. Malignant melanoma and clear cell sarcoma of tendon and aponeuroeses can be differentiated from PEComas based on S-100 positivity; however, up to 11% of PEComas express S-100 as well . The addClinically, most PEComas follow a benign course . MalignaOptimal treatment for PEComas is not well established at this time. Currently, surgery is the mainstay of treatment for primary PEComa at presentation as well as for local recurrences and metastases, with the aim of obtaining clear resection margins. The role of adjuvant therapy remains unclear. Metastases have been successfully managed by resection alone . PrimaryWe have described a case of malignant PEComa-NOS of the uterus with late renal and pulmonary metastases seven years after hysterectomy. Malignant PEComa should be considered in the light microscopic differential diagnosis for all pleomorphic sarcomas. PEComas-NOS show a marked female predominance, are rare but anatomically ubiquitous mesenchymal tumors that are composed of nests and sheets of usually epithelioid but occasionally spindled cells with clear to granular eosinophilic cytoplasm and a focal association with vascular spaces. They usually show immunoreactivity for both melanocytic (HMB-45 and/or Melan-A/Mart-1) and myoid/muscle-specific (actin and/or desmin) markers, which are useful for confirming the diagnosis. The mainstay of treatment is wide excision. Though most PEComas are benign, a subset behaves in a malignant fashion; size >8 cm, high mitotic index, presence of necrosis, marked cytological atypia, and/or infiltrative growth pattern may be associated with malignant behavior. However, since relatively few malignant PEComas have been reported, firm criteria for malignancy have yet to be established and the identity of the normal PEC remains elusive. Further studies on additional cases with longer clinical follow-up would be necessary to accurately predict the biologic behavior of these distinctive tumors. Finally, because PEComas can behave in an aggressive manner, careful follow up is warranted.AML, angiomyolipoma;CCMMT, clear cell myomelanocytic tumour;CCST, clear cell sugar tumour;LAM, lymphangiomyomatosis;PEC, perivascular epithelioid cell;PEComa, perivascular epithelioid cell tumor;TSC, tuberous sclerosis complexThe author(s) declare that they have no competing interests.HBA participated in the histopathological evaluation, performed the literature review, acquired the photomicrographs and drafted the manuscript. AVP conceived and designed the study, gave the final histopathological diagnosis and revised the manuscript for important intellectual content. Both authors read and approved the final manuscript.
Real-time three-dimensional (RT-3D) echocardiography has entered the clinical practice but true incremental value over standard two-dimensional echocardiography (2D) remains uncertain when applied to stress echo. The aim of the present study is to establish the additional value of RT-3D stress echo over standard 2D stress echocardiography. We evaluated 23 consecutive patients referred for dipyridamole stress echocardiography with Sonos 7500 equipped with a phased – array 1.6–2.5 MHz probe with second harmonic capability for 2D imaging and a 2–4 MHz matrix-phased array transducer producing 60 × 70 volumetric pyramidal data containing the entire left ventricle for RT-3D imaging. In all patients, images were digitally stored in 2D and 3D for baseline and peak stress with a delay between acquisitions of less than 60 seconds. Wall motion analysis was interpreted on-line for 2D and off-line for RT-3D by joint reading of two expert stress ecocardiographist. Segmental image quality was scored from 1 = excellent to 5 = uninterpretable. Interpretable images were obtained in all patients. Acquisition time for 2D images was 67 ± 21 sec vs 40 ± 22 sec for RT-3D (p = 0.5). Wall motion analysis time was 2.8 ± 0.5 min for 2D and 13 ± 7 min for 3D (p = 0.0001). Segmental image quality score was 1.4 ± 0.5 for 2D and 2.6 ± 0.7 for 3D (p = 0.0001). Positive test results was found in 5/23 patients. 2D and RT-3D were in agreement in 3 out of these 5 positive exams. Overall stress result (positive vs negative) concordance was 91% (Kappa = 0.80) between 2D and RT-3D. During dipyridamole stress echocardiography RT-3D imaging is highly feasible and shows a high concordance rate with standard 2D stress echo. 2D images take longer time to acquire and RT-3D is more time-consuming to analyze. At present, there is no clear clinical advantage justifying routine RT-3D stress echocardiography use. Two-dimensional dipyridamole stress echocardiography, is an established and validated method for both the diagnosis and prognosis -5 of patOspedale di Savona, Italy with the following criteria: age ≥ 18 years; adequate echocardiogram to assess regional wall motion in 2D and RT-3D . Exclusion criteria included: poor acoustic window, contraindications to dipyridamole, recent (< 1 month) episode of ventricular fibrillation, significant stenosis of the aortic valve, and patient refusal to enter the study. Decisions concerning medical therapy at the time of testing and/or coronary angiography were left to the attending physician. A quantitative luminal narrowing ≥ 50% was considered significant. All patients gave informed written consent prior to dipyridamole stress echocardiography.The study population consisted of 23 consecutive patients with known or suspected coronary artery disease referred for clinically indicated stress echocardiography. Patients were prospectively enrolled in Two-dimensional echocardiography images were performed with Sonos 7500 equipped with a phased array 1.6–2.5 MHz probe with second harmonic capability. In all patients, the four standard views were obtained at baseline and peak stress and were recorded on super-VHS and digitally stored.Real-time three-dimensional echocardiography images were recorded using Sonos 7500 with 2–4 MHz matrix-phased array transducer in a 60 × 70 pyramid-shaped volume containing the entire LV. Volumetric data were obtained only from the apical window and displayed as conventional 2D apical which where digitalized with final interpretation made off-line with the steering and tilting of the image planes for proper alignment and visualization of various scan Figure . AcquisiPatients were asked to abstain from food and drinks containing xanthine for ≥ 24 hrs prior to the study. All patients performed a dipyridamole stress echo. Dipyridamole was given at a maximal dose of 0.84 mg/kg in 10 minutes, unless symptoms of intolerability, positivity or hypotension occurred. Test was considered positive for ischemia if any new or worsening dyssynergy occurred in more than 1 contiguous segment of the same vascular territory. A segment was considered to be viable when it improved by one grade or more at peak stress . The left ventricle was divided into 17 segments as suggested the American heart Association . Segmentt test was used to compare continuous variables. Cohen's coefficient of variation, Kappa was used to assess agreement between 2D and RT-3D results. A Kappa value ≥ 0.45 was considered to be a good agreement while Kappa ≥ 0.75 was considered to be an excellent agreement. A p value < 0.05 was considered significant. All calculations were made using SPSS software .Variables are expressed as mean values ± SD. Student paired-sample The clinical baseline characteristics, risk profile and medical therapy of the study population are outlined in Table Image quality as defined by endocardium border detection was better with 2D than with RT-3D acquisition at peak stress of a volumetric cineloop that contained the entire LV and to review multiple, standard 2D images. The inferior image quality of RT3D might partly be explained by the round and flat face of the transducer which offers poor intercostals skin contact and the low frequencies used . MoreovePrevious studies on three-dimensional imaging used a different technology than real-time three-dimensional echocardiography, these previous techniques were dependant on both ECG and respiratory gating because 3D images were a composite of multiples 2D acquisitions and cardiac cycles, thus highly affected by translation motion, arrhythmia and improper spatial registration . Due to Real-time three-dimensional echocardiography has the prerequisites to analyze segmental wall motion, volume, etc. In fact, it has been demonstrated that ,13-18 usRT3D stress echocardiography has been previously shown to be highly feasible for both dobutamine -18 and eInferior spatial resolution and frame rates compared with 2D echocardiogram are the disadvantages of RT3D echo. These drawbacks may offset the advantages of RT3D echo and could explain why RT3D has not been shown to be superior to 2D echo in previous reports Figure .RT3D has entered the clinical arena but no additional value over conventional 2D echocardiography during stress echocardiography can be demonstrated. Recent ASE and EAE consensus statements on stress echocardiography did not recommend the routine use of this technology during stress echocardiography even though it may shorten significantly time of acquisition counterbalanced by a longer time of data-set analysis. Nonetheless RT3D may play a role in stress echocardiography when wall motion analysis is not the target. The assessment of contractility or the function of the right ventricle can become the perfect clinical setting for this technique. More studies are warranted to assess its real clinical value in different subsets of patients but the evidence is sound enough to state that RT3D is here to stay.SV acquired most images, LP and PS analyzed 3d data, AG and MB contributed to data collection, PB critically revised the manuscript. All Authors have read and approved the final manuscript.AVI 1.avi. stress echo .Click here for file
The detection rate of submucosal veins (SMV) with EUS was 88% for the PH group and68% for the control group. The mean SMV diameter was significantly greater for the PHgroup than for the control group, and no 2-mm or larger SMV was detected in the controlgroup. Serum albumin and cholinesterase levels were significantly higher for the RV(+)patients with SMV 2mm or more in diameter in the PH group than for the RV(–) patients.The spleen index was also significantly higher for the former group. The frequency of RV wassignificantly higher for advanced PH than for mild PH. RV(+) was detected in about 30% ofendoscopically normal patients in the PH group. The results of this study indicate that EUS isuseful in detecting RV and evaluating its pathological condition.The usefulness of endoscopic ultrasonography (EUS) in the evaluation of rectal varices (RV)was determined in 50 patients with portal hypertension (PH) and 25 PH-free controls. F
Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac+ mutants, with and without evidence of descent from the HMS, have similar Lac+ mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac+ mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones.In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in E. coli or only a small fraction of it. First, improved methods allow estimation of mutations per genome accumulated during HMS-generated bursts of mutagenesis and show numbers compatible with fitness after the HMS state. Second, two lines of evidence presented support models in which the HMS is central to this stress-induced mutagenesis pathway. Third, a specific model, with general consequences, for HMS differentiation is discussed.Mutational processes are being discovered in which bacterial, yeast, and human cells under various stresses activate programs that increase mutagenesis, often under the control of cellular stress responses. These programs may potentially increase genetic variability in populations specifically when they are maladapted to their environments, i.e., when they are stressed. When mutation supply is limiting for evolution , these mechanisms might enhance the intrinsic ability of organisms/cells/populations to evolve, specifically during stress. Stress-induced mutagenesis mechanisms recast understanding of, and strategies for combating, problems such as host-pathogen interactions, generation of bacterial antibiotic resistance, cancer progression, and evolution of chemotherapy resistance, all problems of evolution of fitter variant clones fueled by genetic change under stress. A key problem in stress-induced mutagenesis concerns how cells survive the deleterious effects of enhanced mutagenesis. One proposed strategy is the differentiation of a subpopulation of transiently hypermutable cells. This study investigates a previously discovered hypermutable cell subpopulation (HMS) postulated either to underlie most stress-induced mutagenesis in In the + colonies: Lac+ “point mutagenesis” and stress-induced gene amplification. Point mutagenesis dominates during the first week of incubation and creates compensatory -1 frameshift mutations lac region to 20–100 copies represents ∼40% of Lac+ colonies at day eight of incubation, and increases thereafter lac gene produce enough beta-galactosidase activity to restore growth. Both processes are stress-induced and require the RpoS-controlled general-, stationary-phase- or starvation-stress response At least two independent mechanisms produce the stress-induced LacWork from our lab has provided support for a model in which stress-induced point mutagenesis results from DNA polymerase errors made during acts of DNA double-strand-break repair (DSBR), which is switched to a mutagenic mode, using an error-prone DNA polymerase, specifically during stress lac increases mutation rate over 1000-fold; whereas I-SceI-induced DSBs made in another molecule provoke lac reversion only 3-fold. However the DSBs made in a different molecule from lac can again stimulate lac reversion dramatically if one end of the broken DNA molecule contains DNA identical to DNA next to lac, such that homologous repair with the lac region can be initiated First, point mutagenesis requires homologous-recombinational (HR)-DSBR proteins RecA (EG10823), RecBC (EG10824 and EG10825), RuvA (EG10923), RuvB (EG10924), and RuvC (EG10925) + point mutagenesis occurs by the same mechanism as “normal” stress-induced mutagenesis in that both require the HR-DSBR proteins dinB (EG13141), encoding DNA polymerase (Pol) IV dinB, 10- and about 2-fold, respectively. dinB upregulation might account for some or all of the requirement for induction of the SOS and RpoS responses for stress-induced point mutagenesis, though this has not been demonstrated.Second, I-SceI-stimulated stress-induced Lac+ point mutations are indistinguishable in their Lac+ mutation sequences via error-prone HR-DSBR in which DinB/Pol IV has been licensed to participate in the HR-DSBR reaction The similarity of the proteins required for I-SceI-stimulated and “spontaneous” stress-induced mutagenesis argues that both occur by the same mechanism, as does the finding that I-SceI-induced and “normal” stress-induced LacFinally, HR-DSBR is not always mutagenic but rather switches to a mutagenic mode, with DinB/Pol IV participating, under stress. This switch is controlled either by entry of cells into the stationary phase, or, in log-phase cells if the RpoS stationary-phase stress-response transcriptional activator is expressed inappropriately via its coupling to two stress responses (SOS and RpoS). Mutagenesis is potentially also restricted in genomic space via being coupled to potentially localized DNA synthesis during DSBR Thus, mutagenesis is limited to times of stress E. coli+ stress-induced point mutants show, respectively, ∼20 and ∼50 times more loss-of-function mutations in chromosomal genes throughout their genomes than are found in Lac− cells that starved for the same length of time: their Lac− neighbors from the same Petri plates. Those Lac− cells represent the main population whereas some or all of the Lac+ mutants arose from a more mutable subpopulation: a hypermutable cell subpopulation (HMS). The evidence that the hypermutability of this HMS is transient is, second, that once the cells have become Lac+, they do not have elevated spontaneous + mutants were picked and analyzed these colonies were mostly pure, not mosaic, for the unselected mutations that they carried, indicating that they accrued the unselected chromosomal mutations during or before acquiring the Lac+ mutation, not after, further showing that the mutability was transient In this paper, we investigate a third level of restriction/limitation or regulation of mutagenesis: its limitation to a subpopulation of stressed cells while the main population appears to be unaltered. In the Lac system, there is strong evidence that a subpopulation of cells becomes transiently hypermutable, resulting in mutations in genes throughout the genome. First, + point mutants + point mutants E. coli cells derived from the HMS and find a level that need not preclude fitness. Second, we provide two lines of experimental support and mathematical modeling that support the idea that the HMS generates most or all, not just a minority of, Lac+ stress-induced point mutants. Finally, we consider a model for a mechanism by which the HMS is differentiated.Although there is consensus in the field regarding the existence of the HMS, both the extent of HMS-cell mutagenicity and the importance of the HMS to most stress-induced mutagenesis are currently unresolved. First, the HMS could either be important or not. On the one hand, the HMS has been hypothesized to give rise to essentially all stress-induced Lac+ mutants are reported in previous studies, but were not used previously to estimate the numbers of mutations per genome. We used the previous data to estimate numbers of mutations per genome directly from the lactose-selection plates to specific indicator media that, for example, showed a different color colony for fermentation-defective mutants. This technique is likely to miss some mutants that are overlapped with wild-type colonies. By contrast, in the previous Salmonella study + colonies and purified them by streaking, patched them into grids, grew, then replica-plated to media that would indicate auxotrophic mutations by failure of the patch to grow on medium lacking amino acids and bases. This technique is likely to produce fewer false negatives due to overlap of mutant with non-mutant colonies. To understand whether their somewhat different result arose from use of a different organism or the different mutation-detection method, we used their presumably more sensitive method with E. coli to improve estimates of unselected secondary mutations per stress-induced mutant genome.To better understand the potential fitness impact of cells' entering into a transient hypermutable state, we wished to estimate the number of mutations expected per genome in cells that have undergone stress-induced mutagenesis. Numbers of unselected secondary mutations among Lacr genome , and we mutation /Text S1,r genome /Text S1.E. coli, the purify-and-patch method is more sensitive than direct transfer by replica plating for three mutant phenotypes scored (−3) Mal− mutations per Lac+ cell Xyl− mutants per Lac+ point-mutant colony, implying 2.2 mutation clusters in addition to Lac+ per genome (−3) auxotrophic mutants per Lac+ point mutant or the PBAD promoter replacing the phage lambda attachment site (attλ) in the chromosome (“PBAD-only” strain). These strains are negative-control strains for experiments presented below. In BAD-only strain for loss-of-function mutations among Lac+ revertants by direct transfer via replica plating straight from lactose plates onto indicator and selective plates as performed in + mutants with secondary mutations from those previously reported, p = 0.697, (z-test with Yates correction) (E. coli studies The somewhat higher estimates of secondary mutation clusters per genome in this study compared with those estimated from previous oli data is expecrection) . This ru+ per stress-induced-mutant cell genome.Taken together, the data indicate between about one and 3.4 mutation clusters in addition to Lac+ mutants with the linked mutations in the codAB genes (EG11326 and EG11327) 10kb from lac. Previously, loss-of-function mutations in the codAB genes, which confer resistance to the nucleotide analogue 5-fluorocytosine (5-FCR) were shown not to form independently of Lac+ mutations, whereas unlinked chromosomal mutations did, in a study using the direct-transfer-by-replica-plating method E. coli loci confer 5-FCR when mutated, but only codAB mutations confer 5-FCR without also conferring resistance to 5-fluorouracil [5-FU], which is how these mutations were distinguished codAB and lac using the purify-and-patch method for detecting 5-FCR mutants (R (5-FU-sensitive) mutations in codAB are more frequent among Lac+ mutants than are unlinked mutations fusion gene from 30 independent Lac+ point-mutant isolates carrying secondary mutations. We find that the mutation sequence profile is indistinguishable from those previously reported for stress-induced mutants lac frameshift allele endonuclease-defective mutants that cannot make nicks in the transfer origin of on the F', and more than 50-fold above levels in TraI+ cells and are greatly stimulated by creation of DSBs next to + mutagenesis were associated with secondary mutagenesis of unselected genes throughout the genome , or xylose (Xyl−), or a mucoid-colony or auxotrophic phenotypes were not decreased among I-SceI-induced Lac+ point mutants as compared with negative-control strains that did not experience cleavage by I-SceI: the “enzyme-only” or “PBAD-only” controls or carry the chromosomal regulatable promoter without the I-SceI gene, Δattλ::PBAD (“PBAD only”), for secondary chromosomal mutations. The proportion of Lac+ point mutants carrying a chromosomal secondary mutation was no different for cells expressing I-SceI with no cutsite (enzyme only) compared with the PBAD-only strain, p = 0.697, (z-test with Yates correction) . This delac by I-SceI and repair of the break were not sufficient to increase stress-induced Lac reversion; in addition, the cells had to be either in stationary phase, or expressing the stationary-phase- stress-response transcriptional activator protein RpoS (EG10510) + and genome-wide secondary mutations are coupled and induction of I-SceI-DSBs is not sufficient to render the whole population hypermutable.Thus the elevated mutability observed among the DSB-induced Lac+ revertants, not merely a small fraction as had been suggested + reversion mutations was also observed in those Lac+ mutants demonstrably descended from the HMS: those carrying phenotypically-detectable secondary mutations in their genomes , encoding error-prone DNA pol IV, with lac causing a mutator state Mathematical modeling of previous data led two groups to favor the hypothesis that the HMS produced only 10% of stress-induced Lac+ mutants arose from the HMS and finding a lower estimated value than in similar estimates from “double” mutants + single mutants + mutation, a point which has been supported experimentally by evidence that Lac+ colonies with secondary mutations are mostly pure, not mixed for those mutations +. In + mutation. Our model both predicts the apparent lower mutability of single Lac+ mutants seen previously + single mutants and multiple mutants arise from a common population by a common mutation mechanism—not two different mutation mechanisms (one involving dinB amplification and one not) as suggested The hypothesis that only 10% of LacThe existence of a transiently mutable cell subpopulation indicates a differentiated state in a “bi-stable” cell population. We consider a possible model for the origin of the HMS . We sugg+) mutation that allows growth, and relief of their nutritional stress.This model predicts that cells will spend differing lengths of time in the HMS. Pennington and Rosenberg BADI-SceI transcription was repressed by glucose in the medium until stationary phase, when glucose would be exhausted and leaky expression from PBAD would ensue, just prior to plating on the selective lactose medium. Leaky expression from PBADI-SceI continues on the lactose selection pates − stressed-cell population did not experience the same level of secondary mutagenesis as the I-SceI-induced point mutants per genome (Results). We also supported previous findings that mutations at lac occur in clusters lac do + per genome (1 to 3.4 mutations×1.67 mutations per cluster). This is a maximal estimate given that chromosomal mutations might not be clustered similarly, though this hypothesis seems unlikely. Could a developmental program that generates at most 2–6 additional mutations per genome be adaptive for the rare cells that generate an adaptive mutation? This will depend on how many of the additional mutations are not synonymous, and how many of the genes they fall in are relevant to the specific environment the stressed bacterium inhabits. We have no way to assess the latter, but our rough estimate of the former is that about 29.5% of all mutations falling in anywhere in the genome will affect coding 6 = 0.877 , but this too is probably significantly reduced by the likelihood that many of the genes in the genome are irrelevant to fitness in any given environment , 100 µg/ml rifampicin (Rif), 40 µg/ml 5-bromo-4-chloro-3-indoyl β-D-galactoside , on M9 B1 lactose plates, with supplements as above, and suspending in M9 buffer. Aliquots were plated on LBH-Rif-X-gal-Glu medium, incubated overnight at 37°C, and isolated colonies were patched, grown and replica plated as described above.Unselected secondary mutations among Lac+ mutant frequency in the Lac assay, we employed the chromosomal E. coli I-SceI endonuclease system constructed by our lab E. coli genome SceI-endonuclease open reading frame is cloned in front of the E. coli arabinose-inducible PBAD promoter and the expression cassette is present in the E. coli chromosome, replacing the phage lambda attachment site, attλ. We used strains carrying a chromosomal cassette of the PBAD promoter with or without the I-SceI gene and strains with or without the I-SceI cutsite on the F' episome, 4.5 kb from of the lac allele in the mhpA (EG20273) gene To increase Laclac region of Lac+ mutants containing chromosomal secondary mutations was PCR amplified with primers 5′-ATATCCCGCCGTTAACCACC-3′ and 5′-CGGAGAAGCGATAATGCGGTCGA-3′ and sequenced with primer 5′-ATATCCCGCCGTTAACCACC-3′.The Table S1Similar Secondary Mutation Frequencies in New and Old Strains.(0.06 MB DOC)Click here for additional data file.Table S2+ Reversion-Mutation Sequences.Summary of Generation-Dependent Lac(0.06 MB DOC)Click here for additional data file.Text S1Supplementary Text and References.(0.10 MB DOC)Click here for additional data file.
Many studies have examined the relationship between long-term radiographic damage and physical function. However, it is not known if short-term radiographic progression is also associated with physical function.To investigate the longitudinal relationship between physical function and both the level of radiographic damage and the radiographic progression rate in patients with early or advanced active rheumatoid arthritis.The database for the 2-year Trial of Etanercept and Methotrexate with Radiographic Patient Outcomes (TEMPO) was used for this study. Physical function was measured by the Health Assessment Questionnaire (HAQ) score at baseline, 6 months and 1 and 2 years. Radiographs of the hands and feet, taken at the same time points, were scored by the van der Heijde-modified Total Sharp Score (TSS). The HAQ score was modelled using generalised mixed linear modelling by TSS or progression in TSS adjusted for age, sex, treatment and disease activity.After adjustment for age, sex and disease activity, both TSS and the change in TSS (progression rate) were significant determinants of the HAQ score. When radiographic progression was divided into four categories , results showed that HAQ scores tended to be higher with a higher rate of progression. Patients with negative progression scores had lower HAQ scores than patients with positive progression scores.Patients with greater radiographic damage, and those with recent radiographic progression, have a higher degree of disability. Rheumatoid arthritis (RA) affects almost 1% of the adult population.–et alet al reported that joint damage progresses continuously over the first 20 years of RA and that this damage accounts for 25% of the disability seen in patients.Progressive radiographic damage and deterioration in physical function occur in patients with RA who do not receive timely effective treatment. Several long-term prospective studies have looked at the relationship between radiographic damage and physical function in patients with RA.8–13Although studies have shown that a relationship between physical function and radiographic damage exists, they have mostly examined this association looking at radiographic damage over the long term. It is not known whether progression of radiographic damage over short periods is associated with immediate impairment in physical function. This question is particularly relevant today because of the availability of powerful treatments that can prevent radiographic progression if started early enough. For example, in clinical trials, etanercept, either alone or in combination with methotrexate, has been shown to reduce radiographic progression in patients with recent and long-term RA.11Our study used data from the Trial of Etanercept and Methotrexate with Radiographic Patient Outcomes (TEMPO),–16TEMPO was a multicentre, double-blind study that compared the effect of etanercept plus methotrexate versus etanercept alone versus methotrexate alone in 686 patients with active RA of 6 months to 20 years’ duration.14All patients were randomly assigned to one of three treatment groups: etanercept (25 mg twice-weekly subcutaneous doses), methotrexate or combination therapy with etanercept and methotrexate.Physical function was measured by the Health Assessment Questionnaire (HAQ) at baseline and at protocol-specified intervals throughout the study; scores at baseline and 1 and 2 years were used in the analysis. The HAQ is scored on a scale of 0–3, with higher scores indicating increased disability.Radiographs of the hands, wrists and forefeet were taken at baseline, 6 months, 1 year and 2 years. Digitised images were scored by two readers, using the van der Heijde-modified Sharp method.To control for within-patient correlation, generalised mixed linear modelling was used to model the dependent variable HAQ score by the absolute Sharp score (damage score) or the interval change in Sharp score, with age, sex, disease duration, treatment, Disease Activity Score (DAS) and C-reactive protein (CRP) levels as covariates. If interval change in Sharp score was entered as an independent variable, the HAQ score at the end of the interval was chosen as the dependent variable in the model and the HAQ score at baseline was omitted. A random intercept and a compound symmetry covariance structure seemed to best fit the data. Results are expressed as estimated marginal means. In separate analyses, the interaction of disease duration and (change in) Sharp score with respect to HAQ score was tested.Of the 686 randomly assigned patients, 622 (91%) had a baseline and at least one follow-up film and were included in the radiographic analysis. No statistically significant differences were found among the three treatment groups in baseline demographic and disease characteristics. Patients included in the analysis were predominantly women (76.5%), with mean disease duration of 6.35 years, mean HAQ score of 1.72 and mean total Sharp score (TSS) of 33.0 at baseline.In the linear mixed model, which was applied here in order to aggregate data of baseline, year 1 and year 2 data under simultaneous adjustment for within-patient correlation, the TSS was significantly positively associated with the HAQ score independently of the DAS . A numbeThe HAQ score is primarily determined by disease activity. In the context of a clinical trial, it is expected that the disease activity would show important variation that would easily obscure a contributory and independent association with radiographic damage or progression. We first investigated the relationship between the DAS and the HAQ score. After adjustment for age and sex, the relationship between DAS and the HAQ score was almost linear . This liTo visualise the adjusted relationship between the HAQ score and the Sharp score, Sharp scores were divided into six categories of 10 Sharp units each and estimated marginal means were calculated. In an attempt to determine whether the documented association between the Sharp score and the HAQ score, as well as the association between the change in Sharp score and HAQ score, was dependent on disease duration, we tested the following interactions: Sharp score and disease duration and change in Sharp score and disease duration, with disease duration dichotomised at a cut-off level of 3 years, with respect to explaining variation in HAQ score. Both interactions were not statistically significant (results not shown)This analysis provides further evidence for the longitudinal relationship between radiographic damage and physical function. As shown, progression of radiographic damage over short periods of time is associated with worse physical function, which is independent of the effects of inflammatory disease activity (DAS). In addition, patients who had negative radiographic progression tended to have better physical function than patients with zero or low positive progression scores. This finding gains importance in the light of the low radiographic progression rates that were found in the 2-year TEMPO trial.et al also found a significant contribution of joint damage (measured by van der Heijde’s modification of the Sharp score) to explain the variation in physical function.19In a 10-year longitudinal study,There are important differences between these previous studiesIt is not a coincidence that studies using longitudinal data analyses are consistent with the existence of a relationship between radiographic damage and physical function. It has been hypothesised that this relationship exists, but it has turned out to be rather difficult to confirm it statistically in studies using cross-sectional analytical approaches.et al reported that the irreversible part of the HAQ score was between 0% and 33%, increasing with the duration of RA.Physical function is often considered to be a rather static outcome. However, recent experience from randomised clinical trials evaluating highly effective treatments has shown that impairment of physical function in patients with RA is reversible to some extent. In a recent analysis, Aletaha et al showed that the best way to prevent radiographic damage is to start treatment before such damage has occurred.et al showed that the rate of radiological progression is set during the early stages of RA and suggested resetting this progression rate through pharmacological treatment as soon as possible after the disease is recognised.25–Many studies support the use of early treatment of RA. Landewé Rheumatologists frequently question whether the small differences in radiographic progression between trial arms are clinically meaningful. This analysis supports the view that subtle differences in radiographic progression have a measurable impact on physical function, although most probably at a level that is not truly clinically meaningful for the patient over a relatively short period. The question, however, is how physical function would be influenced if mild progression were allowed for years and years, using the argument that small fluctuations are not clinically meaningful.This analysis suggests that in patients with RA, greater radiographic damage and recent radiographic progression correlate with a higher degree of disability, after adjustment for age, sex, disease duration and disease activity.
The fourth author's name was spelled incorrectly. The correct name is: Ethan M. Richards. The correct citation is: Hur C, Hayeck TJ, Yeh JM, Richards EM, Spechler SJ, et al. (2010) Development, Calibration, and Validation of a U.S. White Male Population-Based Simulation Model of Esophageal Adenocarcinoma. PLoS ONE 5(3): e9483. doi:10.1371/journal.pone.0009483.
In the crystal structure, intermolecular N—H⋯O interactions link the molecules into a three-dimensional network. A weak C—H⋯π interaction is also found.In the title compound, C Å b = 9.852 (2) Å c = 14.266 (3) Å V = 1297.8 (5) Å3 Z = 4 Kα radiationMo −1 μ = 0.11 mmT = 294 K 0.30 × 0.20 × 0.20 mm Enraf–Nonius CAD-4 diffractometeret al., 1968T min = 0.971, T max = 0.979Absorption correction: ψ scan (North 2599 measured reflections1378 independent reflectionsI > 2σ(I)1157 reflections with R int = 0.027 3 standard reflections frequency: 120 min intensity decay: 1% R[F 2 > 2σ(F 2)] = 0.050 wR(F 2) = 0.132 S = 1.33 1378 reflections170 parametersH-atom parameters constrainedmax = 0.31 e Å−3 Δρmin = −0.40 e Å−3 Δρ CAD-4 Software (Enraf–Nonius, 1989CAD-4 Software; data reduction: XCAD4 (Harms & Wocadlo, 1995SHELXTL (Sheldrick, 2008SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL and PLATON (Spek, 2009Data collection: 10.1107/S1600536809032991/hk2731sup1.cif Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809032991/hk2731Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:
Vectors of trypanosomiasis – tsetse (Glossinidae) in Africa, kissing-bugs (Triatominae) in Latin America – are very different insects but share demographic characteristics that render them highly vulnerable to available control methods. For both, the main operational problems relate to re-invasion of treated areas, and the solution seems to be in very large-scale interventions covering biologically-relevant areas rather than adhering to administrative boundaries. In this review we present the underlying rationale, operational background and progress of the various trypanosomiasis vector control initiatives active in both continents. Trypanosoma cruzi) was believed to infect over 24 million people , followth ISCTRC in Mombasa, Kenya, in October, 1999. The recommendation was presented to the 36th summit of the OAU in Lomé, Togo, in July 2000. In response, the Heads of State and Government of the 36 member states of the OAU passed resolution AHG/Dec.156 XXXVI recognising the seriousness of the tsetse and trypanosomiasis problem, and calling on member states "to act collectively...... to render Africa tsetse-free within the shortest time possible". With this mandate, the OAU set up the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC), which is now an integral part of the AU Commission for Rural Development.For Africa therefore, the idea of a Pan-African initiative against tsetse and trypanosomiasis was discussed and recommended at the 25th ISCTRC meeting in Ouagadougou in October 2001, and its plan of action endorsed by the OAU summits in Lusaka (OAU Decision AHG/Dec.169/XXXVII) and Durban (OAU/AU Decision CM/Dec.661/LXXVI.2002). The General Conferences of the FAO and IAEA adopted resolutions in support of the initiative /RES/12), as did the World Health Assembly of WHO (Res WHA56.7).PATTEC was formally launched at the 26The PATTEC mandate encompasses the whole of sub-saharan Africa where tsetse are endemic, although it is well recognised that it may not be feasible or necessary to eradicate all tsetse species and populations, and that not all regions are currently amenable to intervention. The PATTEC vision is of long-term activities – extending perhaps over 30–50 years – with the hope that during this period each target area will present a suitable 'window of opportunity' when political and economic stability will permit large-scale interventions against tsetse and trypanosomiasis . No specAlthough AU-PATTEC is still at an early stage, considerable progress has been made, most notably in reviving national programmes in a number of countries. Almost all the tsetse-endemic countries now have designated PATTEC focal points within the Ministry of Livestock and/or Ministry of Health, and a series of multinational interventions has begun with support from the national Governments. In addition, the African Development Bank has provided a series of loans and grants totalling some $72 million to support tsetse elimination activities in Ethiopia, Kenya, Uganda, Mali, Burkina Faso, and Ghana, with several other countries currently negotiating similar arrangements. The new regional programmes include the following:G.p. gambiensis, G.m. submorsitans, G. tachinoides) which is planned to extend progressively also into Cote d'Ivoire, Guinea, and Senegal- The 'Cotton Belt' of Mali, Burkina Faso, and northern Ghana - The Lake Victoria Basin, including parts of Kenya, Uganda, Tanzania, and Ruanda - Southern Rift Valley – southern Ethiopia and neighbouring parts of Sudan of southern Mozambique and northeastern South Africa - The southeastern tsetse pocket of Botswana, Namibia, western Zambia and southern Angola.- The southern tsetse belt and traps for monitoring tsetse densities, appear to have been very successful, with no tsetse encountered since completion of the interventions in June 2006 [G.m. centralis belt of the four countries [Of these, the southern programme is currently the most advanced, and shows a clear example of what can be achieved through multinational cooperation. The southern he 1960s ,32-34 buhe 1960s . In 2000une 2006 . But theountries ,38. The The human trypanosomiases – Chagas disease in Latin America, Sleeping Sickness in Africa – may be eliminated as major public health problems within the next decade or so. In Latin America, the campaigns have focused primarily on vector control – elimination of domestic vector populations by indoor insecticide spraying – although the strategy is now changing to give additional emphasis to detection and treatment of new cases that may occur as a result of adventitious silvatic Triatominae entering houses . In AfriComparing the two situations, both before and after the current initiatives, may be premature in the light of on-going interventions, but seems to reveal a series of key features that may be conducive to success. Of paramount importance is the political mandate, giving legitimacy to large-scale interventions, and greatly assisting – although by no means guaranteeing – the continuity of action required. This has been relatively weak for the Latin American initiatives, and requires constant reaffirmation as national policies change. It is stronger for the PATTEC initiative, and the mandated requirement for annual reporting to the AU Heads of State and Government provides regular opportunities for reaffirmation in the light of progress achieved. The political mandate also influences resource allocation, both in operational funds and the executive personnel who implement the control interventions. It is noteworthy that – so far – almost all funding for the current trypanosomiasis control initiatives, both in Latin America and in Africa, has come from national governments – either from national budgets or repayable loans.Of parallel importance is the academic and research community. Where this is strong in some countries of Latin America, it has greatly helped to promote continuity of control interventions, as well as assisting in problem-solving and in programme monitoring and evaluation. Where it is weak however, or disconnected from the control programmes, it can be a distraction and hindrance . The indThe importance of the political mandate and of the academic community is perhaps equally relevant for the control of any vector-borne disease, but for the trypanosomiases one further factor is of crucial importance. For both African and American trypanosomiasis, the vectors are exquisitely vulnerable to currently available control techniques. The low reproductive rates and limited genetic variability of tsetse and of Triatominae essentially restrict their ability to respond to the changing circumstances imposed by available control interventions . This caThe authors declare that they have no competing interests.CJS and JPK contributed equally to this review.
Wernicke's encephalopathy is an acute, potentially fatal, neuropsychiatric syndrome resulting from thiamine deficiency. The disorder is still greatly under-diagnosed, and failure to promptly identify and adequately treat the condition can lead to death or to the chronic form of the encephalopathy - Korsakoff's syndrome. Wernicke's encephalopathy has traditionally been associated with alcoholism but, in recent years, there has been an increase in the number of clinical settings in which the disorder is observed.We report the case of a 45-year-old Caucasian woman who arrived at the emergency room presenting signs of marked malnutrition and mental confusion, ataxic gait and ophthalmoplegia. Main laboratory test findings included low serum magnesium and megaloblastic anemia. Brain magnetic resonance imaging revealed increased T2 signal in the supratentorial paraventricular region, the medial regions of the thalamus and the central and periaqueductal midbrain. The diagnosis of Wernicke's encephalopathy was made at once and immediate reposition of thiamine and magnesium was started. The patient had a long history of recurrent thoughts of being overweight, severe self-imposed diet restrictions and self-induced vomiting. She had also been drinking gin on a daily basis for the last eight years. One day after admittance the acute global confusional state resolved, but she presented severe memory deficits and confabulation. After six months of outpatient follow-up, memory deficits remained unaltered.In this case, self-imposed long-lasting nutritional deprivation is thought to be the main cause of thiamine deficiency and subsequent encephalopathy, but adjunct factors, such as magnesium depletion and chronic alcohol misuse, might have played an important role, especially in the development of Korsakoff's syndrome. The co-morbidity between eating disorders and substance abuse disorders has emerged as a significant health issue for women, and the subgroup of patients with anorexia nervosa who also misuse alcohol is probably at a particular risk of developing Wernicke-Korsakoff syndrome. The present case report highlights this relevant issue. Wernicke's encephalopathy (WE) is an acute, neuropsychiatric syndrome resulting from thiamine deficiency. According to autopsy-based studies, the disorder is still greatly underdiagnosed in both adults and children: the classic triad of WE - the acute onset of ocular abnormalities, ataxia and a global confusional state - is present in only 16% of affected patients [Thiamine (in its converted form: thiamine pyrophosphate) is necessary to several biochemical pathways in the brain, such as intermediate carbohydrate and lipid metabolism and production of amino acids and glucose-derived neurotransmitters . Although the disorder has been traditionally associated with alcoholism, any condition of unbalanced nutrition that lasts for 2-3 weeks can lead to thiamine depletion and brain lesions - usually in vulnerable regions, with high thiamine content and turnover, in diencephalic and brainstem areas . In receWE remains largely a clinical diagnosis. No specific diagnostic abnormalities have been found in cerebrospinal fluid, brain imaging or electroencephalograms . AlthougWE is a medical emergency. In patients for whom the disorder is suspected, thiamine should be initiated immediately in order to prevent irreversible brain damage which can lead to death, with an estimated mortality rate of about 20%, or to the chronic form of the encephalopathy - Korsakoff's syndrome - in up to 85% of survivors . KorsakoThere have been a few case reports of WE in anorexia nervosa (AN) in literature -9. In mo6/L (NR = 4.2-5.4 × 106/L); folic acid = 2.51 ng/mL (NR = 3.2-17.0 ng/dL), B12 = 300.1 pg/mL (NR = 202-900 mg/dL). The key findings of the serum laboratory tests run were: urea = 102 mg/dL (NR <50 mg/dL); albumine = 2.9 mg/dL (NR = 3.4-4.8 g/dL); triglycerides = 86 mg/dl (NR <150 mg/dL); low-density lipoprotein = 76 mg/dL (NR <100 mg/dL); high-density lipoprotein = 17 mg/dL (NR >40 mg/dL); magnesium = 0.47 mg/dL (NR = 1.3-2.1 mEq/L); and phosphorus = 2.3 mg/dL (NR = 2.7-4.5 mg/dL). The results of the following tests were found to be within the limits of normality: sodium; potassium; creatinine; glucose; amylase; lipase; alanine and aspartate aminotransferase; gamma-glutamyl transpeptidase; alkaline phosphatase; and blood coagulation. The results of serologies for HIV, hepatitis B and C were negative.The patient was a Caucasian 45-year-old, non-smoker, single woman with one son. She had been a model when a teenager but was currently unemployed, arrived at the emergency room presenting mental confusion, ataxic gait and ophthalmoplegia , without any other neurological focal sign. Her physical examination showed marked malnutrition and paleness. Her body mass index was 16.42 . Cerebrospinal fluid analyses were normal. A brain MRI (performed the next day) showed, at T2, a thin hypersignal halo in the supratentorial periventricular region, increased T2 signal in the medial regions of the thalamus and in the central and periaqueductal midbrain. There were enlarged perivascular spaces in the basal nuclei topography, a thinned corpus callosum and widened cerebellar sulci Figures , 4. A heThe diagnosis of WE was made at once and immediate reposition of magnesium and thiamine was started. After 3 hours the patient presented a sudden decrease in the level of consciousness (Glasgow coma score fell from 15 to 10), with pO2 saturation of 99%, blood pressure of 110 × 70 mmHg, serum glucose 73 mg/dL and no hydroeletrolytic imbalance. The patient did not present with cardiac dysrhythmia, dyspnoea, cutaneous rash or flushing. General support measures were maintained and the patient remained under close observation.The patient's mother and son informed us that she has been dieting since she was 30 years old. The patient's goal was to weigh 30 kg and this goal was easily reached. From then on, she would never allow herself to weigh over 40 kg. Periods of amenorrhea were common. Recurrent thoughts about actually being fat led her to severely restrict the diet. Every 40 days she spent 10 days vomiting everything she ate and drank. Such information allowed us to diagnose anorexia nervosa of the binge eating/purging type.In addition, her relatives informed us that the patient had been drinking gin on a daily basis for the last eight years. They told us that she drank about a bottle of gin every three days . There was no evidence that use of alcohol had escalated over the years and she had never tried to quit or cut back the amount she drank. Besides a certain moodiness, there were no other alcohol withdrawal symptoms. Likewise, in the emergency room she did not present any signs of alcohol withdrawal.She was admitted to the psychiatric ward. One day after admission her acute global confusional state resolved, but she presented severe memory deficits and confabulation. Psychiatric hospitalization lasted for a month, during which a daily reposition of 600 mg of thiamin was maintained. Magnesium replacement was made with 450 mg/day of magnesium oxide orally and magnesium sulfate (4 g/d for the first three weeks and 1 g/d in the last week). In a neuropsychological battery (the CAMCOG ) performWE is considered to be a rare complication of AN . In the In this case, self-imposed long-lasting nutritional deprivation is thought to be the main cause of thiamine deficiency and WE, but adjunct factors might have played an important role, especially in the development of KS: magnesium (Mg) depletion and chronic alcohol misuse. Mg as a co-factor has a crucial role in the proper catalytic action of thiamine pyrophosphokinase in the conversion of thiamine into thiamine pyrophosphate. Mg depletion aggravates thiamine deficiency fostering the development of WE . AlcoholThe patient did not present with some of the crucial features of alcohol dependence syndrome but she maintained a pattern of high ingestion of alcohol for many years. The metabolism of alcohol raises the demand for thiamine, at the same time alcohol decreases the amount of thiamine transported across the intestinal mucosa and impairs the conversion of thiamine to thiamine pyrophosphate . Data inLancet Neurology [The patient suffered a sudden decrease in the level of consciousness a few hours after thiamine reposition was started. That raised the question of whether the treatment had an adverse effect on her. Although this hypothesis could not be ruled out, we believe that such an event was part of the consciousness alterations seen in WE, since the patient had no additional sign of an anaphylactic or anaphylactoid reaction. To date, there is no definite consensus on the optimum dose, frequency, route or duration of thiamine treatment for WE but a recent extensive revision on WE published in the journal eurology recommeneurology .et al. [et al. [In the last decade, the co-morbidity between eating disorders and substance abuse disorders has emerged as a significant health issue for women. Patterns of association vary across eating disorder subtypes. In a review of 25 studies, Holderness et al. calculat [et al. reportedThere is a significant co-morbidity between eating disorders and substance related disorders and the subgroup of patients with AN who also misuse alcohol is probably at a particularly high risk of developing Wernicke-Korsakoff syndrome. Highlighting this issue, we presented a clinical case in which AN, combined with magnesium depletion and chronic alcohol misuse, had led to the presentation of the complete Wernicke-Korsakoff syndrome.AN: anorexia nervosa; Mg: magnesium; MRI: magnetic resonance imaging; NR: normal range; WE: Wenicke's encephalopathy.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.LS and LFALS were responsible for the patient's clinical care and the first draft of the case report section. CRD and CGJ were major contributors in writing the manuscript. CEMB was responsible for the clinical care supervision, coordinated the whole publication effort and reviewed all manuscript versions. All authors read and approved the final manuscript.
Sub-Saharan Africa is facing rapidly increasing prevalences of cardiovascular disease, obesity, diabetes and hypertension. Previous and ongoing undernutrition among pregnant women may contribute to this development as suggested by epidemiological studies from high income countries linking undernutrition in fetal life with increased burden of non-communicable diseases in later life. We undertook to study the risks for hypertension, glucose intolerance and overweight forty years after fetal exposure to famine afflicted Biafra during the Nigerian civil war (1967–1970).2) were compared between the three groups. Fetal-infant exposure to famine was associated with elevated systolic and diastolic BP, increased p-glucose and waist circumference , increased risk of systolic hypertension , IGT and overweight as compared to people born after the famine. Limitations of this study include the lack of birth weight data and the inability to separate effects of fetal and infant famine.Cohort study performed in June 27–July 31, 2009 in Enugu, Nigeria. Adults born before (1965–67), during (1968–January 1970), or after (1971–73) the years of famine were included. Blood pressure (BP), random plasma glucose (p-glucose) and anthropometrics, as well as prevalence of hypertension (BP>140/90 mmHg), impaired glucose tolerance , diabetes , or overweight (BMI>25 kg/mFetal and infant undernutrition is associated with significantly increased risk of hypertension and impaired glucose tolerance in 40-year-old Nigerians. Prevention of undernutrition during pregnancy and in infancy should therefore be given high priority in health, education, and economic agendas. Informed consent was obtained from all participants.The Nigerian civil war broke out on 6 July 1967, after the Igbo people in the south-eastern provinces had declared independence as the Republic of Biafra. The war was a culmination of ethnic, economic, and religious tensions among the various peoples of Nigeria. Disapproving of the secession, Nigerian forces rapidly pushed the Biafrans back into a small enclave. Inflow of food to this enclave was cut off. The result was extensive famine among the Igbos, regarded as one of the great nutritional disasters of modern times Of the 1 to 3 million Igbos that are estimated to have lost their lives, only a small fraction (10%) died of military violence. The majority succumbed to starvation. The nutritional emergency was most critical in the Biafran enclave, in which approximately 7 million people - mostly refugees - resided. In August 1968 the first international relief operations were launched but the amount of food provided was clearly not sufficient and the great majority of the Igbos did not get access to this relief food This follow-up study took place between June 27 and July 31, 2009. All shops at the six major market places of Enugu, the former Biafran capital, were systematically covered. People at the markets were actively contacted at their work place. The selection of participants was restricted to men and non-pregnant women who knew they were born in the southeast of Nigeria between 1965 and 1973. All subjects reported year of birth and 73% reported at least month and year. To confirm the birth year specified by the subject, we asked each person for a short review of his or her family history during the Nigerian civil war. The vast majority (more than 90%) of eligible subjects accepted participation in the study. It was not possible to do a systematic categorization of those who declined.Data collection was conducted by MH, PT, PU, as well as doctors and medical students from the University of Nigeria Teaching Hospital (please see acknowledgements). Recruitment was performed by all team members, whereas measurements of blood pressure and p-glucose were obtained by a smaller group (n = 7) of specially trained investigators. Subjects were asked about level of education, current smoking, previously diagnosed hypertension or diabetes mellitus, and treatment for these conditions. Level of education was categorized as none, primary, secondary or higher education. There was no available information on birth weight or infant nutrition.Lite, Abbott Diabetes Care Ltd, Oxon, UK). A random p-glucose of 11.1 mmol/l or higher was classified as diabetes mellitus and impaired glucose tolerance (IGT) was defined as random p-glucose between 7.8 and 11.0 mmol/l 2 and obesity as BMI over 30 kg/m2. Waist circumference was measured with a measuring tape placed around the abdomen at the level of the upper hip bone, and considered as an indicator of central obesity.All measurements were performed according to predefined standard operating procedures. Participants were instructed to rest seated for at least five minutes before blood pressure (BP) and heart rate were measured. Two readings were taken three minutes apart in the left arm using a validated All participants were briefly counselled about their current health status. Hypertensive or glucose intolerant subjects were referred to the University of Nigeria Teaching Hospital for further investigation and treatment.Subjects born between 1965 and 1967 were categorized as being exposed to famine in early childhood. Subjects born between 1968 and January 1970 (end of war) were categorized as being exposed to famine in fetal life and in infancy. Subjects born immediately after the surrender of Biafra were left uncategorized because of uncertainties in their exposure to famine and local variations in nutritional relief. Accordingly, subjects born between 1971 and 1973 and after the transitional period were categorized as being unexposed to famine.Efforts to limit any potential bias included investigator-driven recruitment of participants (in contrast to subjects actively seeking participation in the study), direct assessments of outcomes with non-operator dependent methods and predefined standardized operational procedures applied by all data collectors.We aimed to include 630 individuals in each group, based on an assumption of a standard deviation of 19 mmHg and a group difference in systolic blood pressure of 3 mmHg or more, given 80% power and a statistical significance level of 0.05. Because of exhausted recruitment at the six study markets, data collection was ended when 1,339 subjects had been included in the study. All subjects were included in logistic regression analyses according to year of birth whereas in group wise logistic regression analyses, the 173 subjects born during the transition from famine to nutritional relief, i.e., from February to December 1970, were excluded.Data are presented as means (SD), proportions (%) and odds ratios (95% CI). Group differences were tested using ANOVA and chi-squared test. Logistic and linear regression analyses were used to calculate odds ratios (OR) and for evaluation of relations between exposure , potential confounders and outcomes . As several previous reports have shown that the associations between early life exposures and adult outcomes vary in relation to sex and adult body size Age differed among the three groups due to inclusion criteria. The proportion of men varied between 66 and 74% in the three groups and was highest in the group born after the famine. Smoking occurred predominantly in men and the proportion of smokers differed in the three groups, SBP and DBP were higher after fetal-infant exposure to famine, as compared to the other two groups, i.e., those exposed in early childhood as well as those that were born after the famine, i.e., were unexposed, In linear regression analyses, SBP and DBP were associated with BMI and male sex , but not with smoking. After adjusting for BMI odds ratios for SBP and DBP in the hypertensive range were of the same magnitude as before adjustment and still significantly higher for the group exposed to fetal-infant famine. In addition, analyses stratified on sex showed that exposure to famine in fetal life and infancy was a risk factor for SBP and DBP in the hypertensive range in both women and men. The OR (95% CI) for severe hypertension in the group exposed to fetal-infant famine was 2.82 (1.18–6.73) in men and 2.30 (0.65–8.10) in women, Odds ratios for high SBP (≥140 mmHg) according to each year of birth and with 1972 as reference year (OR = 1), are presented in Odds ratios for high SBP (≥140 mmHg) in relation to exposure group and stratified on current overweight (BMI≥25), are presented in Random p-glucose was higher in adults exposed to famine in fetal-infant life, In linear regression analyses, random p-glucose was associated with BMI and male sex . In analyses adjusted for BMI, fetal-infant exposure to famine remained as a risk factor for IGT and diabetes in Nigerian subjects in their forties. Stratified analyses on sex showed that the OR (95% CI) for diabetes after exposure to fetal-infant famine was 3.84 (0.95–15.5) in men and 2.06 (0.48–8.86) in women, The group born after the famine was significantly taller than the other two groups, whereas there were no group differences in weight, in utero and in infancy. Comparing unexposed offspring with that of starving pregnant women, fetal-infant undernutrition was associated with significant increases in the prevalence of hypertension and impaired glucose tolerance or diabetes (from 8.0 to 13%). Famine in early childhood was also associated with an increased prevalence of adult blood pressure in the hypertensive range (from 9.5 to 16%). Given the additive effects of early famine and adult overweight (This study showed higher BP, higher p-glucose and higher weight in middle-aged Nigerian people exposed to severe undernutrition erweight , early uOur results are in line with previous epidemiological and experimental studies suggesting that fetal undernutrition contributes significantly to cardiovascular disease risk in adult life Gender differences in developmental programming have previously been described The strengths of this study include the design with prospectively set inclusion criteria and active enrolment of a large cohort of customers and traders at markets, i.e., places where many people in the urban areas gather and work, as other sectors of employment and sites for purchasing of everyday goods are not very developed in this part of the world. Thus we believe that the study cohort is representative for urban settings in sub-Saharan Africa and at highest risk for the present epidemic in non-communicable diseases. Categorization was based on date of birth – i.e., exposure to famine in early life. In addition, since subjects born in the transitional period were excluded, there was no late gestational overlap with famine in the unexposed group. The follow-up time was sufficiently long to establish relations to outcomes that are directly related to adult cardiovascular disease. Finally, we addressed the possibility that smoking confounded our results Although heritability for birth weight has been estimated to range from 25% to 40% It is likely that survival of the healthiest pregnant women occurred during the Biafran famine. The most severe cases of fetal and infant undernutrition are also likely to have died prior to follow-up, either in early childhood or from the increasing cardiovascular morbidity reported from adults residing in the area Before the war, Biafra differed from most developing nations because it had a good supply of food and water, and sound public health policies with many physicians, nurses, hospitals, and clinics The external validity of data from a convenience sample from market places can also be discussed. The method of recruiting participants will have missed subsistence farmers and others not attending the markets, whose nutritional status and other factors are likely to differ from market people. We note that the prevalences of hypertension, diabetes and obesity reported herein are similar to those reported from comparable urban and rural Nigerian cohorts The study design, i.e., comparing long-term outcomes in people born before, during or after famine, has previously been used in utero may programme abnormal glucose homeostasis and vascular endothelial dysfunction, whereas results are less consistent with regard to programming of high blood pressure Previous studies indicate that a nutritional insult - during gestation or the first few months of postnatal life - may be important for later outcome and disease risk The implications of our findings are important. On a population level, a 3.3 mmHg increase in mean SBP and 2 mmHg in DBP can be translated into an estimated increase in cardiovascular deaths by 25% and stroke by 32% In summary, our study demonstrates a fetal-infant contribution to adult hypertension and glucose intolerance in an African cohort. It can be assumed that fetal and infant undernutrition is a significant contributor to the increasing prevalence of hypertension and glucose intolerance also in other parts of sub-Saharan Africa. Therefore, prevention of fetal and infant undernutrition should be given high priority in national health, education, and economic agendas to limit the increase of non-communicable diseases in many African countries. Given that the highest risk for hypertension was found in those undernourished in early life and then growing overweight, it is appropriate to consider that preventing excess growth in later childhood may be as important for reducing adult ill-health as supporting fetal-infant growth.
Tightly-focused, near infrared wavelength light (1064 nm) is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW). Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of Cell immobilization by chemical or physical means alters the physiochemical microenvironment and may give rise to changes in electrochemical potentials across the cell membrane that, in turn affect cellular functions Optical tweezers are proving to be of widespread utility in contemporary research in a number of fields, particularly in the biomedical sciences E. coli, P. fluorescens, S. epidemis, B. subtilis and E. cloacaAmong several spectroscopy techniques, Raman spectroscopy is a particularly potent tool to probe the biochemical composition of cells; it has proved to have the potential of being able to differentiate between, for example, normal and malignant cells The relatively recent marriage between the techniques of optical trapping and Raman spectroscopy −1; the intensity of this band decreases with alcohol concentration Applications of Raman Tweezers to studies of red blood cells (RBCs) have attracted considerable contemporary attention We describe in the following details of a successful coupling in our laboratory of a Raman spectrometer with an optical trap that allows, without physical contact, simultaneous optical trapping, optical manipulation, and recording of Raman spectra of micron-sized objects floating in a medium. The trapping is accomplished using tightly-focused, 1064 nm wavelength laser light while Raman spectra are recorded by exciting the trapped particle by 785 nm laser light. We have recorded Raman spectra of single, living red and white blood cells under physiological conditions, achieving an enhancement of overall spectral quality that has enabled identification of several additional lines in the Raman spectra of trapped cells.The experimental work involved extraction of blood samples from healthy volunteers. In the present series of experiments, two of the co-authors (AB and EZ) were such volunteers from whom the blood is drawn. Approval for this was obtained from the University Ethics Committee, Manipal University, on 11 December 2008 (reference UEC/35/2008 and UEC/36/2008) and written, informed consent was obtained from the two co-authors whose blood was taken. All the experimental work reported in this paper relied only on blood samples taken from the co-authors, AB and EZ.1 ml of whole blood was extracted from co-authors AB and EZ and was centrifuged at 3000 rpm for 5 minutes to remove the serum. The RBCs were diluted in Phosphate Buffered Saline (PBS) to get a suspension of living RBCs. About 10 µl of serum was again added to 1 ml of suspension to ensure that the RBCs remained intact for an extended period of time. For collecting while blood cells (WBC) same centrifugation procedure is followed. About 20 µl of serum near the buffy coat layer was pippetted out and diluted in 1 ml of PBS to obtain a solution of live WBCs.−2, leading to localized thermal effects such as the formation of bubbles We first discuss a number of considerations that need to be addressed in the process of designing an apparatus that would find practical application in the biomedical sciences. On the trapping of biological matter, an important question relates to potential laser-induced damage within the trapping volume. Such potential hazards are expected to be wavelength and power dependent. For instance, photo-chemically induced alterations in the properties of trapped cells might include damage to DNA, alterations in cell metabolism and chemical effects like oxidation E. coli and CHO cells in the earlier reports have indicated that wavelengths in the close vicinity of 1 µm are expected to cause minimal laser-induced cell photodamage 2) which has high reflectivity at 1064 nm wavelength directed the sufficiently expanded laser beam to the back aperture of our microscope objective. A CCD camera was attached to one of the exit ports of the microscope, enabling visualization and recording of optically trapped micron-sized objects. Manipulation of the trapped objects was achieved simply by means of a controllable x–y translational stage.The laser beam was amenable to manipulation by a 1∶1 telescopic arrangement comprising two convex lenses of equal focal length, f, at a distance of 2f from each other as well as from the back focal plane of the microscope objective. A dichroic mirror that was focused onto the sample by the same microscope objective. The choice of wavelength was dictated by the following considerations. The efficiency of Raman scattering exhibits a λMight it be possible to utilize the 785 nm light for both trapping and excitation purposes? We have considered this possibility and, in some cases, the answer is in the affirmative. However, cognizance has to be taken of the fact that the stiffness of the optical trap strongly depends on the laser beam quality. The diode lasers presently available for Raman excitation have distinctly inferior beam quality to that obtained from a laser like the Nd:YAG laser that we utilize for trapping. Conseqently, use of the Raman laser for trapping results in considerably less efficient trapping. However, this can be tolerated under some circumstances. For instance, in some preliminary experiments we did use only the 785 nm laser beam for both trapping and excitation of RBCs, albeit less efficiently. This scheme certainly cannot be applied to WBCs whose refractive index (1.360±0.004) is somewhat lower than that of RBCs (1.399±0.006); hence a more efficient trap is required when work is to be conducted on WBCs.1). The combination of two dichroic mirrors was carefully chosen such that they transmitted Raman signals to the spectrometer and visible light to the CCD camera so as to facilitate visualization of trapped samples. In our experiments with the 785 nm laser we observed that the output beam contained an additional weak line around 790 nm which might interfere with the Raman signals. To circumvent this, we passed the laser beam through a holographic band pass filter BF that effectively blocked the extraneous 790 nm line. The 785 nm beam was first expanded using a beam expander (BE2) so as to overfill the back aperture of the microscope objective and then reflected into the microscope by the 785 nm dichroic mirror with 1024x256 pixels, operating at 140 K.A 1∶1 telescopic arrangement allowed us to exercise proper control over the Raman probe beam. Using the telescopic arrangements both the laser foci was brought to the same point in the sample plane so as to enable simultaneous trapping and excitation. A backscattering configuration was used to collect the scattered signals from the trapped particle, as indicated in −1. Values of laser power used to trap cells and to record Raman spectra were measured by locating an integrating sphere just after the microscope objective in our set-up (−1 (with the spectrometer slit width kept at 100 µm) by measuring the FWHM of the 997 cm−1 Raman band of the polystyrene bead spectrum. The inset to Alignment and calibration of our set-up was accomplished using polystyrene beads of average diameter 3 µm . A bead suspended in deionised water was trapped, typically using a trapping power of ∼5 mW, and the Raman spectrum was recorded by exciting it with 785 nm laser light. Raman spectrometer optics were aligned for maximum signal-to-noise ratio in measured spectra. r set-up . The speIt is important to note here that laser power levels that were incident on the trapped cell were kept low enough to ensure that cell viability was likely to be maintained. In experiments whose results we report in the following, we typically made measurements on a single trapped cell for periods as long as ∼1 hour; in the course of this time we recorded Raman spectra every 5–10 minutes. The fact that recorded spectra was consistent during the entire stretch of the experiment helped allay concerns regarding cell viability. Moreover, other optical trap work conducted in our laboratory over several years −1 was overcome by using a fused silica coverslip which leaves only a peak at 798 cm−1 in the Raman spectrum. We first focus attention on spectra of trapped red blood cells (RBCs) of which typical spectrum is presented in Raman spectra of optically trapped single RBCs and WBCs were measured with our apparatus using trapping and excitation power levels that are significantly less than earlier work A single living RBC was trapped, typically using ∼5 mW power, and Raman excited using typical power levels of ∼5 mW. Our recorded spectrum tends to cover a significantly larger region than that of earlier micro-Raman work −1 and the CH2/CH3 deformation modes primarily from amino acid side chains at 1445 cm−1. The Raman spectrum of RBC seems to be dominated by peaks corresponding to various vibrational modes of heme. The bands obtained here are well resolved and the apparatus that we have set up is clearly sensitive to even faint peaks. The richness of the spectrum brings to the fore the fact that Raman studies of live cells in suspension are more informative as far as molecular concentration and conformation are concerned than micro-Raman studies where the cells are chemically bound to a glass coverslip.The spectra contain a mixture of bands from the heme group and various protein components. The protein bands include the amide at 1649 cm−1, 962 cm−1, 936 cm−1, 898 cm−1, 860 cm−1, 635 cm−1, 620 cm−1, 589 cm−1, 545 cm−1, 517 cm−1, 490 cm−1 and 473 cm−1. Most of these peaks are amenable to assignment on the basis of the conventional Raman measurements on hemoglobin conducted by Hu and coworkers −1, 898 cm−1, 635 cm−1, 517 cm−1 and 490 cm−1 were not assigned in the studies of Hu and coworkers, though these lines may be contributed by the hemoglobin itself. We have assigned all these peaks in the present study using standard literature on vibrational spectroscopy of proteins A comparison of our spectra with those reported by Wood and coworkers The presence of all these peaks in the present measurements on trapped RBCs may be a reflection of the relatively larger sample volume that is probed in our apparatus in comparison to that in the case of micro-Raman studies. Furthermore, the enhanced resolution in the present set of measurements may also be a contributory factor. In the micro-Raman study, the Raman probe beam is inevitably perpendicular to the plane of RBCs whereas in the case of trapped RBCs the orientation is parallel, and the probe beam passes through larger cell volume than is the case in micro-Raman measurements. In other words, the larger optical density in our experimental arrangement might contribute to higher signal sensitivity coupled with enhanced resolution that is achieved in the present experiments.The concentration of hemoglobin in normal red blood cell is typically 32% to 36%; the domination of hemoglobin in IR spectra has been noted before and it has been postulated that this could be ascribed to excitonic coupling between aligned porphyrins in the highly aggregated heme environment of the RBC . Further work needs to be undertaken to gain a deeper insight.−1 to 1800 cm−1 and the peaks that we observed, along with their assignments, are presented in −1, 1321 cm−1, 1138 cm−1 and 489 cm−1 are missing in whole blood spectra. These “missing lines” in whole blood spectra are most likely a consequence of the fact that the whole blood Raman spectrum is the ensemble average of many randomly oriented RBCs within the focused laser spot size (30 µm diameter in these measurements). However, further work needs to be undertaken to verify this conjecture of ours.A comparative analysis was also carried out by recording Raman spectra of blood samples after removal of serum as shown in White blood cells were also probed in the present series of experiments. Relatively little work has been reported Granulocyte and lymphocytes were trapped using typical power levels of ∼5 mW. Some un-trapped and trapped cells are shown in the inset to −1, 1128 cm−1, 998 cm−1, 853 cm−1, 755 cm−1 and 513 cm−1 corresponding to proteins and amino acids are more intense in the granulocyte spectrum than the lymphocyte spectrum. On the other hand, the features at 1486 cm−1, 1087 cm−1, 783 cm−1, 725 cm−1 and 683 cm−1 corresponding to the vibrations of nucleic acid bases and DNA backbone stretching are more prominent in the lymphocyte spectrum. This may be because of the increased nucleus-to-cytoplasm ratio in lymphocytes. However, the Raman line at 975 cm−1 assigned to deoxyribose is very intense in granulocyte spectra (in comparison with lymphocyte spectra). The reason for these remains unclear at present. The above results seem to be more prominent in the Raman spectra recorded with 30 mW power , cell-drug interactions, cell under stress, radiation induced cell damage, cell undergoing malignant conditions and many more. Work on cells under stress and malaria-infected RBCs is currenty underway in our laboratory.